U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 5–B11

Determination of Pesticides and Pesticide Degradates 

in Filtered Water by Direct Aqueous-Injection Liquid 

Chromatography-Tandem Mass Spectrometry

Chapter 11 of

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis 

Determination of Pesticides and Pesticide 

Degradates in Filtered Water by Direct 

Aqueous-Injection Liquid ChromatographyTandem Mass Spectrometry

By Mark W. Sandstrom, Leslie K. Kanagy, Cyrissa A. Anderson, and 

Christopher J. Kanagy

Chapter 11 of

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

Techniques and Methods 5–B11

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Director

U.S. Geological Survey, Reston, Virginia: 2015

For more information on the USGS—the Federal source for science about the Earth, its natural and living 

resources, natural hazards, and the environment—visit http://www.usgs.gov or call 1–888–ASK–USGS.

For an overview of USGS information products, including maps, imagery, and publications, 

visit http://www.usgs.gov/pubprod/.

Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the 

U.S. Government.

Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials 

as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner.

Suggested citation:

Sandstrom, M.W., Kanagy, L.K., Anderson, C.A., Kanagy, C.J., 2015, Determination of pesticides and pesticide 

degradates in filtered water by direct aqueous-injection liquid chromatography-tandem mass spectrometry: U.S. 

Geological Survey Techniques and Methods, book 5, chap. B11, 54 p., http://dx.doi.org/10.3133/tm5B11.

ISSN 2328-7055 (online)
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Background............................................................................................................................................1

Purpose and Scope ..............................................................................................................................2

Methods of Study..................................................................................................................................2

Approach.......................................................................................................................................2

Previous Studies ...................................................................................................................................2

Acknowledgments................................................................................................................................3

Analytical Method..........................................................................................................................................3

Method Number, Schedule, and Code ..............................................................................................3

Scope and Application.........................................................................................................................3

Nomenclature...............................................................................................................................4

Summary of Method.............................................................................................................................4

Safety Considerations..........................................................................................................................4

Interferences.........................................................................................................................................4

Equipment and Supplies ......................................................................................................................5

Sample Collection and Preparation Supplies .........................................................................5

Instrumentation.....................................................................................................................................6

Reagents.................................................................................................................................................6

Neat Reagents..............................................................................................................................6

Reagent Solutions........................................................................................................................7

Standards...............................................................................................................................................7

Sample Collection, Filtration, Preservation, Shipment, and Holding Times ................................8

Sample Filtration ..........................................................................................................................8

Field Quality Control Samples....................................................................................................8

Sample Preservation...................................................................................................................8

Sample Shipment and Holding Time.........................................................................................9

Sample Preparation..............................................................................................................................9

Analysis by Liquid Chromatography-Tandem Mass Spectrometry—Setup and Data 

Acquisition................................................................................................................................9

Overview........................................................................................................................................9

Separation by Liquid Chromatography.....................................................................................9

Tandem Mass Spectrometry (MS/MS) Analysis ..................................................................10

Development of the MRM Method ................................................................................10

Dynamic MRM Conditions...............................................................................................10

Mass Calibration...............................................................................................................11

Sample Analysis Sequence .....................................................................................................11

Use of Isotope-Labeled Internal Standards ..........................................................................11

Quantitation and Calculation of Results..........................................................................................12

Qualitative Determination..................................................................................................................12

Retention Times..........................................................................................................................12

Qualifier Ion Ratios ....................................................................................................................12

Quality Assurance and Quality-Control Criteria ............................................................................14
iv

Routine laboratory QC Samples ..............................................................................................14

Batch QC Samples............................................................................................................14

Instrument Laboratory QC Samples...............................................................................17

CCV Standards .........................................................................................................17

Instrument Detection Level Standards ................................................................17

Third-Party Check Standards ................................................................................17

Reporting of Results ...........................................................................................................................17

Results and Discussion of Method Validation Data...............................................................................18

Data-Quality Objectives.....................................................................................................................18

Data Analysis.......................................................................................................................................18

Assessment of Suitability for the DAI LC-MS/MS Method..........................................................18

Analyte Response as a Function of Concentration.......................................................................19

ESI+ Analytes..............................................................................................................................19

ESI− Analytes..............................................................................................................................19

Method Detection Level Determination..........................................................................................21

Procedure....................................................................................................................................21

Method Detection Level Study Design...................................................................................21

Results of Method Detection Level Determination ..............................................................21

Bias and Variability from Matrix-Spike Recovery Tests...............................................................22

Qualification of Matrix-Spike Sample Results ......................................................................23

ESI+ Mode Analytes..................................................................................................................23

ESI− Mode Analytes..................................................................................................................27

Performance of Laboratory Quality Control Samples During Implementation of Method .....27

Third-Party Check Standards ..................................................................................................29

Laboratory Reagent Blank Samples .......................................................................................29

Laboratory Reagent Spike Samples .......................................................................................29

Application to Environmental Samples—Paired-Sample Stream Study...................................30

Field-Matrix Spikes....................................................................................................................30

Study Design......................................................................................................................30

Spike Recovery in Stream Samples...............................................................................30

Relative Percent Difference of Spike Recovery ..........................................................35

Field Blank Samples ..................................................................................................................36

Environmental Sample Results................................................................................................36

Interlaboratory Comparison of Methods ...............................................................................36

Linear Regression of Concentrations............................................................................37

Detection Frequency in Stream Samples .....................................................................37

Stability Studies...................................................................................................................................40

Stability Study Design ...............................................................................................................40

Maximum Holding Times..................................................................................................40

Stability Study Results ..............................................................................................................41

Tolerable Range of Variation...........................................................................................41

Stability at 4 °C Pesticides ESI+ Mode Analytes.........................................................41

Stability at 4 °C Pesticides ESI− Mode Analytes.........................................................42

Stability at 4 °C Degradates ESI+ Mode Analytes.......................................................42

Stability at 4 °C Degradates ESI− Mode Analytes.......................................................42

Stability at −10 °C Pesticides ESI+ Mode Analytes ....................................................42
v

Stability at −10 °C Pesticides ESI− Mode Analytes ....................................................43

Stability at −10 °C Degradates ESI+ Mode Analytes ..................................................43

Stability at −10 °C Degradates ESI− Mode Analytes ..................................................43

Pesticide Stability Using Dechlorination Reagent (Ascorbic Acid) ESI+ Mode 

Analytes ................................................................................................................43

Pesticide Stability Using Dechlorination Reagent (Ascorbic Acid) ESI− Mode 

Analytes ................................................................................................................44

Pesticide Degradate Stability Using Dechlorination Reagent (Ascorbic Acid) 

ESI+ Mode Analytes ...........................................................................................44

Pesticide Degradate Stability Using Dechlorination Reagent (Ascorbic Acid) 

ESI− Mode Analytes ...........................................................................................44

Pesticide Stability Using Antimicrobial Reagent (Citric Acid) ESI+ Mode 

Analytes ................................................................................................................44

Pesticide Stability Using Antimicrobial Reagent (Citric Acid) ESI− Mode 

Analytes ................................................................................................................45

Pesticide Degradate Stability Using Antimicrobial Reagent (Citric Acid) 

ESI+ Mode Analytes ...........................................................................................45

Pesticide Degradate Stability Using Antimicrobial Reagent (Citric Acid) 

ESI− Mode Analytes ...........................................................................................45

Summary and Recommendations Based on Stability Study Results .......................45

Problematic Compounds.............................................................................................................................45

Summary and Conclusions.........................................................................................................................46

References Cited..........................................................................................................................................48

Glossary.........................................................................................................................................................51

Appendix 1. Supporting Figures and Tables.........................................................................................53

Supporting Figures..............................................................................................................................53

Supporting Tables ...............................................................................................................................54

Figures

1. Example of a calibration curve for metolachlor determined by direct

aqueous-injection liquid chromatography-tandem mass spectrometry showing

response of metolachlor relative to the internal standard metolachlor-d6 plotted 

as a function of concentration, an expanded view of the lowest section of the

calibration curve, and low concentration standard of metolachlor showing 

qualifier and quantifier multiple reaction monitoring ions for metolachlor......................13

2. The qualifier ion response ratio of atrazine and three atrazine degradates

determined by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in positive electrospray ionization mode spiked at different

concentrations in different matrices and reagent water.....................................................15

3. Chromatograms of the multiple reaction monitoring ions for atrazine determined

by direct aqueous-injection liquid chromatography-tandem mass spectrometry at

different concentrations in four different matrices ..............................................................16

4. Response factors as a function of concentration for metolachlor and degradates

determined by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in positive electrospray ionization mode.......................................................21
vi

5. Recovery of atrazine and degradates as a function of background environmental

sample concentration determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in 146 field-matrix-spike samples

collected during the National Water Quality Assessment Program field study from

May to September 2012 .............................................................................................................25

6. Recovery of metolachlor determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization

mode in samples spiked at different concentrations in four different matrices..............26

7. Recovery of 2,4-D determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization

mode in samples spiked at different concentrations in four different sample 

matrices........................................................................................................................................28

8. Recovery of atrazine and degradates determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in 146 field-matrix-spike samples

collected during the National Water Quality Assessment Program field study from

May to September 2012 .............................................................................................................31

9. Comparison of selected pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry gas chromatography-mass

spectrometry in 150 samples from 48 stream sites collected during National Water

Quality Assessment Program field comparison study from May to September 2012 .....39

10. Comparison of selected pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry and liquid chromatography-mass

spectrometry in 150 samples from 48 stream sites collected during National Water

Quality Assessment Program field comparison study from May to September 2012 .....39

Tables

1. Information about pesticide compounds in direct aqueous-injection liquid

chromatography-tandem mass spectrometry method...................................................... link

2. Concentrations of isotope-labeled pesticide compounds in internal standard 

solution ...................................................................................................................................... link

3. High-performance liquid chromatography acquisition parameters ............................... link

4. Electrospray ionization positive mode pump gradient ...................................................... link

5. Electrospray ionization negative mode pump gradient..................................................... link

6. Optimum dynamic multiple reaction monitoring parameters for the determination 

of pesticide compounds by direct aqueous-injection liquid chromatographytandem mass spectrometry ................................................................................................... link

7. Tandem mass spectrometry acquisition parameters ........................................................ link

8. Example of a sample sequence for determination of pesticide compounds in an

analytical batch of 75 environmental samples and associated quality-control

samples by direct aqueous-injection liquid chromatography-tandem mass

spectrometry ............................................................................................................................ link

9. Assignment of pesticide compounds and surrogates to isotope-labeled internal

standards for positive electrospray ionization and negative electrospray ionization

mode analyses ......................................................................................................................... link

10. Acceptance criteria for retention times in direct aqueous-injection liquid

chromatography-tandem mass spectrometry calculated from reproducibility of

pesticide compound retention times in environmental water spike samples at

concentrations from 10 to 5,075 nanograms per liter........................................................ link
vii

11. Acceptance criteria for relative ion intensity ratios for mass spectrometric

techniques used for identification of organic contaminants in food products ............. link

12. Acceptance criteria of qualifier ion ratios for analytes determined in positive

electrospray ionization mode calculated from reagent water and matrix-spike 

samples ..................................................................................................................................... link

13. Acceptance criteria of qualifier ion ratios for pesticide compounds determined in

negative electrospray ionization mode calculated from reagent water and

matrix-spike samples .............................................................................................................. link

14. Quality-control criteria for calibration standards, continuing calibration standards,

and retention times for analyte identification from recent U.S. Environmental

Protection Agency methods that use by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry .................................................................... link

15. Summary of response factor and parameters of linear fit of response factor by

nominal concentration for positive electrospray ionization analytes............................. link

16. Summary of response factor and parameters of linear fit of response factor by

nominal concentration for negative electrospray ionization mode analytes ................ link

17. Summary of method detection levels, laboratory reporting levels, and low- and

high-calibration standards for pesticide compounds determined by direct 

aqueous-injection liquid chromatography-tandem mass spectrometry........................ link

18. Estimated method detection levels for pesticide compounds determined by direct 

aqueous-injection liquid chromatography-tandem mass spectrometry in positive 

electrospray ionization mode calculated according to U.S. Environmental Protection 

Agency procedure from analysis of seven replicate 5- and 10-nanograms per liter 

standards determined in seven consecutive analytical batches.................................... link

19. Estimated method detection levels for pesticide compounds determined by direct 

aqueous-injection liquid chromatography-tandem mass spectrometry in positive 

electrospray ionization mode calculated according to U.S. Environmental 

Protection Agency procedure from analysis of eight replicate 5-, 10-, 25-, 50-, 

and 100-nanograms per liter standards determined in one analytical batch................ link

20. Estimated method detection levels for pesticide compounds determined by direct 

aqueous-injection liquid chromatography-tandem mass spectrometry in negative 

electrospray ionization mode according to U.S. Environmental Protection Agency 

procedure from replicate analysis of 5-, 10-, 50- and 100-nanograms per liter 

standards in seven consecutive analytical batches ......................................................... link

21. Environmental samples collected for validation studies for direct aqueousinjection liquid chromatography-tandem mass spectrometry pesticide method......... link

22. Recovery of pesticide compounds in different water matrices and concentrations

determined by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in positive electrospray ionization mode.................................................... link

23. Recovery of pesticide compounds in different water matrices and concentrations

determined by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in negative electrospray ionization mode................................................... link

24. Recovery of analytes in third-party check standards analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in positive

electrospray ionization mode from September 2012 to March 2013............................... link

25. Recovery of analytes in third-party check standards analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in negative

electrospray ionization mode from September 2012 to March 2013............................... link

26. Occurrence and concentrations of pesticide compounds in laboratory reagent

blank samples analyzed by direct aqueous-injection liquid chromatographytandem mass spectrometry between June 19, 2012, and March 3, 2013....................... link
viii

27. Recovery of pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in laboratory reagent-spike samples

collected during implementation of the method from May 2012 to June 2013.............. link

28. Recovery of pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in 146 field-matrix-spike samples

collected during National Water Quality Assessment Program field comparison

study from May to September 2012 ...................................................................................... link

29. Recovery of pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in five field-matrix-spike samples

fortified at 4,747 nanograms per liter collected during National Water Quality

Assessment Program field comparison study from May to September 2012................ link

30. Relative difference in recovery of pesticides determined by direct aqueous-injection 

liquid chromatography-tandem mass spectrometry in 50 field-matrix-spike and 

spike-replicate samples collected during National Water Quality Assessment 

Program field comparison study from May to September 2012....................................... link

31. Summary of concentrations of pesticides determined by direct aqueous-injection

liquid chromatography-tandem mass spectrometry in 43 field blank samples

collected during National Water Quality Assessment Program field comparison

study from May to September 2012 ...................................................................................... link

32. Summary of concentrations of pesticides determined by direct aqueous-injection

liquid chromatography-tandem mass spectrometry in 150 stream-water samples

collected during National Water Quality Assessment Program field comparison

study from May to September 2012 ...................................................................................... link

33. Parameters for linear fit of concentrations determined by direct aqueous-injection

liquid chromatography-tandem mass spectrometry and gas chromatography-mass

spectrometry in environmental samples collected during National Water Quality

Assessment Program field comparison study from May to September 2012................ link

34. Parameters for linear fit of concentrations determined by direct aqueousinjection liquid chromatography-tandem mass spectrometry and liquid

chromatography-mass spectrometry in environmental samples collected during

National Water Quality Assessment Program field comparison study from May 

to September 2012 ................................................................................................................... link

35. Consistency of detections or nondetections of pesticide compounds in 150

paired-stream samples determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry and gas chromatography/mass

spectrometry during National Water Quality Assessment Program field 

comparison study from May to September 2012................................................................ link

36. Sign test that number of detections in 150 paired-stream samples are the same

for pesticide compounds analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry and gas chromatography/mass

spectrometry ............................................................................................................................ link

37. Concentrations of pesticide compounds determined in National Water Quality

Laboratory (NWQL) schedule 2033 gas chromatography/mass spectrometry in

paired samples reported as nondetections in NWQL laboratory schedule 2437

direct aqueous-injection liquid chromatography-tandem mass spectrometry ............ link

38. Concentrations of pesticide compounds determined in National Water Quality

Laboratory (NWQL) schedule 2437 direct aqueous-injection liquid

chromatography-tandem mass spectrometry in paired samples reported as

nondetections in NWQL laboratory schedule 2033 gas chromatography/mass

spectrometry ............................................................................................................................ link
ix

39. Consistency of detections or nondetections of pesticide compounds in 

150 paired-stream samples determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry and liquid chromatography/mass

spectrometry during National Water Quality Assessment Program field 

comparison study from May to September 2012................................................................ link

40. Sign test that number of detections in 148 paired-stream samples are the same 

for pesticide compounds analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry and liquid chromatography/mass

spectrometry ............................................................................................................................ link

41. Concentrations of pesticide compounds determined in National Water Quality

Laboratory (NWQL) schedule 2060 liquid chromatography/mass spectrometry in

paired samples reported as nondetections in NWQL laboratory schedule 2437

direct aqueous-injection liquid chromatography-tandem mass spectrometry ............ link

42. Concentrations of pesticide compounds determined in National Water Quality

Laboratory (NWQL) schedule 2437 direct aqueous-injection liquid

chromatography-tandem mass spectrometry in paired samples reported as

nondetections in NWQL laboratory schedule 2060 liquid chromatography/mass

spectrometry ............................................................................................................................ link

43. Stability (holding time) study design for pesticide compounds determined by 

direct aqueous-injection liquid chromatography-tandem mass spectrometry in 

laboratory reagent-water-spike samples............................................................................ link

44. Summary of concentrations and recovery of 29 continuing calibration verification

standards analyzed by direct aqueous-injection liquid chromatography-tandem

mass spectrometry in positive electrospray ionization during stability studies to

calculate tolerable range of variation and lower limit of recovery for holding-time

determination ........................................................................................................................... link

45. Summary of concentrations and recovery of 29 continuing calibration verification 

standards analyzed by direct aqueous-injection liquid chromatography-tandem 

mass spectrometry in negative electrospray ionization during stability studies to

calculate tolerable range of variation and lower limit of recovery for holding-time

determination ........................................................................................................................... link

46. Stability of pesticides in reagent water stored at 4 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization 

mode .......................................................................................................................................... link

47. Stability of pesticides in reagent water stored at 4 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization 

mode .......................................................................................................................................... link

48. Stability of degradates in reagent water stored at 4 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization 

mode .......................................................................................................................................... link

49. Stability of degradates in reagent water stored at 4 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization 

mode .......................................................................................................................................... link

50. Stability of pesticides in reagent water stored at –10 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization

mode .......................................................................................................................................... link
x

51. Stability of pesticides in reagent water stored at –10 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization 

mode .......................................................................................................................................... link

52. Stability of degradates in reagent water stored at –10 degrees Celsius for various

times for as many as 28 days and analyzed by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization 

mode .......................................................................................................................................... link

53. Stability of pesticide degradates in reagent water stored at –10 degrees Celsius 

for various times for as many as 28 days and analyzed by direct aqueous-injection

liquid chromatography-tandem mass spectrometry in negative electrospray

ionization mode ........................................................................................................................ link

54. Stability of pesticides in reagent water treated with ascorbic acid and stored at 

4 degrees Celsius for various times for as many as 28 days and analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in positive

electrospray ionization mode ................................................................................................ link

55. Stability of pesticides in reagent water treated with dechlorination reagent

(ascorbic acid) stored at 4 degrees Celsius for various times for as many as 28

days and analyzed by direct aqueous-injection liquid chromatography-tandem

mass spectrometry in negative electrospray ionization mode........................................ link

56. Stability of degradates in reagent water treated with ascorbic acid and stored at

4 degrees Celsius for various times for as many as 28 days and analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in positive

electrospray ionization mode ................................................................................................ link

57. Stability of pesticide degradates in reagent water treated with dechlorination

reagent (ascorbic acid) stored at 4 degrees Celsius for various times for as many

as 28 days and analyzed by direct aqueous-injection liquid chromatographytandem mass spectrometry in negative electrospray ionization mode.......................... link

58. Stability of pesticides in reagent water treated with citric acid pH 3.8 and stored at

4 degrees Celsius for various times for as many as 28 days and analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in positive

electrospray ionization mode ................................................................................................ link

59. Stability of pesticides in reagent water treated with citric acid pH 3.8 and stored at

4 degrees Celsius for various times for as many as 28 days and analyzed by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in negative

electrospray ionization mode ................................................................................................ link

60. Stability of degradates in reagent water treated with citric acid pH 3.8 and stored

at 4 degrees Celsius for various times for as many as 28 days and analyzed by

direct aqueous-injection liquid chromatography-tandem mass spectrometry in

positive electrospray ionization mode ................................................................................. link

61. Stability of pesticide degradates in reagent water treated with citric acid pH 3.8

and stored at 4 degrees Celsius for various times for as many as 28 days and

analyzed by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in negative electrospray ionization mode................................................... link

62. Summary of validation study results from selected tables............................................... link
xi

Supporting Figures

S1. The qualifier ion response ratio of pesticide compounds determined by LC-MS/MS

in positive electrospray ionization mode fortified at different concentrations in

different matrices in reagent water...................................................................................... link

S2. The qualifier ion response ratio of pesticide compounds determined by LC-MS/MS

in negative electrospray ionization mode fortified at different concentrations in

different matrices and in reagent water.............................................................................. link

S3. Graphs of response factor and parameters of linear fit of response factor by

nominal concentration for direct aqueous-injection liquid chromatography-tandem

mass spectrometry method positive electrospray ionization mode analytes ............... link

S4. Graphs of response factor and parameters of linear fit of response factor by

nominal concentration for direct aqueous-injection liquid chromatography-tandem

mass spectrometry method negative electrospray ionization mode analytes.............. link

S5. Recovery of pesticide analytes determined by liquid chromatography-tandem

mass spectrometry in positive electrospray ionization mode fortified at different

concentrations in four different matrices............................................................................ link

S6. Recovery of pesticide analytes determined by liquid chromatography tandem

mass spectrometry in negative electrospray ionization mode fortified at different

concentrations in four different matrices............................................................................ link

S7. Recovery of pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in field-matrix-spike samples by 

site in 200 field-matrix-spike samples that were prepared for the National Water 

Quality Assessment Program field study from May to September 2012 ........................ link

S8. Pesticide compound recovery as a function of background environmental 

sample concentration determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in 146 field-matrix-spike samples 

that were prepared for the National Water Quality Assessment Program field 

study from May to September 2012 ...................................................................................... link

S9. Recovery of pesticides in reagent water after storage for as many as 28 days 

with different treatments determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization 

mode .......................................................................................................................................... link

S10. Recovery of pesticide degradates in reagent water after storage for as many as

28 days with different treatments determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in positive electrospray ionization 

mode .......................................................................................................................................... link

S11. Pesticide degradates found in reagent water fortified with pesticides after storage 

for as many as 28 days with different treatments determined by direct aqueousinjection liquid chromatography-tandem mass spectrometry in positive 

electrospray ionization mode ................................................................................................ link

S12. Recovery of pesticides in reagent water after storage for as many as 28 days 

with different treatments determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization 

mode .......................................................................................................................................... link

S13. Recovery of pesticide degradates in reagent water after storage for as many as

28 days with different treatments determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry in negative electrospray ionization 

mode .......................................................................................................................................... link
xii

S14. Pesticide degradates found in reagent water fortified with pesticides after storage

for as many as 28 days with different treatments determined by direct

aqueous-injection liquid chromatography-tandem mass spectrometry in negative

electrospray ionization mode ................................................................................................ link

Supporting Tables

S1. Groups of pesticide compounds used to prepare intermediate standard solutions 

of pesticide compounds for direct aqueous-injection liquid chromatographytandem mass spectrometry method..................................................................................... link

S2. Preparation of primary dilution standard solutions in methanol used to make

calibration standards for direct aqueous-injection liquid chromatography-tandem

mass spectrometry method ................................................................................................... link

S3. Examples of National Water Information System qualifier codes used by analyst

with direct aqueous-injection liquid chromatography-tandem mass spectrometry

method to qualify samples or analytes not meeting quality-control criteria................. link

S4. Summary of quality-control criteria for determination of pesticides by direct

aqueous-injection liquid chromatography-tandem mass spectrometry........................ link

S5. Pesticide compounds identified as high priority for method development but

not included in direct aqueous-injection liquid chromatography-tandem mass

spectrometry method for various reasons .......................................................................... link

S6. Summary of response factor and parameters of linear fit of response factor by

nominal concentration for direct aqueous-injection liquid chromatography-tandem

mass spectrometry method positive electrospray ionization mode analytes ............... link

S7. Summary of response factor and parameters of linear fit of response factor by

nominal concentration for direct aqueous-injection liquid chromatography-tandem

mass spectrometry method negative electrospray ionization mode analytes.............. link

S8. Recovery of pesticides determined by direct aqueous-injection liquid

chromatography-tandem mass spectrometry method in field-matrix-spike samples

collected during National Water Quality Assessment Program field comparison

study from May to September 2012 excluded from data summaries because

environmental concentration was greater than spike level............................................. link

S9. Summary of water quality parameters measured in streams when direct

aqueous-injection liquid chromatography-tandem mass spectrometry method

samples were collected during National Water Quality Assessment Program field

comparison study from May to September 2012................................................................ link

S10. Summary of ancillary water quality parameters for each stream site when direct

aqueous-injection liquid chromatography-tandem mass spectrometry method

samples were collected during National Water Quality Assessment Program field

comparison study from May to September 2012................................................................ link

S11. Degradates found in reagent water samples spiked with pesticides spiked at

250 nanograms per liter and stored at 4 degrees Celsius for as many as 28 days

and analyzed by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in positive electrospray ionization and negative electrospray

ionization modes ...................................................................................................................... link

S12. Degradates found in reagent water samples spiked with pesticides spiked at 

250 nanograms per liter and stored at –10 degrees Celsius for as many as 133 days

and analyzed by direct aqueous-injection liquid chromatography-tandem mass

spectrometry in positive electrospray ionization and negative electrospray

ionization modes ...................................................................................................................... link
xiii

Conversion Factors and Abbreviations

SI to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

micrometer (μm) 0.3937 x 10-6 inch (in.)

Volume

liter (L) 33.82 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

liter (L) 61.02 cubic inch (in3

) 

microliter (μL) 0.3382 x 10-4 ounce, fluid (fl. oz)

milliliter (mL) 0.03382 ounce, fluid (fl. oz)

Flow rate

liter per minute (L/min) 33.81 fluid ounce per minute (oz/min)

milliliter per minute (mL/min) 0.3381 fluid ounce per minute (oz/min) 

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound avoirdupois (lb)

milligram (mg) 0.3527 x 10-4 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F-32)/1.8

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 

25 °C).

Concentrations of chemical constituents in water are given in nanograms per liter (ng/L).

Concentration equivalent units (assuming 1 liter equals 1 kilogram):

ppm: mg/kg, mg/L, μg/mL

ppb: μg/L, ng/mL

ppt: ng/L, pg/mL

NOTE TO USGS USERS: Use of liter (L) as a special name for cubic decimeter (dm3) is restricted 

to the measurement of liquids and gases. No prefix other than milli should be used with liter. 
Abbreviated Water-Quality Units

pg picogram

g/L gram per liter

ng/L nanogram per liter

ng/mL nanogram per milliliter

v/v volume-to-volume

μg/mL microgram per milliliter

μg/L microgram per liter

μL microliter

μm micrometer

μS/cm microsiemens per centimeter

Abbreviations

ACS American Chemical Society

ASTM American Society for Testing and Materials

CAL calibration [standard]

CASRN Chemical Abstracts Service Registry Number

CCB continuing calibration blank

CCV continuing calibration verification [standard]

DAI direct aqueous injection

EPA U.S. Environmental Protection Agency

ESI electrospray ionization

ESI− negative electrospray ionization

ESI+ positive electrospray ionization

FEP fluorinated ethylene-propylene

GC-MS gas chromatography-mass spectrometry

HPLC high-performance liquid chromatography

IDL instrument detection level

IS internal standard

LIMS laboratory information management system

LC-MS liquid chromatography-mass spectrometry

LC-MS/MS liquid chromatography-tandem mass spectrometry

LRL laboratory reporting level

MDL method detection level

xiv
xv

MRM multiple reaction monitoring

MSPK matrix spike

MS/MS tandem mass spectrometry

m/z mass-to-charge ratio

NAWQA National Water Quality Assessment Program

NWIS National Water Information System

NWQL National Water Quality Laboratory

PFA perfluoroalkoxy alkane

PBLNK laboratory reagent blank

pKa logarithmic measure of the acid dissociation constant

PSPK laboratory reagent spike

PTFE poly(tetrafluoroethylene), a fluoropolymer of tetrafluoroethylene

QC quality control

QIR qualifier ion ratio

RPD relative percent difference

RSD relative standard deviation

SIM selected-ion monitoring

SMILES simplified molecular-input line-entry system

SPE solid-phase extraction

TPC third-party check

USGS U.S. Geological Survey

WBLK wash blank
Abstract

A liquid chromatography-tandem mass spectrometry 

(LC-MS/MS) method was developed for determination of 

229 pesticides compounds (113 pesticides and 116 pesticide degradates) in filtered water samples from stream and 

groundwater sites. The pesticides represent a broad range of 

chemical classes and were selected based on criteria such as 

current-use intensity, probability of occurrence in streams 

and groundwater, and toxicity to humans or aquatic organisms. More than half of the analytes are pesticide degradates. 

The method involves direct injection of a 100-microliter (μL) 

sample onto the LC-MS/MS without any sample preparation 

other than filtration. Samples are analyzed with two injections, 

one in electrospray ionization (ESI) positive mode and one in 

ESI negative mode, using dynamic multiple reaction monitoring (MRM) conditions, with two MRM transitions for each 

analyte. The LC-MS/MS instrument parameters were optimized for highest sensitivity for the most analytes. This report 

describes the analytical method and presents characteristics of 

the method validation including bias and variability, detection 

levels, and holding-time studies.

Mean recoveries of most analytes (223 of 229) were 

within data-quality objectives of 100±30 percent at spike 

concentrations above method detection levels (MDLs) in all 

four matrices. The calculated MDLs ranged from 1 to 103 

nanograms per liter (ng/L) for 182 analytes analyzed in the ESI 

positive mode, and from 2 to 106 ng/L for 42 analytes analyzed 

in the ESI negative mode. Five analytes had MDLs between 

100 and 250 ng/L. The stability studies in reagent water demonstrated that the largest number of the pesticide compounds 

(227 of 229) were stable after 14 days of storage at 4 degrees 

Celsius, so these were selected as the practical holding time 

and storage temperature for routine sample processing. The use 

of antimicrobial reagent citric acid to adjust the sample pH to 

about 4 also resulted in lower recoveries of some analytes, so it 

should not be used as a routine sample preservative.

Introduction

Analytical methods that are capable of determining a 

broad range of pesticide compounds (pesticide active ingredients and degradation products of these pesticides) at low 

concentrations are needed for monitoring water quality of the 

Nation’s stream and groundwater resources. 

Background

The U.S. Geological Survey (USGS) recently reevaluated the pesticides monitored in national studies and prioritized 

them based on a variety of factors including potential for occurrence and persistence in streams and groundwater and toxicity 

to humans and aquatic organisms (Norman and others, 2012). 

This prioritization resulted in a list of 257 pesticide compounds 

that were considered high priority (tier 1) for method development and future monitoring studies. The high-priority pesticide compounds included 78 new compounds not previously 

monitored in USGS analytical methods for determination of 

pesticides in water (based on their unique National Water Information System [NWIS] method code) as well as many pesticide compounds determined infrequently or in small research 

studies. In addition, 96 pesticide compounds are in commonly 

used USGS analytical methods (National Water Quality Laboratory [NWQL] laboratory schedules 2033 and 2060) that will 

be important for long-term monitoring studies. 

Current NWQL analytical methods utilize solid-phase 

extraction (SPE) for sample concentration followed by both 

gas chromatography/mass spectrometry (GC-MS) (NWQL 

laboratory schedule 2033) and liquid chromatography/

mass spectrometry (LC-MS) (laboratory schedule 2060) for 

quantitation. Although these methods have suitable detection 

levels for environmental studies, comprehensive analysis of all 

high-priority pesticide compounds is expensive because multiple preparation steps and multiple analytical instruments are 

Determination of Pesticides and Pesticide Degradates 

in Filtered Water by Direct Aqueous-Injection Liquid 

Chromatography-Tandem Mass Spectrometry

By Mark W. Sandstrom, Leslie K. Kanagy, Cyrissa A. Anderson, and Christopher J. Kanagy
2 Determination of Pesticides and Pesticide Degradates in Filtered Water

needed. In 2009, a new generation of liquid chromatographytandem mass spectrometry (LC-MS/MS) analytical instruments became commercially available that provided an 

alternative approach to determine a wide range of pesticide 

compounds (Greulich and Alder, 2008). The instruments 

had increased sensitivity and selectivity that allows for the 

determination of a broad range of analytes at environmentally 

relevant concentrations without any sample preparation.

Existing NWQL pesticide methods have several disadvantages such as bias and variability introduced by the 

extraction and evaporation procedures and the presence of 

interfering peaks due to the low selectivity of these detection 

techniques. Because of their high selectivity and sensitivity, 

tandem mass spectrometers can overcome these problems. 

In particular, high-performance liquid chromatographyelectrospray ionization-tandem mass spectrometry with 

dynamic multiple reaction monitoring (MRM) can be used 

without additional sample preparation.

Purpose and Scope

The purpose of the report is to describe the analytical 

procedure for a new direct aqueous-injection (DAI) LC-MS/MS 

method for determination of pesticide compounds in filtered 

water samples and provide characteristic performance measures 

for their validation in the method including bias and variability, 

method detection level (MDL), and stability studies. The 

bias and variability data were determined in four water types 

(reagent water, groundwater, and two different streams). MDLs 

were determined in reagent water using the U.S. Environmental 

Protection Agency (EPA) procedure (40 CFR part 142, appendix B). Stability studies were conducted in reagent water at two 

temperatures (4 °C and –10 °C) for 28 days to determine sample 

holding-time protocols. The applicability of the method to a 

variety of stream samples and comparison with two other USGS 

methods for determination pesticides in water is described.

Methods of Study

Approach

The approach was to first obtain standards for as many 

of the high-priority ranked (tier 1) pesticide compounds as 

possible. There were 99 degradates ranked as high priority 

because they had been identified in previous USGS studies 

(Norman and others, 2012). In addition, any other degradates of these high-priority pesticides available from the EPA 

National Pesticide Standard Repository were also obtained. 

These degradates are available from the standards repository because they are included in the tolerance limits for each 

pesticide that may be found on foods based on potential risks 

to human health (40 CFR parts 136 and 180, respectively). 

Although they are of concern for risk to human health in food, 

the degradates were not evaluated for likelihood to be present 

in streams and groundwater based on persistence, sorption, or 

toxicity to aquatic organisms. However, they were included 

in the method because their parent pesticide had a high usage 

rate, which is one of the factors used to prioritize degradates 

for monitoring studies (Sinclair and others, 2006). Pesticides 

were divided into 24 main analytical groups consisting of 

1–31 pesticides per group. Analytical groups were based on 

pesticide use (herbicide, insecticide, fungicide) and chemical 

class (for example, triazine, acetanilide, and so forth).

The method was developed and validated using two 

identical Agilent 6460 LC-MS/MS instruments. One MS/MS 

instrument was operated in positive and one in negative 

electrospray ionization mode to obtain optimum performance 

for different groups of analytes. The first tasks were selection 

of precursor and product ions and optimization of MS/MS 

parameters (fragmentor voltage and collision energy). Analytes were optimized in the positive ionization mode first. If 

response was low (less than about 103 area counts for integrated precursor ion) or previous literature indicated good 

performance in negative mode, analytes were optimized in 

negative ionization mode.

MS/MS instrument conditions and LC parameters were 

optimized for highest sensitivity of most analytes. Validation 

tests were conducted, including bias and variability in different matrices and range of concentrations, MDL, and stability 

studies (including assessment of preservation reagents).

Previous Studies

Methods based on GC-MS operated in the selected-ion 

monitoring (SIM) mode, combined with solid-phase extraction (SPE), have provided adequate sensitivity and selectivity needed for the determination of low concentrations of 

pesticide compounds in water samples. The GC-MS methods developed by the EPA for organic compounds in source 

water and water after treatment for drinking (Eichelberger 

and others, 1995) and by the USGS for studies of pesticides 

in streams and groundwater (Zaugg and others, 1995; Sandstrom and others, 2001) have been successfully used for more 

than two decades. However, a disadvantage of these GC-MS 

methods is that they are not suitable for polar or nonvolatile 

compounds, so it is difficult to have one method covering a 

wide range of pesticide compounds. In addition, because most 

degradates have a more polar character than the pesticide, 

LC-MS and LC-MS/MS are more suitable techniques for the 

determination of transformation products because the polar 

compounds are not amenable to volatilization needed for 

GC-MS without derivatization (Zwiener and Frimmel, 2004a).

The sensitivity of commercially available LC-MS/MS 

instruments priced for routine high-throughput use has 

improved over the last 5 years. Instrumental limits of quantitation of 1 picogram could be expected for electrospray ionization (ESI) in 2008 (Bester, 2008), and many mulitresidue 

methods for determination of pesticides by LC-MS/MS have 

recently been developed. A comparison of the scope, sensitivity, and selectivity of LC-MS/MS compared to GC-MS for 

more than 500 pesticide compounds showed better performance for LC-MS/MS for all except the organochlorine 
Analytical Method 3

pesticides (Alder and others, 2006). Multiple reaction monitoring with a tandem mass spectrometer is more sensitive and 

selective technique compared to SIM in a single quadrupole 

mass spectrometer because multiple product ions from one 

or more precursor ions are measured rather than one ion (Le 

Grand and others, 2012). In addition, the effect of matrix 

interferences are reduced in MS/MS compared to MS because 

different molecules that share the same transition are more 

rarely found than molecules producing fragments of identical 

mass. As a consequence, peak identification, integration, and 

data processing are much easier and faster in LC-MS/MS and 

require less manual corrections compared to GC-MS (Lehotay 

and others, 2005). 

The combination of the sensitivity of modern 

LC-MS/MS instruments and selectivity of the tandem 

mass spectrometry led to the development of direct injection methods that do not need the preconcentration steps 

of SPE (Backe and Field, 2012). A method using DAI of a 

100-micrograms per liter (μL) sample LC-MS/MS in ESI 

negative mode (ESI−) was used to validate acetanilide degradates at the 50-nanogram per liter (ng/L) level in different 

water matrices (Fuhrman and Allan, 2003). Another study 

demonstrated that quantitative results for 300 pesticides 

in drinking water at 100 ng/L could be achieved by DAI 

LC-MS/MS with a 100-μL sample injection without any 

sample enrichment or cleanup (Greulich and Alder, 2008). 

EPA method 538 uses DAI LC-MS/MS for determination 

of 11 pesticides in water at 11 to 1,500 ng/L (Shoemaker, 

2009). Other applications of DAI LC-MS/MS methods for 

pesticides in water used two injections, one for ESI positive (ESI+) analytes and one for ESI− analytes, including 

the determination of atrazine, simazine, propazine, and their 

respective dealkylated chlorotriazine metabolites; ametryn 

and prometryn and their respective dealkylated thiomethyltriazine metabolites; and S-metolachlor and its ethanesulfonic 

and oxanilic acid degradates at 50 ng/L (Huang and others, 

2008); and 31 triazine, organophosphate, and acid pesticides 

at 15 ng/L (Díaz and others, 2008).

For MS/MS, at least two transition ions and the molecular ion are necessary to meet the identification criteria for an 

organic contaminant in food according to European Union 

guidelines (Zwiener and Frimmel, 2004b). These measures 

plus chromatographic retention-time specifications help avoid 

false positives in mass spectrometry, as demonstrated in the 

case of pesticides in water (Pozo and others, 2006) and steroid 

hormones in wastewater (Schlusener and Bester, 2005). 

A dynamic (scheduled) MRM acquisition method monitors only the transitions within the target time window for each 

analyte, rather than a time segment of the chromatogram. For 

determination of a large number of pesticides in multi-residue 

LC-MS/MS analysis, dynamic MRM acquisition provides 

lower detection levels because the mass spectrometer spends 

more time with each MRM transition (increased dwell time, 

duty cycle, and thus data quality) without impacting on the 

cycle time, therefore assuring that enough data points are 

acquired (Fillâtre and others, 2010). 

Acknowledgments

Alfred Lück (ETH, Switzerland) provided valuable assistance in the initial optimization of the LC-MS/MS parameters 

for pesticides. Robert Kent, Jerri Davis, and James Fallon 

kindly collected and filtered extra sample bottles for our validation studies.

Analytical Method

Method Number, Schedule, and Code

The analytical method and validation data described 

in this report were evaluated by the USGS Office of Water 

Quality and approved as USGS method O-2437-15 on June 1, 

2015. Pesticide compounds are analyzed using DAI LC-MS/

MS for filtered water using NWQL schedule 2437 and laboratory method code LCM60. The laboratory method code is used 

in the USGS National Water Information System (NWIS) data 

system (http://nwis.waterdata.usgs.gov/nwis/qw) to describe 

methods used for determination of water-quality parameters. 

In addition, the NWIS water-quality software (QWDATA) 

uses a reference table of parameter-method code pairs (called 

analyteID) for reporting rounded values to data users (http://

help.waterdata.usgs.gov/codes-and-parameters/codes#WQ).

Scope and Application

Method O-2437-15 is used to determine 229 pesticide 

compounds (113 pesticides and 116 pesticide degradates) 

in filtered stream and groundwater samples (table 1). The 

range of concentrations of calibration standards is from 1 to 

10,000 ng/L. The detection levels in reagent water range from 

1 to 250 ng/L, with a median of 3 ng/L for pesticide compounds determined in ESI+ mode, and from 2 to 250 ng/L, 

with a median of 40 ng/L for pesticide compounds determined 

in the ESI− mode. 

Information about the pesticide compounds summarized 

in table 1 includes pesticide compound names, USGS parameter codes, analyte identifications numbers (IDs), MDLs, 

laboratory reporting level (LRL), and the lab code. Lab codes 

are used for grouping of analytes in the NWQL laboratory 

information management system (LIMS) system and are 9030 

for the ESI+ analytes, 9031 for the ESI− analytes, and 3406 

for the sample volume. Other information about the pesticides 

includes the Chemical Abstracts Service Registry Number 

(CASRN1) if available, molecular weight, molecular formula, analytical use (pesticide, degradate, internal standard), 

and SMILES (simplified molecular-input line-entry system) 

notation. The SMILES is a specification in the form of a line 

1

 This report contains CAS Registry Numbers®, which is a registered trademark of the American Chemical Society. CAS recommends the verification of 

the CASRNs through CAS Client ServicesSM.
4 Determination of Pesticides and Pesticide Degradates in Filtered Water

notation for describing the structure of molecules using short 

ASCII strings. SMILES strings can be imported by most molecule editors for conversion back into two-dimensional drawings or three-dimensional models of the molecules (Weininger, 

1988; O’Boyle, 2012). The analytical method group, pesticide 

group, and validation quality code were used in this report for 

discussion of validation results and sorting of pesticide compounds in figures and tables. The pesticide group is the name 

of the active form of pesticide applied in the environment and 

provides a way to link pesticide degradates to the active form 

of pesticide that was used. The validation quality codes are 

unique to method validation qualification of data in this report 

and can be A, acceptable; E, estimated; or D, deleted from 

method in May 2013. Finally, information about whether the 

pesticide compound was new or in previous USGS methods 

based on their unique NWIS analyteID is shown.

The new method includes many high-priority (tier 1) 

pesticide compounds that were in previous USGS pesticide 

methods that will be useful for long-term trend analysis. For 

example, 37 pesticide compounds in the new method are 

in method O-1126-95, 38 in method O-2002-01, and 42 in 

method O-2060-01 determined at the NWQL, and 17 pesticide compounds in method O-2138-02 and 20 in method 

O-2139-03 determined at the Kansas Organic Geochemistry 

Research Laboratory (some pesticide compounds are in more 

than one previous method). Other high-priority (tier 1) analytes were not in any previous USGS methods, and represent 

new analytes for USGS studies. These include 22 pesticide 

active ingredients and 56 pesticide degradates (table 1).

Nomenclature

Terms used for the analytes in this report are “pesticide” for the active ingredient, “degradate” for the pesticide metabolite or degradate, or “pesticide compound” for 

both. Synonyms for these terms used in literature include 

parent for pesticide and daughter or transformation product 

for the degradate.

Common names for pesticide compounds in this report 

(table 1) generally are the same as those used in NWIS. 

Some alternatives to the NWIS names were selected for 

this report to make the names used for a chemical class 

more consistent or the text more readable. For example, the 

names of all organophosphate degradates used the “oxon” 

format rather than “oxygen analog.” Similarly, the acetanilide degradate names all used the abbreviations SA for sulfonic acid, SAA for sulfinylacetyl acid, and OA for oxanilic 

acid. For the triazine degradates, deethylatrazine is used 

as a synonym for the atrazine degradate 2-chloro-4-isopropylamino-6-amino-s-triazine (CIAT), deisopropylatrazine 

is used as a synonym for the atrazine degradate 2-chloro4-ethylylamino-6-amino-s-triazine (CEAT), and didealkylatrazine is used as a synonym for the atrazine degradate 

2-chloro-4,6-diamino-s-triazine (CAAT) in this report.

Summary of Method

Samples are filtered at the field site using a 0.7-micrometer (μm) nominal glass fiber filter and shipped to the laboratory. Prior to analysis at the laboratory, a 900-μL subsample 

is removed and placed in a 2-milliliter (mL) analytical vial. 

A solution of isotope-labeled (enriched) pesticides is added 

to the samples for use as internal standards, and 90 μL of 

methanol is added to modify the sample matrix for optimal 

chromatographic performance. Pesticides are determined in 

the samples in two groups, one by the MS/MS operated in 

positive electrospray ionization (ESI+) mode, the other in 

negative electrospray ionization (ESI−) mode. For each group 

an aliquot of the sample (100 μL) is injected onto a liquid 

chromatographic column (C18 phase), and the analytes are 

separated using binary mobile phase of formic acid/ammonium 

formate-methanol for ESI+ mode or acetic acid-acetonitrile for 

ESI− mode. A dynamic multiple reaction monitoring (MRM) 

method is used on the MS/MS to collect specific quantification 

and qualifier ions for each analyte. Analyte identification is 

based on agreement between known standards with respect to 

chromatographic retention time and the ratio of the two characteristic MRMs. The concentration of each identified component 

is determined using the internal standard technique.

Safety Considerations

All procedures that require the use of solvents, such 

as preparation of mobile phases and calibration standards, 

should be conducted in a fume hood. Eye protection, nitrile 

gloves, and protective clothing should be worn when handling 

reagents, solvents, or standards.

Liquid waste produced during sample preparation and analysis, including solvents (methanol, acetonitrile) used for rinsing 

glassware, unused mobile phase mixtures, and mobile phase eluting from the high-performance liquid chromatography (HPLC) 

instrument not transferred to the MS/MS instrument, is collected 

in thick-walled plastic carboys. Solid waste, including analytical 

vials containing samples, spike mixtures, and methanol, is stored 

in glass carboys. All liquid and solid waste is stored and disposed 

according to the NWQL waste handling procedures. 

Interferences

All glassware must be thoroughly cleaned to prevent contamination and avoid interferences. All glassware is washed 

with detergent, rinsed with tap water, and finally rinsed with 

methanol. Nonvolumetric glassware such as sample containers, analytical vials, and micropipette bores and stainlesssteel spatulas must be either solvent-rinsed (with methanol) 

or baked in a furnace at 450 °C for 2 hours (h). Volumetric 

glassware must be solvent rinsed (with methanol followed by 

solvent used for standard). Clean glassware is covered with 

aluminum foil for storage.
Analytical Method 5

Disposable items made of high-purity polypropylene such 

as transfer pipette tips, syringes, and syringe-tip filters are suitable for this tandem mass spectrometry method. These items 

must be pre-rinsed with solvent or sample prior to use.

Method interferences may be caused by contaminants 

in solvents, reagents, disposable transfer pipets, gloves, and 

other laboratory supplies. These consumable items should be 

demonstrated to be free from interferences by routine analysis 

of laboratory reagent blanks. Identification of contaminants 

or interferences in MRM chromatograms will require topical blanks that examine only specific aspects of the method 

(solvents, vials) to identify and remove them.

Good laboratory practices must be followed at all times 

when preparing reagents and samples. Analysts and sample 

collectors should wear nitrile gloves for safety reasons and to 

avoid contamination of the samples. All reagent and sample 

preparation steps must be conducted in a clean environment, 

and care must be taken to prevent contamination while preparing samples and standards. Material that comes in contact 

with samples must only be made of glass, metal, ceramics, or 

fluoropolymers (for example, fluorinated ethylene-propylene 

[FEP]). Flexible plastic materials containing plasticizers such 

as pipette bulbs, polypropylene squeeze bottles, and Tygon 

tubing must be replaced with plasticizer-free material such 

as mechanical dispensers, FEP squeeze bottles, and FEP 

and perfluoroalkoxy alkane (PFA) tubing. Some high-purity 

plastic materials can be used if shown to be contamination 

free using this method, for example, disposable pipette tips, 

filters, and syringes constructed of high-purity polypropylene. All standard solution preparations are described in 

the standards laboratory notebook, with concentrations, lot 

numbers, and expiration dates listed in the notebook and on 

the vial label.

Equipment and Supplies

The equipment and supplies obtained from vendors listed 

below were tested during development of this method. Specific brand names and catalog numbers are shown for illustration, and equivalent vendors can be used where appropriate.

•	 Standard solution containers—Amber borosilicate glass 

vials fitted with polytetrafluoroethylene (PTFE)-lined 

screw cap, 20-mL (C&G Containers Inc., Lafayette, 

La., container 109149)

•	 Analytical vials—Amber glass vials, 2-mL 

(National Scientific, Rockwood, Tenn., Target DP, 

C4000-2W), with 11-millimeter (mm)-diameter 

polypropylene screw caps lined with PTFE/red 

silicone septa (Agilent Technology, Wilmington, 

Del., catalog number [cat. no.] 5182-0717, PTFEsilicone-PTFE liner). These caps appear to minimize 

errors in the HPLC autosampler and stoppage of the 

analytical sequence.

•	 Analytical balance—Capable of weighing to nearest 

0.01 milligram (Mettler Toledo, Columbus, Ohio, 

model XS205, or equivalent)

•	 Digital microdispenser—Various volume ranges 

including 10 μL, 25 μL, and 100 μL (VWR Scientific, Radnor, Pa., cat. no. 53506-121), with disposable glass bores. These are used for preparation of 

mixed compound intermediate standards and calibration standards.

•	 Digital pipette—Various volume ranges including 

10 mL (Rainin Pipettes, Oakland, Calif., model EDPPlus 1000, 100 μL to 10,000 μL) and 20 mL (Rainin 

Pipettes, Oakland, Calif., model EDP-Plus 2000, 

200 μL to 20,000 μL) electronic pipettes, with disposable polypropylene tips. These pipettes are used 

to prepare mixed intermediate-calibration solutions 

and to transfer samples from collection containers to 

analytical vials. The pipette tips are rinsed once with 

clean solvent or the sample to pre-wet the tip and 

remove any contaminants.

•	 Transfer pipette—Manual macro pipette, variable volume, 0.5 to 5 mL for use with glass Pasteur pipettes 

(VWR International, Radnor, Pa., Socorex model 

835.02PP, Wheaton part number W810316)

•	 Disposable glass pipets—Disposable Pasteur pipets, 

glass, 14.6-centimeter (cm) long × 7-mm diameter 

(VWR International, Radnor, Pa., model 14672-200)

•	 Vortex mixer—Vortex Genie 2 (Scientific Industries, 

Bohemia, N.Y., model G560)

•	 Wash bottle—Nalgene wash bottles, Teflon FEP, Narrow Mouth, 250-mL (Thermo Scientific, Waltham, 

Mass.). These “squeeze” bottles are used to dispense 

acetonitrile, methanol, laboratory reagent water, and 

other solvents into glass beakers and flasks.

•	 Spatula—Rounded spatula/spoon tip, stainless steel, 

(VWR International, Radnor, Pa., model 82027-522). 

These spatulas are used to transfer neat standards to 

volumetric flasks.

•	 Automatic ampule sealer—Ampulmatic, model number 

290001 (Bioscience Inc., Bethlehem, Pa.)

Sample Collection and Preparation Supplies

•	 Syringe—Disposable, high-purity polypropylene, 

30-mL, male Luer-lock tip (Norm-Ject 30 mL, 

polypropylene; no rubber, latex or silicone oil [VWR 

International, Radnor, Pa., model no 80076-426 or 

USGS OneStop number Q763FLD]).
6 Determination of Pesticides and Pesticide Degradates in Filtered Water

•	 Syringe-tip filters—Disposable, 25-mm diameter, 

polypropylene housing with female Luer-lock inlet 

and male Luer outlet and glass fiber filter (Whatman 

GF/F with graded multifiber [GMF], 0.7-μm nominal 

pore diameter, Whatman GD/X syringe [GE Healthcare Bio-Sciences, Pittsburgh, Pa., cat. no. 6902-2504 

or USGS OneStop number Q762FLD]). The filter 

is specifically designed for high-particulate-loaded 

samples. Constructed of a pigment-free polypropylene housing with a prefiltration stack of Whatman 

GMF 150 (graded density) and GF/F glass microfiber media. GMF 150 and GF/F are produced from 

100-percent borosilicate-glass microfiber. The graded 

density GMF 150 medium has a coarse top layer 

meshed with a fine bottom layer that retains particles 

to 1.0 μm. A GF/F filter then retains particles down to 

0.7 μm.

•	 Stainless-steel bore—disposable bore, 5.08-cm length, 

18-gauge with Luer fitting (USGS OneStop number 

Q764FLD).

•	 The syringe, syringe-tip filter, and stainless steel bore 

are available as a sampling kit (USGS OneStop number Q765FLD).

•	 Sample containers—Amber borosilicate-glass vials 

fitted with PTFE-lined screw cap, 40-mL (C&G 

containers, Inc. [Lafayette, La., container number 

107941] or 20-mL [C&G Containers, Inc., container 

109149]), pre-cleaned by vendor protocol B (volatile 

organic compounds, wash and rinse with American 

Society for Testing and Materials (ASTM) type 

1 water [ASTM Standard D-1193-91, West Conshohocken, Pa.]). Vials are shipped in a foam sleeve 

that can be used for storage of vials in a freezer.

Instrumentation

•	 LC-MS/MS system—The samples were analyzed 

using an Agilent model 6460A triple quadrupole 

(MS/MS) mass spectrometer equipped with electrospray ionization with Agilent jet stream technology 

(Agilent Technology, Wilmington, Del.). The MS/MS 

was coupled to an Agilent model 1200 Series LC 

system. The LC system consisted of a binary pump 

(G1312B), vacuum solvent degasser (G1379B), automatic liquid sampler (G1367D), thermostated sample 

tray (G1330B), thermostated column compartment 

(G1316B), solvent degasser (G1379B), and MassHunter data acquisition system. The sampler included 

a high-volume kit (G1367-68713) for injection volumes greater than 40 μL. The thermostated column 

compartment allows column temperatures to as much 

as 100 °C and pressures to as much as 600 bar. Narrow bore capillary tubing (0.12-mm interior diameter) 

was used to connect the LC system components to 

maximize chromatographic peak height and resolution. Qualitative and quantitative data analyses were 

performed using the Agilent MassHunter data system 

(version B.04.00).

Reagents

Neat Reagents

• Nitrogen gas—High-purity nitrogen (99.5 percent 

pure), dry, supplied as much as 30 liters per minute 

(L/min), is used as a drying gas, sheath gas, nebulizing 

gas, and to pressurize the calibrant delivery system. A 

high-capacity gas conditioner attached to the gas inlet 

tubing of the LC-MS/MS is used to remove hydrocarbon contamination from the nitrogen source for collision cell reagent gas (Agilent Technology, Wilmington, 

Del., part no. BHT-4).

• Laboratory reagent water—High-purity organic-free 

reagent water was obtained by passing deionized water 

through a Solution 2000 ultrapure water purification 

system that contains charcoal prefilters and a dualwavelength ultraviolet oxidizer lamp for removal of 

trace organic compounds (Aqua Solutions Inc., Jasper, 

Ga., Model 2121AL)

• Solvents—acetonitrile, isopropanol, dimethylsulfoxide, 

and methanol (B&J Brand, pesticide grade, Honeywell-Burdick and Jackson, Muskegon, Mich.)

• Ammonium formate (NH4CHO2, CASRN 540-69-2)—

LC-MS Ultra grade (Fluka cat no. 14266, SigmaAldrich, St. Louis, Mo.)

• Ammonium acetate (NH4C2H3O2, CASRN 

631-61-6)—LC-MS Ultra grade (Fluka, cat no. 14267, 

Sigma-Aldrich, St. Louis, Mo.)

• Formic acid—LC-MS Ultra grade (Fluka cat no 14265, 

Sigma-Aldrich, St. Louis, Mo.)

• Acetic acid—Eluent additive for LC-MS grade (Fluka 

cat no 49199, Sigma-Aldrich, St. Louis, Mo.)

• Citric acid monohydrate (C6H8O7 H2O, CASRN 

5949-29-1)—American Chemical Society (ACS) 

reagent grade (cat. no. C-1909, Sigma-Aldrich, St. 

Louis, Mo.)

• Trisodium citrate dihydrate (C6H5Na3O7 2H2O, 

CASRN 6132-04-3)—ACS reagent grade (cat. no. 

ED-355, Sigma-Aldrich, St. Louis, Mo.)

• Ethylenediaminetetraacetic acid (EDTA) trisodium salt 

hydrate (C10H13N2Na3O8 H2O, CASRN 85715-

6-02)—ACS reagent grade (cat. no. C-1909, SigmaAldrich, St. Louis, Mo.)
Analytical Method 7

• L-Ascorbic acid (CASRN 50-81-7)—High purity (cat. 

no. 255564, Sigma-Aldrich, St. Louis, Mo.)

• Electrospray calibrant mix—Pre-mixed solution of 

perfluorinated compounds that are distributed over the 

mass range (112 to 2,834 mass-to-charge ratio [m/z]) 

for tuning the electrospray MS/MS (Agilent Technology, Wilmington, Del., part no. G1969-85000 EI-L)

Reagent Solutions

• 1-molar (M) ammonium formate solution—Dissolve 

65.69 grams (g) of ammonium formate in 1 liter (L) of 

laboratory reagent water.

• 1-M formic acid solution—Dilute 38.8 mL of 

98-percent formic acid with laboratory reagent water 

to a final volume of 1 L. 

• ESI positive mode mobile phase A—Dilute 10 mL of 

1-M ammonium formate solution and 12 mL 1-M formic acid solution with lab laboratory reagent water to 

a final volume of 1 L (0.05 percent volume-to-volume 

[v/v] ammonium formate [0.010 M; pH 6.3–7.3] and 

0.05 percent [v/v] formic acid [0.012 M; pH = 1.88; 

logarithmic measure of the acid dissociation constant 

[pKa] = 3.75]) in water. 

• ESI negative mode mobile phase A—Dilute 2 mL of 

acetic acid with laboratory reagent water to a final 

volume of 1 L (0.2 percent [v/v] acetic acid in water; 

0.0334 M; pH = 3.12; pKa = 4.76). 

Standards

Analytical reference standards were obtained primarily from the EPA National Pesticide Standards Repository 

(Ft. Meade, Md.). If standards were not available from the 

Standards Repository, they were obtained from EQ Laboratories (Athens, Ga.) or Sigma-Aldrich (St. Louis, Mo.). Isotopelabeled reference standards were obtained from Cambridge 

Isotope Laboratories (Andover, Mass.) and CDN Isotopes Inc. 

(Quebec, Canada).

Concentrated standard solutions of individual analytes 

and internal standards were prepared at concentrations of 

about 100 micrograms per milliliter (μg/mL) by accurately 

weighing about 1 milligram (mg) of each analyte to the nearest 

0.1 mg into a 10-mL glass volumetric flask and dissolving in 

methanol to volume. Some analytes were unstable in methanol 

so were prepared in acetonitrile (paraoxon-methyl, parathionmethyl, diazinon oxon, and all sulfonylureas) or acetone 

(desulfinylfipronil amide, fipronil sulfide). For MS/MS MRM 

parameter optimization, standards were prepared at a concentration of 1 μg/mL by dilution of the concentrated standards in 

methanol in an analytical vial. The concentrated standard solutions were mixed using a vortex mixer (for 15–30 seconds [s]) 

and transferred to 20-mL amber glass storage containers. The 

concentrated standard solutions are stored in a refrigerator at 

4 °C ±2 °C and are stable for a least 1 year.

Internal standard (IS) solution.—The internal standard 

solution consists of isotope-labeled pesticide compounds at 

varying concentrations from 6 to 800 nanograms per milliliter (ng/mL) (table 2). These labeled compounds are used as 

internal standards for quantitation of method analytes. Their 

recovery is calculated relative to a single internal standard 

(atrazine-d5 in ESI+; ibuprofen-13C in ESI−) to provide matrixrelated method-performance information, similar to a surrogate 

analyte. The concentration of each labeled analyte in the solution was selected at about the mid-point of the calibration range 

to provide good analytical signal. We found that trace amounts 

of pyrimidinol, a degradate of diazinon-d10, were present in 

the neat labeled standard of diazinon-d10 as an impurity and 

caused measurable concentrations of pyrimidinol in samples, 

so the concentration of this IS was lowered to try to minimize 

this contamination.

The IS solution is prepared by transferring appropriate volumes of the 100-μg/mL standard solutions to a 50-mL 

volumetric flask and diluting to volume with methanol–

acetonitrile (80:20). The IS solution is mixed using a vortex mixer (15–30 s) and transferred to 12-mL amber-glass 

storage containers. The solutions are stored in a freezer at 

–10 °C ±2 °C and are stable for a least 1 year. Samples are 

prepared for analysis by adding 10 μL of this solution to 

900 μL of sample in an analytical vial, resulting in expected 

nominal concentrations of the IS compounds from 60 to 

10,000 ng/L (table 2).

Mixed intermediate standard solutions.—Pesticide compounds were divided into 24 main analytical groups consisting 

of 1–31 pesticide compounds per group. Analytical groups 

were based on pesticide use (herbicide, insecticide, fungicide), 

chemical class (acetanilide, triazine, acid), and solvent used. 

Pesticides and degradates within each group were also separated to monitor the stability of pesticides and the formation 

of degradates during storage. Analytical groups for the mixed 

intermediate solutions are shown in supporting table S1. 

Mixed intermediate solutions of these groups were prepared 

by transferring appropriate volumes of the 100-μg/mL concentrated standard solutions to a 5-mL or 10-mL volumetric flask 

and diluting to volume with methanol–acetonitrile (50:50) to 

make a final concentration of 5 to 12.5 μg/mL. Stability of the 

mixed intermediate standard solutions for some groups was 

found to be better in acetonitrile–methanol (75:25) and for 

diflufenzopyr in acetone/DMSO (80:20). Each mixed intermediate solution is agitated using a vortex mixer (15–30 s) 

and transferred to 20-mL amber glass storage containers. The 

mixed intermediate solutions are stored in a freezer at –10 °C 

±2 °C, and are stable for a least 1 year (based on initial verification during implementation of the method). 

Primary dilution standard solutions.—A solution containing 

all pesticide compounds was prepared by combining appropriate 

volumes (140–350 μL) of the 5–12.5-μg/mL mixed intermediate solutions in a 25-mL volumetric flask and diluting to volume 
8 Determination of Pesticides and Pesticide Degradates in Filtered Water

with methanol–acetonitrile (80:20) to make a final concentration 

of 175 ng/mL. This primary dilution standard solution is used 

to prepare lower concentration standard solutions using digital 

pipettes and disposable glass pipettes to add appropriate amounts 

of the primary dilution standard and methanol to 2-mL analytical 

vials (see supporting table S2). For example, appropriate volumes (43–171 μL) of the 175-ng/mL primary dilution standard 

solution and methanol (1,329–1,457 μL) are added to 2-mL 

analytical vials to make standard solutions with concentrations of 

20, 10, and 5 ng/mL. Appropriate volumes of these standards are 

further diluted to prepare solutions with concentrations of 1, 0.5, 

0.1, and 0.05 ng/mL (supporting table S2). The primary dilution 

standard solutions are stored in a freezer at –10 °C ±2 °C and are 

stable for at least 6 months.

Calibration standards.—Calibration standards are prepared in reagent water with methanol (10 percent) at 12 concentration levels (1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1,000, 

5,000, and 10,000 ng/L) for each analytical batch. The calibration standards are prepared by adding appropriate volumes 

(20–57 μL) of the 0.01–175-ng/mL primary dilution standard 

solutions to 900 μL of laboratory reagent water in an analytical vial to make final concentrations from 1 to 10,000 ng/L. A 

10-μL aliquot of the mixed IS solution is added to the calibration solutions, and sufficient methanol (33–90 μL) is added 

to make the final volume 1,000 μL. The resulting calibration 

standards contain 10 percent methanol necessary for optimal 

chromatographic performance of early eluting analytes.

Spiking solution.—A spiking solution for preparing 

laboratory reagent spikes and field-matrix spikes was prepared 

by combining appropriate volumes (200–1,250 μL) of the 

5–12.5-μg/mL mixed intermediate solutions in a 100-mL volumetric flask and diluting to volume with methanol–acetonitrile 

(80:20) to make a final concentration of 25 ng/mL. This 

spiking solution is mixed using a vortex mixer (15–30 s) and 

transferred to 20-mL amber glass storage containers. The spike 

solution is stored in a freezer at –10 °C ±2 °C and is stable 

for a least 1 year. Reagent spikes and matrix-spike samples 

are prepared for analysis by accurately adding 100 μL of this 

solution using a fixed-volume 100-μL micropipettor to about 

10 mL of sample in a sample container. The sample weight 

of field-matrix-spike samples is determined by weighing the 

filled vial to the nearest 0.1 g. The resulting nominal concentrations of the analytes are 250 ng/L.

Spike solutions for preparation of field-matrix spikes at the 

sample collection site are made using 2-mL flame-sealed glass 

ampoules. The spiking solution (25 ng/mL) is transferred to each 

glass ampoule using a 500-μL glass syringe, and then the ampoules 

are sealed using an automatic ampule sealer. The ampoules must 

be stored in a refrigerator at 4 °C ±2 °C or freezer and used prior to 

their expiration date (about 1 year after preparation).

Sample Collection, Filtration, Preservation, 

Shipment, and Holding Times

Samples must be collected using techniques designed to 

collect a representative, unbiased sample. The National Field 

Manual for the Collection of Water Quality Data describes protocols and provides guidelines for USGS personnel who collect 

water-quality data (U.S. Geological Survey, variously dated).

Sample Filtration

Stream and groundwater samples are filtered at the field 

site using small disposable 25-mm-diameter syringe-tip filters 

(Wilde and others, 2014). About 10 mL of sample is pushed 

through the filter and collected in a glass 20-mL sample 

container. A disposable 20-mL polypropylene syringe is used 

to push the sample through the filter. The syringe and filter are 

prerinsed with about 15 mL of sample to clean. Alternatively, 

if using a pump for groundwater sampling, various reducing 

fittings made of PTFE and FEP can be used to connect the 

Luer inlet of the filter to the discharge of the pump. Other filtration procedures suitable for trace organic compounds (metal 

or FEP plate filters with 147-mm- and 47-mm-diameter GF/F 

grade glass fiber filters can also be used (Radtke and others, 

2004, with updates through 2009)

Field Quality Control Samples

Collection and analysis of quality-control (QC) samples 

such as field-matrix spikes and field blanks are essential components of USGS water-quality field studies (U.S. Geological Survey, 2006). Matrix spikes prepared at the collection 

or shipping site are recommended for the DAI LC-MS/MS 

method in order to monitor degradation during shipping and 

storage as well as matrix effects because the samples are typically not preserved. Field-matrix spikes are prepared by using 

a 100-μL micropipette to accurately add the spiking solution to about 10 mL of sample in a 20-mL sample container 

(Sandstrom and Lewis, 2009). The sample weight is accurately 

determined at the NWQL and reported to the data user (NWIS 

parameter 3406) for calculation of spike recovery.

Field blanks are prepared using pesticide-grade water 

processed through sample collection, splitting, and filtration 

equipment.

Sample Preservation

Samples collected from sites potentially impacted by a 

water treatment plant that might have residual chlorine are 

dechlorinated with ascorbic acid. These samples should be 

collected in containers that contain ascorbic acid added to 

the container as a dry solid so that the final concentration is 

0.10 g/L (1 mg for 10 mL of sample). Note that preservative 

studies described in this report show that ascorbic acid is not 

an ideal dechlorination reagent because it causes low recovery 

and higher variability for some of the pesticide compounds. 

Research studies are needed to identify an effective dechlorination reagent that is suitable for all pesticide compounds.

Samples collected from autosamplers require preservation or chilling to 4 °C to prevent microbial degradation while 
Analytical Method 9

the sample is stored in the autosampler. One approach tested 

during development of the DAI LC-MS/MS method is the use 

of methanol as a microbial inhibiter. High concentrations of 

alcohol are considered toxic to most microorganisms (Malcolm Pirnie Inc., 1999), and the DAI LC-MS/MS analytical 

method conveniently uses methanol added to the samples at a 

concentration of 10 percent for optimal HPLC separation. For 

use as an antimicrobial, the sample containers are pre-filled 

with methanol so that, after filling with sample, the concentration of methanol is 10 percent, suitable for use as a microbial 

inhibitor. These samples do not have methanol added at the 

laboratory prior to analysis and so require different sample 

preparation steps (NWQL labcode 8237).

Sample Shipment and Holding Time

After samples are collected and filtered, they are put on 

ice in a cooler. Sample containers are shipped to the laboratory 

in ice-chilled (4–6 °C) coolers. Holding-time studies described 

in this report demonstrate that all pesticide compounds are 

stable for 14 days when kept chilled at 4 °C. The samples 

should be shipped overnight to the NWQL to allow efficient 

management of samples to meet the 14-day holding time.

Sample Preparation

Upon arrival at the laboratory, the samples in 20-mL glass 

containers are stored in a refrigerator (4 °C) until analysis. 

Holding-time studies described in this report show that samples 

should be analyzed within 14 days of sample collection.

A batch of samples and associated QC samples and 

calibration samples is prepared for each analytical run. If the 

samples are frozen, the sample is allowed to thaw at room 

temperature prior to analysis (about 4 h). The sample batch 

is prepared by sequential addition of the internal standard, 

methanol, and sample to the 2-mL amber glass analytical vials 

in parallel—each additional step is performed on all samples 

before moving to the next step. To prepare the samples, a digital micropipette equipped with glass bore is used to transfer 

10 μL of the internal standard mixture to all analytical vials, 

followed by 90 μL of methanol to all vials. A digital pipette 

fitted with disposable polypropylene tip is then used to remove 

a 900-μL aliquot of each sample from the sample container 

and transfer to the appropriately-labeled analytical sample 

vials. The methanol is used to match the organic solvent 

concentration (10 percent) of the mobile phase in order to 

optimize the peak shape of early eluting analytes in the liquid 

chromatographic run. 

Laboratory reagent blank samples and laboratory reagent 

spike samples (1 per batch of 50 samples) are prepared in 

20-mL sample containers to match the sample processing steps. 

These laboratory reagent blanks and reagent spikes are prepared by adding exactly 10 mL of the reagent water to 20-mL 

sample containers. The laboratory reagent spike is prepared at 

an expected concentration of 250 ng/L by adding 100 μL of the 

spiking solution to the sample in the 20-mL sample container. 

Aliquots of 900 μL of these samples are then transferred to the 

analytical vials during preparation of the sample batch. Similarly, if requested, laboratory matrix spikes can be prepared 

the same way as the reagent spikes by addition of the spiking 

solution to the environmental sample in the 20-mL sample 

container. These samples, like the field-matrix spikes, have 

the sample weight determined by weighing the sample plus 

container and subtracting the container tare weight. 

After data acquisition on the instrument, the original 

sample containers are stored in a freezer (–10 °C) until data 

analyses and reviews are completed. The glass containers 

will not break in a freezer as long as the sample is filled to no 

more than about two-thirds the capacity of the vial. In addition, when the method was developed using 40-mL containers, 

placing the containers in foam sleeves prevented breakage 

by slowing the sample’s freezing rate when transferred to the 

freezer, as well as when the sample was thawed.

Analysis by Liquid Chromatography-Tandem 

Mass Spectrometry—Setup and Data 

Acquisition

Overview

Samples for analysis by DAI LC-MS/MS are placed in 

2-mL analytical vials with isotope-labeled internal standards 

and 10 percent methanol. A volume of 100 μL is injected 

directly into the LC-MS/MS system to maximize sensitivity. 

The analytes are separated using an HPLC fitted with a 

reversed-phase column and water–methanol (ESI+) or water–

acetonitrile (ESI−) gradient elution. After LC separation, the 

analytes are transferred in a flowing stream to the MS/MS 

instrument (for tandem mass spectrometric analysis) where the 

compounds undergo electrospray ionization in the positive or 

negative ion mode followed by MRM of two unique precursor 

to product ion transitions. Data analysis is performed using 

MassHunter software (Agilent), and custom reports written in 

Excel (Microsoft) at the NWQL are used to summarize salient 

analytical-run and quality-control (QC) information and prepare results for transfer to the laboratory information management system (LIMS).

Separation by Liquid Chromatography

The samples along with laboratory QC samples and 

calibration standards in the 2-mL analytical vials are placed in 

a sample tray of the autosampler, which is chilled to 4 °C by a 

Peltier cooling unit to minimize degradation of the analytes. A 

100 μL aliquot of the sample is injected into the HPLC to start 

chromatographic separation.

The HPLC parameters are shown in table 3. An Agilent 

Zorbax C18 column (3.0×100 mm, 1.8 micrometers) is used 

to separate analytes in both ESI+ and ESI− modes. The guard 

column is an Agilent 1290 Infinity in-line filter with 0.3-μm 
10 Determination of Pesticides and Pesticide Degradates in Filtered Water

stainless-steel frit (part no. 5067-4638). The binary mobile 

phase consisting of aqueous (A) formic acid/ammonium 

formate solution and solvent (B) methanol is used for the ESI+ 

mode separation. The flow rate is 0.6 mL/min except for the 

initial 4 min where the flow rate is 0.4 min. During method 

development, we determined that a slower initial flow rate gave 

narrower peak widths for early eluting peaks. The column temperature was set to 50 °C. The gradient for the mobile phase 

composition for ESI+ is shown in table 4. The total run time 

was 25 min, plus 5 min for post-run equilibration time. 

The binary mobile phase consisting of aqueous (A) acetic 

acid solution and solvent (B) acetonitrile is used for the ESI− 

mode separation. The flow rate is 0.6 mL/min, and the column 

temperature was set to 60 °C. The gradient for the mobile 

phase composition for ESI− is shown in table 5. The total run 

time was 17 min, plus 3 min for post-run equilibration time.

Tandem Mass Spectrometry (MS/MS) Analysis

The LC-MS/MS system is operated in the dynamic MRM 

mode during data acquisition to achieve maximum sensitivity and selectivity of the MS/MS technology. In this mode, 

the ion source creates charged ions from the sample flowing 

from the LC column, and a precursor ion is selected by the 

first quadrupole of the MS. The second quadrupole serves as a 

collision cell focusing and transmitting the ions while introducing the collision gas (nitrogen) to fragment the precursor 

ion. The third quadrupole is used to select the characteristic 

fragment ions. For the method described here, two characteristic fragment ions are selected, one for quantitation and one for 

qualification. These characteristic precursor/product ion pairs 

are called MRM transitions. 

Development of the MRM Method 

The analytical method was first developed by selection 

and optimization of the MRM conditions for each analyte. 

The MS/MS was operated in both positive and negative 

electrospray ionization modes for different groups of analytes. 

Standards of 1 μg/mL in methanol were infused into the MS 

(no LC column) for selection of precursor and product ions 

and optimization of MS/MS parameters (fragmentor voltage 

and collision energy). Analytes were optimized in the positive 

ionization mode first. If response was low (integrated area of 

the precursor ion less than about 1,000) or previous literature 

indicated good performance in negative mode, then analytes 

were optimized in negative ionization mode. After MS/MS 

parameters were optimized for each analyte, individual standards of 0.1 μg/mL in reagent water were injected into the LC 

system with the analytical column connected to determine the 

analyte retention time and chromatographic performance on the 

LC column.

The optimized fragmentor voltages and other dynamic 

MRM parameters (collision energy and product ions) are 

shown in table 6. For most analytes in the ESI+ mode (171 

of 185), the protonated molecular ion [M+H]+ was most 

abundant and was selected as precursor ion. For 10 pesticide 

compounds, the ammonia adducts [M+NH4]+ were most 

abundant and selected (oxamyl, aldicarb, isoxaflutole, 

flumiclorac-pentyl, lactofen, piperonyl butoxide, propargite, cis-permethrin, trans-permethrin, bifenthrin). For some 

compounds the precursor ions were ion fragments of the 

molecular ion (for example, famoxadone, desiodoflubendiamide, fenbutatin oxide, fentin, terbacil). Generally, the two 

most abundant product ions were selected for quantitation and 

qualification MRM transitions, and their collision energies 

were optimized. For four analytes, there was only one major 

product ion, so the qualifier MRM selected was precursorto-precursor type (for example, 1H-1,2,4-triazole, 70 → 70; 

methomyl oxime, 106 → 106; phorate oxon, 245 → 245; 

parathion-methyl, 264 → 264).

For most analytes in the ESI− mode (38 of 44), the 

deprotonated molecular ion [M-H]- was most abundant and 

was selected as precursor ion (table 6). Generally, the two 

most abundant product ions were selected for quantitation and 

qualification MRM transitions, and their collision energies 

were optimized. For six pesticide compounds, there was only 

one major product ion, so the qualifier MRM selected was 

precursor-to-precursor type (for example, sec-acetochlor-OA 

206 → 206; metribuzin DK, 183 → 183; sec-alachlor OA, 220 

→ 220; hexazinone TP E, 240 → 240; hexazinone TP D, 224 

→ 224; MCPA, 199 → 199).

The retention times (table 6) show that the LC conditions 

were sufficient to separate analytes that shared MRM transitions and provide two unique MRM transitions for all analytes. 

In the few cases where analytes co-eluted and shared the same 

precursor ion, there were unique product ions that allow identification and quantitation (for example, azinphos-methyl and 

phosmet, ESI+, retention time = 12.4 min). The acetanilides 

acetochlor SA and alachlor SA were exceptions and co-elute 

in ESI− mode at 6.8 minutes and have common precursor and 

product ions. During development of the method, a number of 

modifications to LC conditions including column type, column 

temperature, and mobile phase were tested, yet all were unable 

to achieve separation. Additional low-abundance qualifier 

ions (acetochlor SA, 314 → 162; alachlor SA, 314 → 160, 

314 → 176) were later added to the method to enable separate 

identification (table 6), at the cost of higher detection levels 

for these analytes.

Dynamic MRM Conditions

The dynamic MRM methods used to acquire analytical results contained 423 transitions for ESI+ mode and 120 

transitions for ESI− mode. In the dynamic MRM acquisition 

method, the dwell times are determined by the MS/MS instrument software depending on the number of concurrent MRM 

transitions and retention time window set for each analyte. The 

retention time windows shown in table 6 were manually minimized to maintain dwell times longer than 10 microseconds 

for optimal signal-to-noise measurement of chromatographic 

peaks while still allowing for small shifts in retention time.

Once the dynamic MRM method was developed, other 

operational parameters were optimized by analysis of a 10,000-

ng/L mixed calibration standard. These parameters were varied, 
Analytical Method 11

and those that resulted in the highest response for the largest 

number of analytes were selected. The optimized MS/MS 

instrument acquisition parameters for electrospray ionization 

are shown in table 7.

Mass Calibration

The mass spectrometer is tuned and calibrated with the 

electrospray calibrant mix and procedures specified by the 

manufacturer. Routine use only requires resolution and mass 

axis verification for the resolution used in the method (Unit, 

Wide, or Widest resolution) and is done using the Checktune 

software of the instrument. The Autotune software is used to 

adjust and optimize ion transmission and update the electron 

multiplier voltage every 1 to 2 months, or if the Checktune 

fails. The ion response drifts lower over time, and the Autotune software restores the response.

Sample Analysis Sequence

Samples are analyzed in a sequence of instrument blanks, 

calibration standards, laboratory QC samples, and environmental samples called an analytical batch (table 8). A wash blank 

(WBLK) sample is used to verify that previous instrument 

cleaning was sufficient, followed by a continuing calibration 

blank (CCB) sample. Calibration (CAL) standards are analyzed 

at the beginning of each batch of samples to develop a calibration curve. Depending on the potential for carryover, one (ESI+ 

analytes) or two (ESI− analytes) CCB samples follow the highest calibration sample, followed by a series of continuing calibration verification (CCV) standards, about 15 environmental 

samples, and then by laboratory QC samples. The laboratory 

QC samples include laboratory reagent blanks and laboratory 

reagent spikes prepared in sample containers, similar to the 

environmental samples. Low concentration CAL standards are 

used as instrument detection level (IDL) standards to check 

LC-MS/MS response during and at the end of the analytical 

batch. A typical analytical batch consists of about 109 analytical 

vials with 75 environmental samples, 2 laboratory QC samples, 

and 32 instrument QC samples (blanks, calibration standards, 

CCVs). 

The analytical batch is analyzed using an acquisition 

method set up with the MassHunter acquisition software. 

The list of samples and associated sample information for 

the analytical batch (created from the LIMS data system) 

is transferred to the LC-MS/MS prior to data acquisition to 

create a worklist file for data acquisition. The samples are 

assigned to sample types used by the MassHunter software 

for later data analysis (table 8). Sample vials are placed in the 

sample trays to match locations indicated on the worklist. The 

LC-MS/MS system is prepared to analyze samples by making sure adequate mobile phases are available, temperatures 

are set, column pressure indicates no leaks or blockage of 

the LC column, and the LC column has equilibrated with the 

initial mobile phase. Then the analytical sequence is started 

to acquire data for all samples. Data acquisition for a batch of 

75 environmental samples plus associated QC samples takes 

about three days for the ESI+ mode (30 min per sample) and 

two days for the ESI− mode (20 min per sample).

Data users should use caution when retrieving laboratory 

QC data from the NWQL LIMS database because the type of the 

QC samples can be different from those used during data acquisition. In particular, the blanks used to check for carryover after 

high-calibration samples and at the start of the batch as well as 

the instrument blanks (IBLNK) are all identified as IBLNK-type 

samples in the LIMS database. The instrument blanks are most 

important for evaluating routine sample contamination, whereas 

the others are useful for evaluations of specific sources of contamination, but they should not all be combined for a summary of 

blank contamination that might be representative of the environmental samples.

Use of Isotope-Labeled Internal Standards

Isotope-labeled standards are used for quantitation of the 

pesticides using the internal standard technique. In this technique 

response of the pesticide relative to the internal standard—

measured as chromatographic peak area for the quantitation 

MRM—is determined over the calibration range. The internal 

standards improve quantitation because they compensate for 

matrix effects and injection variables compared to an external 

standard calibration. The internal standards cover a range of pesticide chemical classes and retention times and are chosen to best 

represent the diverse range of pesticides in the method. 

Table 9 shows the internal standards used for the pesticide 

compounds. Internal standards selected were stable-isotopelabeled pesticides when available, with sufficient mass difference (>3 m/z) between the pesticide and internal standard to 

avoid fragmentation of both to the same product ion. Internal 

standards from the same chemical class as the pesticide compounds were used if possible, although other considerations 

such as retention time were used to assign pesticide compounds 

to internal standards. Internal standards reduce the effect of ion 

suppression during analysis because any matrix components 

co-eluting with the analyte will be identical for the internal standard allowing the analyte-to-internal-standard response ratio to 

compensate for any ion suppression that may be present (Furey 

and others, 2013). However, because this method uses internal 

standards for analytes that elute at retention times different from 

the internal standard, there might be analyte responses that are 

not compensated for ion suppression if the matrix interference 

is related to a specific time of the chromatographic run. Matrix 

spikes are useful to monitor and identify this type of matrix 

interference.

The recovery of the internal standards is calculated 

and reported to provide a way to monitor ion suppression or 

enhancement of a group of analytes related to the internal 

standard, similar to a surrogate. Atrazine-d5 is used to calculate 

recovery of ESI+ mode internal standards as surrogates, and 

ibuprofen-13C3 is used to calculate recovery of ESI− mode 

internal standards as surrogates. 
12 Determination of Pesticides and Pesticide Degradates in Filtered Water

Quantitation and Calculation of Results

Data from the analytical batch is analyzed using the 

MassHunter quantitative analysis software to prepare a quantitative method. The quantitative method includes information 

about samples and QC sample types (CAL, CCVs, blanks, and 

so forth) and concentrations, as well as analyte information 

such as whether it is an analyte, surrogate, or internal standard. 

In addition, analyte retention times, qualifier ion ratios, and 

QC limits are updated to assist in data analysis and review. 

The time required for quantitative analysis, peer review, and 

transfer of results to the LIMS for a typical batch of 75 environmental samples is about 14 days for the ESI+ mode and 6.5 

days for the ESI− mode. 

Calibration is performed using the peak areas and the 

internal standard technique. A series of 12 CAL standards, 

ranging from 1 to 10,000 ng/L are analyzed at the start of each 

batch. The number of calibration standards used for fitting 

the calibration response varies from 5 to 12, depending on 

the sensitivity of the analyte. The lowest calibrator used is 

less than the LRL. Calibration curves are generated using the 

MassHunter software using quadratic curve fit, ignore origin, 

and 1/x weighting settings. An example of a calibration curve 

is shown in figure 1A. The 1/x weighting is used to provide a 

better fit of the curve and less bias at the lowest concentration 

CAL standards. There is negligible change in the bias at the 

highest concentration CAL standards using this weighting.

The calibration curve is evaluated by the overall linear 

fit of the quadratic curve (the statistical r2 value) and the bias 

of individual standards (measured as residuals in percent in 

MassHunter software). The fit of the quadratic curve must 

be greater than 0.990. The bias of each CAL standard in 

the curve relative to the nominal concentration must be less 

than ±30 percent, except at the lowest level where bias is 

±50 percent. In some batches, especially in the ESI− mode, a 

CAL standard might have larger bias, but the overall fit must 

still be greater than 0.990. If the overall calibration curve 

criteria cannot be met, it might be difficult to achieve other 

data-quality requirements, such as CCV and IDL sample 

acceptance criteria. In that case, corrective action is taken to 

reanalyze the CAL standards, or perform maintenance on the 

LC-MS/MS instrument.

Qualitative Determination

Identification of pesticides in samples is done by evaluation of retention times and ion ratios of the characteristic MRM 

transitions. Expected values for retentions times and qualifier 

ion ratios were established when the method was developed 

and are usually updated when new calibration samples are analyzed. When a batch of samples is analyzed, the MassHunter 

software uses peak identification criteria based on retention 

time and qualifier ion ratios to identify and integrate chromatographic peaks. The analyst reviews the chromatographic peaks 

of both quantitative and qualifier MRM transitions and verifies 

the identifications. An example of a chromatogram with the 

MRM quantifier and qualifier ions for a calibration standard of 

atrazine is shown in figure 1C. Manual integrations might be 

required for peaks with low response in noisy background, or 

alternatively, ignoring integrated peaks that are very small and 

cannot be differentiated from background noise. The signal to 

noise ratio, measured as peak height relative to height of background signal, needs to be above 3 for peak to be integrated 

and accepted by the analyst.

Retention Times

The acceptance criteria for retention times were calculated from the reproducibility of the analytes spiked at different concentrations in groundwater and stream samples during development of the method. The environmental samples 

typically provide more variability encountered in retention 

time than in reagent water. An acceptance window was set as 

3 times the standard deviation of the calibration standard retention time (Lehotay and others, 2008). Typical retention times 

and acceptance ranges of the analytes are shown in table 10. 

The acceptance ranges vary with retention time, from less than 

0.5 percent after 7 min, and wider acceptance ranges from 2 

to 10 percent in the first 6 min of the analysis. These empirical 

determinations of retention time of the analytes determined in 

matrix spikes are used as identification criteria. For example, 

both MRM transitions must have a retention time within the 

mean ±3 standard deviations (in percent) acceptance range to 

be considered for qualitative identification. These retentiontime acceptance ranges are entered into the MassHunter software and are used to display outliers in quantitative analysis 

and review.

Qualifier Ion Ratios

The acceptance ranges for qualifier ion ratios (QIR) vary 

depending on the relative response of the qualifier and quantifier 

MRM transitions. The relative abundance is the area of the most 

abundant (quantifier) ion divided by the qualifier ion expressed 

as a percentage; the acceptance criteria is wider for ions with 

small area relative to the quantifier ion. General guidelines for 

acceptance used by European Union for identification of veterinary drug residues in food (The Commission of the European 

Communities, 2002) and widely used by LC-MS/MS analysts 

are shown in table 11. 

These guidelines are generally narrower than can be 

achieved in all matrices and across all concentrations for many 

analytes, and the criteria should not be taken as absolute limits 

for all applications. For this reason most laboratories require 

complementary interpretation by an experienced analyst to 

determine whether the criteria are appropriate for the sample 

by comparison with other QC samples (European Commission, 2012). A more practical approach is to calculate acceptance criteria for QIRs determined in calibration standards and 

matrix spikes to better represent the desired concentrations 

and matrices of the samples (Lehotay and others, 2008). This 

approach was taken for the DAI LC-MS/MS method, and the 
Analytical Method 13

Figure 1. Example of (A) a calibration curve for 

metolachlor determined by direct aqueous-injection 

liquid chromatography-tandem mass spectrometry 

(DAI LC-MS/MS) showing response of metolachlor 

relative to the internal standard metolachlor-d6

 plotted 

as a function of concentration, (B) an expanded view of 

the lowest section of the calibration curve, and (C) low 

concentration standard (10 nanogram per liter [ng/L]) 

of metolachlor showing qualifier (284 → 176, shaded 

blue) and quantifier (284 → 252, shaded gray) multiple 

reaction monitoring (MRM) ions for metolachlor. Dotted 

lines show acceptance criteria for qualifier ion ratio 

based on ±15 percent.

A

Metolachlor - 10 Levels, 10 Levels Used, 10 Points, 10 Points Used, 0 QCs

Concentration, in nanograms per liter

−500 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 5,500 6,000 6,500 7,000 7,500 8,000 8,500 9,000 9,500 10,000 10,500

x101

−0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9 y = 2.069307E-008 * x ^ 2 + 0.008643 * x - 0.003682

R^2 = 0.99964911

Type:Quadratic, Origin:Ignore, Weight:1/x

Relative response

B

Metolachlor - 10 Levels, 4 Levels Used, 10 Points, 4 Points Used, 0 QCs

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11

x10−2

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

Relative response

Concentration, in nanograms per liter

y = -1.420816E-004 * x ^ 2 + 0.007696 * x - 9.436926E-005

R^2 = 0.99238737

Type:Quadratic, Origin:Ignore, Weight:1/x

C

CAL500-1 [Metolachlor]

14.6 14.7 14.8 14.9 15 15.1 15.2 15.3 15.4

x105

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2 284.1 -> 252.1 Final Conc.=534.3 ng/L

284.1 -> 176.0 Ratio=25.8

Acquisition time, in minutes

Area counts
14 Determination of Pesticides and Pesticide Degradates in Filtered Water

acceptance criteria for qualifier ion ratios determined for the 

pesticide compounds are shown in tables 12 and 13. 

These qualifier ion ratios were determined in reagent water 

(laboratory reagent spike [PSPK], CCV, and CAL standards) 

and the three environmental matrices (two streams and a 

groundwater) used in the matrix validation study at four concentrations (10, 100, 500, and 5,075 ng/L). The variability is larger 

in some cases in reagent water because this dataset included 

some CAL standards at lower concentrations (<10 ng/L) than 

the matrix samples. Statistical tolerance intervals of qualifier ion 

response ratios determined by DAI LC-MS/MS in both ESI+ 

and ESI− mode were calculated for 99 percent of the ratios (at 

confidence [1-alpha] of 0.95). This means that, with 95 percent 

confidence, the tolerance interval given by the upper and lower 

limits will enclose 99 percent of the QIRs. An upper limit of 

75 percent and lower limit of 5 percent were used to provide 

useful and meaningful limits. The acceptance criteria based on 

the calculated tolerance intervals were used if larger than the 

EU limits. These empirical determinations of qualifier ion ratios 

are used as guides for identification criteria and are entered into 

the MassHunter software to help speed up initial determination of the analyte identification by displaying outliers. For 

example, the qualifier ion ratio must be within the acceptance 

criteria window to be considered for qualitative identification; 

otherwise, results are flagged for review by the analyst. The 

qualitative identification of a pesticide compound in a sample 

would then be based on interpretation by a skilled analyst of the 

comparison of ion ratios with calibration and IDL standards at a 

similar concentration.

Graphs of the QIRs of all pesticide compounds determined 

by DAI LC-MS/MS fortified at different concentrations (10, 

100, 500, and 5,075 ng/L) in the different matrices studied in 

the validation experiments and in CAL and CCV standards in 

reagent water are shown in supporting information figures S1 

and S2. An example of a graph of the QIRs of atrazine and atrazine degradates is shown in figure 2. These graphs show that the 

MRM ratios are within the EU limits at higher concentrations 

(>50 ng/L) but exceed them at lower concentrations. The empirical-tolerance interval limits are wider and encompass most of 

the qualifier ion ratios at lower concentrations. Examples of the 

corresponding chromatograms of quantifier and qualifier multiple reaction monitoring (MRM) ions for atrazine at different 

concentrations (unspiked, 10, 100, 500, and 5,075 ng/L) in four 

different matrices are shown in figure 3. These chromatograms 

show that the relative abundance of the qualifier ion is more 

variable in different matrices at the lowest concentration.

Quality Assurance and Quality-Control Criteria

General guidelines for acceptance criteria for QC 

samples, calibration, and other quality-control procedures 

applicable to the NWQL were used for this DAI LC-MS/MS 

method (Maloney, 2005). More relevant QC criteria for calibration standards, continuing calibration standards, and retention times for analyte identification were used from recent EPA 

methods that use LC-MS/MS for quantitative determination 

of organic compounds in drinking water samples (Shoemaker 

and Bassett, 2005; Shoemaker, 2009; Smith and others, 2011). 

These are summarized in table 14. If a QC sample is outside 

these limits, analysts might have difficulty meeting other QC 

requirements. Some examples of corrective actions that are 

taken for samples not meeting the QC requirements are shown 

in supporting table S4.

Results from this method are sometimes reported using 

NWIS water-quality-database remarks or value qualifier codes 

to qualify sample results when quality-control criteria are not 

met or there are other problems identified that might affect 

accuracy of the sample result. These codes are shown in supporting table S3.

Routine laboratory QC Samples

Routine laboratory QC samples include instrument QC 

samples (CAL standards, CCV standards, instrument blanks, 

third-party check standards) and batch QC samples (laboratory reagent spikes or preparation spikes [PSPK] and laboratory reagent blanks) analyzed with environmental samples. 

All instrument QC samples are prepared using about 1 mL 

of sample in 2-mL analytical vials. All laboratory batch QC 

samples are prepared using a 10-mL sample contained in a 

20-mL analytical vial.

Batch QC Samples

Laboratory reagent-water spikes and laboratory reagentwater blanks provide information about overall method 

performance in a control matrix and potential contamination 

from all processing and analysis steps in the laboratory. These 

QC samples are prepared with each batch of environmental 

samples analyzed—usually 1–2 laboratory spikes and 1 laboratory blank sample is prepared in each batch (table 8). The 

QC samples provide information about bias and variability 

due to the sample container and sample processing steps, in 

addition to the instrument analysis steps.

The recovery of the analytes in the laboratory reagent 

spikes is calculated and compared to the acceptance criteria. 

For the DAI LC-MS/MS method, initial acceptance criteria 

were set to 100±30 percent (70 to 130 percent). Analytes with 

recoveries outside acceptance limits are reported to the NWIS 

water-quality database qualified with an “E” (estimated) 

remark and a value qualifier explaining that the spike failed 

acceptance criteria (supporting table S4) in environmental 

samples that have detected concentrations of the analyte.

The laboratory reagent blanks provide information about 

contamination during sample preparation and analysis in the 

laboratory. Detections of analytes in the laboratory reagent 

blanks are used together with instrument blanks to evaluate levels of contamination or carryover. Analytes that are 

detected in the blanks are reported to the NWIS water-quality 

database censored (reported as nondetected at the LRL) or 

qualified with a “v” value qualifier in samples with detected 

concentrations, depending on the concentration in the sample 

relative to the concentration in the blank.
Analytical Method 15

Figure 2. The qualifier ion response ratio (QIR) of atrazine and three atrazine degradates determined by direct aqueous-injection 

liquid chromatography-tandem mass spectrometry (DAI LC-MS/MS) in positive electrospray ionization (ESI+) mode spiked in 

different matrices (Santa Ana River below Prado Dam, California; Mississippi River below Grafton, Illinois; groundwater from Well 

1N/14W-8K1S, Calif.) at 10, 100, 500, and 5,075 nanograms per liter (ng/L) and in reagent water (calibration [CAL] and continuing 

calibration verification [CCV] standards at 1, 2.5, 5, 10, 25, 50, 100, 500, 5,000, and 10,000 ng/L). Samples were excluded (shown 

as open symbols) from evaluation if the fortification concentration was less than the method detection level (MDL). Dotted lines 

show European Union QIR ratio limits, and dashed lines show limits based on the statistically calculated tolerance interval. Dotted 

vertical line shows calculated MDL.

A

C

B

D

2-Hydroxyatrazine

0

20

40

60

80

100

120

140

160

180

200

1 0.6 10 6432 100 503020 1,000 200 400 10,000 4,000

Concentration, in nanograms per liter

Qualifier ion response ratio

0

20

40

60

80

100

120

140

160

180

200

1 0.6 10 6432 100 503020 1,000 200 400 10,000 4,000

Concentration, in nanograms per liter

2-Hydroxy-4-isopropylamino-6-amino-s-triazine

Qualifier ion response ratio

2-Hydroxy-6-ethylamino-4-amino-s-triazine

0

20

40

60

80

100

120

140

160

180

200

1 0.6 10 6432 100 503020 1,000 200 400 10,000 4,000

Concentration, in nanograms per liter

Qualifier ion response ratio

Atrazine

0

20

40

60

80

100

120

140

160

180

200

1 0.6 10 6432 100 503020 1,000 200 400 10,000 4,000

Concentration, in nanograms per liter

Qualifier ion response ratio
Figure 3. Chromatograms of the multiple reaction monitoring (MRM) ions for atrazine determined by direct aqueous-injection liquid chromatographyspectrometry (DAI LC-MS/MS) at different concentrations (unspiked, 10, 100, 500, and 5,075 nanograms per liter [ng/L]) in four different matrices (reagfrom well 1N/14W-8K1S, stream 1 [Mississippi River below Grafton, Illinois], and stream 2 [Santa Ana River below Prado Dam, California]). Quantifier igray and qualifier ion (216 → 104) shown in blue. Dashed lines show acceptance criteria for qualifier ion peak height relative to quantifier ion (qualifie

concentrations of atrazine were 5 and 2 ng/L in groundwater well 1N/14W-8K1S and Santa Ana River below Prado Dam samples. An unidentified interunspiked sample from Mississippi River below Grafton, Illinois. (CMick here to locate full-size, high-resolution image.)
Analytical Method 17

Instrument Laboratory QC Samples

Instrument QC samples include CAL standards, CCV 

samples, third-party check (TPC) standards, IDL standards, 

and instrument blanks prepared in 1-mL reagent water contained in a 2-mL analytical vial.

CCV Standards

Continuing calibration verification (CCV) standards 

are calibration standards at one concentration level analyzed 

throughout the analytical batch (table 8). They are used to 

verify continuing accuracy of the calibration curve during 

analysis of the entire batch of samples. Low- and mediumlevel CCVs are placed in the middle and end of the batch 

sequence. Acceptance criteria for recovery of all CCVs are 

initially ±30 percent and later updated based on annual summaries of CCV sample results. Analytes with recoveries outside acceptance limits are reported to the NWIS water-quality 

database qualified with an “E” (estimated) remark and a value 

qualifier explaining that the spike failed acceptance criteria 

(supporting table S4) for environmental samples that have 

detected concentrations of the analyte.

Instrument Detection Level Standards

Low-level CCVs (below the LRL) are used as IDL 

standards in the batch. These are analyzed at the end of the 

batch to verify that the detection levels are maintained during 

analysis of the batch of samples (table 8). The IDL is verified 

if all results meet qualitative identification criteria of the 

method that (1) both quantifier and qualifier MRM transitions are present at the correct retention time, and (2) that 

both MRM transitions have signal to noise ratio greater 

than 3 (based on visual evaluation of peak height relative to 

baseline). The MRM qualifier ion ratio can exceed limits that 

are used for qualitative identification of pesticide compounds 

in other non-spiked samples (for example, identification of 

unknowns) because the MRM ratios of the IDL are expected 

to be more variable at low concentrations and at the end of 

the analytical batch. If the IDL is not verified, there might 

have been a decrease in sensitivity during analysis of the 

batch, potentially requiring reporting all nondetections with a 

raised reporting level. 

Third-Party Check Standards

Third-party check standards are analytical standards prepared from an independent source—different from that used 

to prepare the calibration and spike standards. A third-party 

check standard is usually analyzed with each batch of calibration standards and QC and environmental samples. During 

development and implementation of the DAI LC-MS/MS 

method, third-party check standards were not available for all 

analytes. However, spike solutions for NWQL schedules 2033 

and 2060 contained many analytes in common and thus were 

suitable for use as third-party check standards. These spike 

standards are in ethyl acetate (2033) and methanol (2060) and 

can be directly added to the analytical vials. The spike mixture 

is diluted so that 100 μL added to the 0.9-mL reagent-water 

sample contained in the 2-mL analytical vial will result in 

an expected concentration of 250 ng/L. Acceptance criteria 

for recovery for all third-party check standards is ±30 percent. Analytes with recoveries outside acceptance limits are 

reported to the NWIS water-quality database qualified with an 

“E” (estimated) remark and a value qualifier explaining that 

the TPC failed acceptance criteria (supporting table S4) for 

environmental samples that have detected concentrations of 

the analyte.

Reporting of Results

Analyte concentrations in each sample are determined 

using the calibration curves after the MRM chromatogram 

has been reviewed to confirm qualitative identification of the 

analyte. All concentrations that meet identification criteria are 

reported to the NWQL LIMS, provided other quality-control 

criteria, primarily laboratory blank results, are met (Childress 

and others, 1999). Concentrations of analytes below the MDL 

are reported with a remark code “E” (estimated) to reflect 

the larger uncertainty of the concentration determined, and 

the appropriate NWIS value-qualifier code (“b”: value was 

extrapolated at low end; “t”: value below the MDL; supporting information tables S3 and S4). The “E” remark does not 

indicate that there is more uncertainty in the identification of 

an analyte with a concentration below the MDL because all 

qualitative identification criteria must be met before a concentration can be reported. 

Samples with results that exceed the highest CAL standard should be either diluted or reanalyzed. In some cases 

where this is impractical, sample concentrations that exceed 

the highest CAL standard can be reported with the appropriate NWIS value-qualifier code (“a”: value was extrapolated at 

high end; supporting information tables S3 and S4). 

Final concentrations are reported in nanograms per 

liter rounded to the precision of the standard deviation of 

the replicate determinations of spike samples. For pesticide 

compounds with MDLs less than 10 ng/L, concentrations are 

reported with a rounding unit of 0.1—to 1 decimal place, but 

no more than 4 significant figures. For pesticide compounds 

with MDLs between 10 ng/L and 100 ng/L, concentrations are 

reported with a rounding unit of 1—no digits to the right of 

the decimal place, but no more than 4 significant figures. For 

pesticide compounds with MDLs above 100 ng/L, concentrations are reported with a rounding unit of 10—no digits to the 

right of the decimal place and no significant figures between 

1 and 9, but no more than 3 significant figures. Results for 

analytes not detected in the samples are reported as less than 

the laboratory reporting level (<LRL). 
18 Determination of Pesticides and Pesticide Degradates in Filtered Water

Results and Discussion of Method 

Validation Data

The method validation experiments consisted of studies 

that determined calibration linearity and response, bias and 

variability in different matrices at different concentrations, 

method detection levels, and stability of samples during storage prior to analysis and a comprehensive field study of the 

method performance in 47 streams, with comparison of this 

method to two existing NWQL pesticide analytical methods. 

The analytes included in the method validation were the prioritized tier 1 pesticide compounds that provided reasonable 

signal response under the MS/MS acquisition and chromatographic conditions used during testing in the validation studies. A number of analytes that had responses in both ESI+ and 

ESI− acquisition modes were tested in both modes. After initial testing, the acquisition mode providing the best response 

was selected. There were about 90 analytes from the initial 

prioritized tier 1 pesticide compounds that were not included 

in the method validation because they had low signal response 

or no retention on the reversed-phase LC system. These were 

mainly organochlorine, pyrethroid and dinitroaniline pesticides, as well as highly polar compounds. These analytes are 

shown in supporting table S5.

Data-Quality Objectives

The data-quality objectives of the validation experiments 

were that bias must be within 30 percent of the expected value 

(recovery of 100 percent ±30 percent) and variability less than 

or equal to 30-percent relative standard deviation. Analytes not 

meeting these objectives in more than one matrix or stability test were removed from the method. Some analytes where 

these objectives were not met in one matrix are reported to 

NWIS with an estimated remark (“E”).

Data Analysis

JMP software version 9.0 (SAS Institute Inc., 2010) 

was used for statistical analysis. Mean, standard deviation, 

relative standard deviation, median, and selected quantiles 

were summarized for pesticide compounds in the different 

validation experiments. Symmetric confidence intervals for 

the mean were computed using the Student’s t statistic. The 

critical values for t are computed at one-half the desired level 

of confidence (alpha level) for a two-sided test. The width of 

the confidence interval is a function of these critical values, 

the standards deviation of the data, and the sample size (Helsel 

and Hirsch, 1992). Control limits for the pesticide compounds 

were calculated using the median and F-pseudosigma of spike 

recoveries. The F-pseudosigma is the interquartile range 

divided by 1.349 and is equal to the standard deviation of a 

normal distribution but is not as strongly influenced by outliers 

(Helsel and Hirsch, 2002).

The relative percent difference (RPD) of replicate sample 

results was calculated using the following equation:

e r

e r

−

 +



 





×

2

100

(1)

where

e is the environmental sample result and

r is the replicate sample result.

The RPD was not calculated if one of the sample results was 

missing or reported as not detected.

Specification limits for qualifier ion response ratios (QIR) 

were developed using tolerance intervals. A tolerance interval 

contains at least a specified proportion of the population; it is 

a confidence interval for a specified proportion of the sample 

population (Hahn and Meeker, 1991). Tolerance intervals of 

QIR were calculated for 99 percent of the ratios (at confidence 

[1-alpha] of 0.95). This means that, with 95 percent confidence, the tolerance interval given by the upper and lower 

limits will enclose 99 percent of the QIRs.

The sign test was used to compare two sets of results 

for paired samples in the paired-sample stream study. The 

sign test is a non-parametric alternative to the paired t-test for 

paired data, where the data are from a population that is neither normal nor even symmetrical (Miller and Miller, 1993). 

The sign test is used in this case to determine whether the 

median difference between paired observations equals 0. The 

number of detections was not symmetrical in all cases, so the 

more powerful Wilcoxon Signed rank test could not be used 

(Miller and Miller, 1993), even though these test results are 

shown in the tables for comparison.

The p value is the probability that a difference as large as 

or larger than that seen in the experiment would have occurred 

by chance alone if the treatment groups were in fact not different and depends on the experimental design, statistical test 

used, and variability of the data (Lovell, 2013). In this report 

a p value less than 0.05 is used to highlight test results that 

might have chemical significance.

Assessment of Suitability for the DAI LC-MS/MS 

Method

Some analytes with very low response or limited range of 

calibration were identified as possible candidates for elimination from the method; five analytes (diquat; the kresoximmethyl degradates BF490-1, BF490-2, and BF490-9; and 

paraquat) were removed from the LC-MS/MS method prior to 

matrix-effects studies method for this reason. Eight other analytes (diflufenzopyr, diketonitrile isoxaflutole, dimethenamid 

OA, dimethenamid SA, dimethenamid SAA, fipronil, fipronil 

amide, and fipronil sulfide) performed better in ESI− mode 

and thus were removed from the ESI+ mode method (and 

added to the ESI− mode), whereas six analytes (chlorimuronethyl, chlorsulfuron, famoxadone, novaluron, propanil, and 
Results and Discussion of Method Validation Data 19

prosulfuron) performed better in ESI+ mode so were removed 

from the ESI− mode method (and added to the ESI+ mode). 

During the validation tests of bias and variability in different 

matrices and stability during storage, a number of analytes 

were found to greatly exceed quality-control requirements. 

These analytes were deleted from the method (shown with a 

“D” validation quality code in table 1), including eight analytes 

in the ESI+ mode (3-ketocarbofuran, 7-hydroxycarbofuran, 

aminopyralid, bifenazate, bifenazate diazene, flumicloracpentyl, formetanate, and phosmet oxon) and three in the ESI− 

mode (ammelide, deisopropyliprodione, and iprodione). Iprodione is unstable in methanol (Anisuzzaman and others, 2008), 

which is used for preparation of the calibration standards, and 

overall performance was highly variable even though we used 

iprodione-d5 as in internal standard. Other analytes had high 

bias and variability in one or two of the matrices tested and so 

were qualified with the estimated “E” validation quality code 

(table 1). There are seventeen qualified analytes in the ESI+ 

mode (1H-1,2,4-triazole, 2-(1-hydroxyethyl)-6-methylaniline 

(HEMA), 3-hydroxycarbofuran, lactofen, asulam, bifenthrin, 

butralin, carbendazim, diazinon oxon, famoxadone, fenbutatin 

oxide, flumetsulam, naled, novaluron, oxyfluorfen, parathionmethyl, and phosmet) and five analytes in the ESI− mode 

(alachlor-SA, chlorosulfonamide acid, chlorthal-monomethyl, 

dicamba, and hexazinone TP D). Results from the validation 

tests for the deleted and qualified analytes are included in the 

following sections to demonstrate the reasons for their deletion 

or qualification. Deleted analytes are shown in the tables with 

the validation quality code “D,” whereas qualified analytes are 

shown in in tables with the validation quality code “E,” and a 

different symbol in some figures.

Analyte Response as a Function of Concentration

ESI+ Analytes

The response factors (peak area divided by nominal 

concentration) were measured as a function of concentration 

to evaluate any significant change in response as a function 

of concentration. In addition, the response factors provide 

an indication of the magnitude of the signal in electrospray 

ionization that reflects detection levels, with higher response 

factors leading to lower detection levels. Response factors 

for the ESI+ analytes were calculated from seven replicate 

determinations of 10 calibration standards (1, 2.5, 5, 10, 50, 

100, 500, 1,000, 5,000, and 10,000 ng/L) analyzed consecutively. An example of the response factors as a function of 

concentration for metolachlor and metolachlor degradates 

is shown in figure 4 (graphs of response factors of all ESI+ 

analytes are shown in supporting figure S4). A linear fit was 

applied to the response factors as a function of concentration (table 15; fig. 4). Response factors were excluded from 

the linear fit and summaries if the calibration standard was 

excluded from the calibration curve because qualifier ion 

response did not meet identification criteria (shown as open 

circles in the figures).

Response factors varied from 2 to 4,782, with an overall 

median of 255. Analytes with median response factors greater 

than 1,000 typically had narrow, symmetrical peaks that were 

well-resolved from the background and calculated MDLs 

(discussed in the “Method Detection Level Determination” 

section of this report) less than 10 ng/L. Analytes with median 

response factors less than 10 typically were wide and difficult 

to identify from the background signal and had calculated 

MDLs greater than 10 ng/L. There were 30 analytes from 

different pesticide groups that had median response factors 

greater than 1,000 (supporting table S6).

Analytes with median response factors less than 10 

included some of those later deleted from the method because 

of poor performance (7-hydroxycarbofuran, aminopyralid), 

were deleted from the ESI+ mode and moved to the ESI− 

mode (fipronil, diketonitrile isoxaflutole), or qualified with 

an estimated remark (asulam,1H-1,2,4-triazole, oxyfluorfen, 

diazinon oxon, parathion methyl). Response factors for cispermethrin and trans-permethrin were low because the initial 

quantification MRM selected (408–355) had low response; 

after this experiment a different MRM (408–183) (table 6) that 

provided higher responses (about 500) was used for analysis.

For many of the analytes, the response factors were constant as a function of concentration over 4 orders of magnitude, with the slopes of the linear fit not significantly different than 0 (supporting table S6). In most cases, the response 

factors were more variable at the lowest concentrations, 

resulting in lower confidence that the slopes were not different than 0 (shown in bold and red highlight). For example, the 

response factors for metolachlor hydroxy morpholinone are 

about 340 at all concentration levels above 10 ng/L but varied 

between 20 and 600 below 10 ng/L (fig. 4), and the slope of 

the linear fit of response factors was significantly different 

from 0 (p = 0.0041). However, the overall magnitude of the 

change in response factor was small (slope = 0.006). For most 

analytes, there was no decline in response factor at higher 

concentrations, which had been reported previously for some 

analytes using DAI LC-MS/MS (Greulich and Alder, 2008), 

perhaps due to instrumental differences in electrospray interface and (or) electronics for handling overlapping MRMs.

ESI− Analytes

The negative ion response factors (peak area divided 

by nominal concentration) were also measured as a function 

of concentration. An example of the response factors as a 

function of concentration for metolachlor SA and metolachlor 

OA determined in ESI− mode is shown in figure 4 (graphs of 

response factors of all ESI− analytes are shown in supporting 

table S7).

Median response factors varied from near 0 to about 

230, with an overall median of 9. These are 1–2 orders of 

magnitude lower than the ESI+ response factors. There were 

six pesticide compounds with median response factors greater 

than 100, and these have calculated MDLs (discussed in the 

“Method Detection Level Determination” section of this 
20 Determination of Pesticides and Pesticide Degradates in Filtered Water

A

C

E

B

D

F

Nominal concentration, in nanograms per liter

Response factor

Response factor = 1,761+ 0.0080 x Nominal

concentration ng/L 

Hydroxymetolachlor

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Nominal concentration, in nanograms per liter

Response factor

Response factor = 4,471- 0.0207 x Nominal 

concentration ng/L 

Dechlorometolachlor

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Nominal concentration, in nanograms per liter

Response factor

Response factor = 573 + 0.0114 x Nominal 

concentration ng/L 

2-(2-ethyl-6-methyl-phenyl)aminopropan-1-ol

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Nominal concentration, in nanograms per liter

Response factor

Response factor = 2194 + 0.0426 x Nominal 

concentration ng/L 

Metolachlor

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Nominal concentration, in nanograms per liter

Response factor

Response factor = 326 + 0.0057 x Nominal 

concentration ng/L 

Metolachlor hydroxy morpholinone

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Metolachlor SA

Nominal concentration, in nanograms per liter

Response factor

Response factor = 3.5 - 8.1e-6 x Nominal

concentration ng/L 

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000
Results and Discussion of Method Validation Data 21

report) less than 10 ng/L. There were eight pesticide compounds with median response factors less than 1 (table 16). 

Pesticide compounds with very low response factors are likely 

to have more problems with interferences at low concentrations that prevent identification.

The response factors were constant as a function of concentration over 4 orders of magnitude, with the slopes of the 

linear fit not significantly different than 0 (table 13). In many 

cases the response factors were variable at all concentrations 

(for example, acetochlor SA) because of the analytical variability of the analyte.

Method Detection Level Determination

Procedure

The MDL was determined from an estimate of the standard 

deviation of low concentrations samples based on the EPA test 

procedure guidelines for analysis of pollutants (40 CFR, part 136).

The MDLs were calculated in equation 2:

MDL = s × t(n–1, 1–α = 0.99) (2)

where

n is the number of replicate determinations at 1 

to 5 times the estimated MDL,

s is the standard deviation of measured 

concentrations of n spike determinations,

t is the Student’s t-value at n–1 degrees of 

freedom and 1–α (99 percent) confidence 

level, and

α is the level of significance.

When n = 49 (seven replicates in seven batches) and 

α = 0.01, t = 2.40, and when n = 8 (seven replicates in one 

batch) and α = 0.01, t = 2.998.

The LRLs are initially calculated as twice the MDL, 

based on Childress and others (1999). After this, the LRLs 

might be adjusted to higher levels if the qualitative identification of a calibration standard at LRL concentration cannot 

be achieved.

Method Detection Level Study Design

Replicate samples of reagent water—spiked at 5 and 

10 ng/L for ESI+ analytes and at 5, 10, 50, and 100 ng/L 

for ESI− analytes—were analyzed to determine the MDL. 

Seven replicates at each concentration were analyzed in seven 

analytical batches over a period of 2–3 days using corresponding calibration curves for each analytical batch (8 analytical 

batches were analyzed at 10 ng/L in ESI− mode). The purpose 

of analyzing samples in different batches was to provide an 

estimate of analytical variability that incorporates more of 

the measurement variability that is typical for routine analyses in a production laboratory (Childress and others, 1999). 

For some ESI+ analytes, the 10 ng/L spike level was too low 

for determination of MDLs, so an additional eight replicate 

reagent water samples were prepared at 25, 50, and 100 ng/L 

and analyzed in one batch.

Results of Method Detection Level Determination

Method detection levels, LRLs, and lowest and highest 

calibration standards are summarized in table 17.

The calculated MDLs ranged from 1 to 103 ng/L for 183 

analytes in ESI+ mode. Two analytes had MDLs of 250 ng/L 

(oxyfluorfen and parathion methyl). The median MDL was 

3 ng/L, with 75 percent of the MDLs less than 5 ng/L and 

90 percent less than 11 ng/L. The calculated MDLs in ESI− 

mode were about an order of magnitude higher and ranged 

from 2 to 106 ng/L for 42 analytes. Two analytes had MDLs 

of 250 ng/L (chlorthal-monomethyl and dicamba). The median 

MDL was 40 ng/L, with 75 percent of the MDLs less than 

72 ng/L and 90 percent less than 106 ng/L.

These MDLs are comparable to or less than the MDLs 

of NWQL’s existing analytical methods for pesticides. There 

are 70 pesticide compounds in the DAI LC-MS/MS method 

(schedule 2437) in common with laboratory schedule 2033 

Figure 4 above and prevous page. Response factors (peak area 

divided by nominal concentration) as a function of concentration 

(1, 2.5, 5, 10, 50, 100, 500, 1,000, 5,000, and 10,000 nanograms per 

liter [ng/L], seven replicates at each concentration) for metolachlor 

and degradates determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/

MS) in positive electrospray ionization (ESI+) mode. Calibration 

standards were excluded from fit if the qualifier ion response 

did not meet identification criteria (shown as open circles in the 

figures). Metolachlor SA and metolachlor OA are determined in 

negative electrospray ionization (ESI−) mode, the other pesticide 

compounds in ESI+ mode.

G Metolachlor OA

Nominal concentration, in nanograms per liter

Response factor

1

10

5

3

100

50

30

1,000

500

300

10,000

5000

3000

0.1 1 0.2 0.5 10 642 100 4020 1,000 300 10,000 3,000 100,000

Response factor = 10.6 + 0.0002 x Nominal

concentration ng/L 
22 Determination of Pesticides and Pesticide Degradates in Filtered Water

that uses C-18 SPE and GC-MS. The median and 90th percentile MDLs for these compounds are 2.5 and 10 ng/L in 

schedule 2437 compared to 6.5 and 10 ng/L in the GC-MS 

method. There are a few exceptions; for example, EPTC, 

parathion-methyl, and oxyfluorfen have MDLs greater than 

100 ng/L in the schedule 2437 compared to less than 10 ng/L 

in the GC-MS method. Phosmet, azinphos-methyl, dicrotophos, fenamiphos sulfoxide, fenamiphos sulfone, carbaryl, and 

carbofuran have MDLs less than 10 ng/L in schedule 2437 

and between 30 and 100 ng/L in the GC-MS method. During 

development of schedule 2437, most of the pyrethroids tested 

were eliminated from further testing because of low sensitivity, except for cis-permethrin, trans-permethrin, and bifenthrin, which had a reasonable response in LC-MS/MS. Their 

calculated MDLs are comparable to the GC-MS method (less 

than 10 ng/L). There are 37 pesticide compounds in schedule 

2437 method in common with laboratory schedule 2060 that 

uses Carbopack SPE and LC-MS (Furlong and others, 2001). 

The median and 90th percentile MDLs for these compounds 

are 4.5 and 44 ng/L in schedule 2437 method compared to 20 

and 60 ng/L in the LC-MS method (schedule 2060 MDLs for 

water year 2012). There are some exceptions; for example, 

chlorthal-monomethyl and dicamba have MDLs greater than 

100 ng/L in the DAI LC-MS/MS method compared to 20 and 

70 ng/L in the LC-MS method. Overall, the calculated MDLs 

show that schedule 2437 is suitable for determination of pesticide compounds in environmental samples.

The lowest calibration standard concentrations in table 17

are those that meet qualitative identification criteria, which 

vary according to ionization efficiency of each analyte and 

conditions of the mass spectrometer. The highest calibration 

standard concentration is 10,000 ng/L for most analytes, except 

for 1H-1,2,4-triazole, where the highest concentration calibration standard was 1,000 ng/L. Usually the LRL was set at twice 

the MDL except for a few cases the LRL was set higher than 

twice the calculated MDL to make sure the lowest calibration 

standard was below the LRL (2-aminobenzimidazole, acephate, 

desisopropylaltrazine, formetanate, methidathion).

The low-concentration replicate determinations used 

for calculating MDLs for ESI+ mode analytes are shown in 

table 18. The MDLs were calculated for both the 5- and 10-ng/L 

concentration levels. According to the EPA procedure, MDLs 

are considered valid if the nominal spike concentration was 1 

to 5 times the calculated MDL and if the detection frequency 

was greater than 50 percent. For many analytes, the calculated 

MDLs were similar using either the 5- or 10-ng/L concentration 

level because the standard deviations were similar. Constant 

standard deviation of the low-concentration spikes is one of the 

assumptions of the EPA procedure (Childress and others, 1999). 

The detection frequency of the low-concentration spikes is also 

shown in table 18. The detection frequency of low-concentration spikes (49 replicates) that have valid MDLs were from 

86 to 100 percent. The MDL selected was the lowest nominal 

spike level that met the criteria of the EPA procedure. Other less 

sensitive analytes (28 of the 182 pesticide compounds shown 

in bold in table 18) required separate MDL calculation using 

higher concentration spikes because they had lower detection 

frequencies and calculated MDLs higher than the nominal spike 

concentration.

The higher concentration replicate determinations used for 

calculating MDLs for the less sensitive ESI+ mode analytes are 

shown in table 19. These MDLs were calculated from replicate 

analysis (n=8) of 5 to 100 ng/L standards in one analytical 

batch—a smaller number of replicates than the previous experiment but still valid for the EPA MDL procedure. The MDL 

selected was the lowest nominal spike level that met the criteria 

of the EPA procedure. For the analytes that have calculated 

MDLs less than 10 ng/L, the MDLs are similar to those shown 

in table 18, which shows that the variability of eight replicates 

determined in one batch was similar to 49 replicates determined 

in seven consecutive batches. In other words, the batch-to-batch 

analytical variability is small for the DAI LC-MS/MS method. 

The calculated MDLs for cis-permethrin and trans-permethrin 

shown in table 19 are lower than those in table 18 because a 

more sensitive MRM transition (408 → 183) was used for data 

acquisition for these analytes. The results shown in table 19 

also provide summaries of bias and variability at multiple lowconcentration levels in reagent water and supplement the matrix 

experiment data discussed in the “Field-Matrix Spikes” section 

of the report. 

The low concentration replicate determinations used 

for calculating MDLs for ESI− mode analytes are shown 

in table 20. The MDLs were calculated for all four concentration levels (5, 10, 50, and 100 ng/L) analyzed and were 

considered valid if the nominal spike concentration was 1 to 

5 times the calculated MDL and if the detection frequency 

was greater than 50 percent. For many analytes, the valid 

MDLs used the 50- and 100-ng/L concentration levels 

because the lower concentrations were not detected or had 

high bias and variability. The detection frequency of lowconcentration spikes (49 replicates; 57 replicates in eight 

analytical batches at 10 ng/L) that have valid MDLs were 

from 92 to 100 percent. 

Bias and Variability from Matrix-Spike Recovery 

Tests

The performance of the method in different matrices and 

over a range of concentrations was evaluated in a matrix-spike 

study. Recovery of all analytes spiked into four water matrices—reagent water, streams from Illinois and California, and 

groundwater from California—was measured at four concentration levels (10, 100, 500, and 5,075 ng/L). The study design 

included three replicates at the two higher levels and four 

replicates for the two lower levels. The purpose of separating 

the low and higher level concentrations in two studies was to get 

more information about bias a variability across a wider range 

of concentrations than is typically done in method development studies conducted in the USGS (U.S. Geological Survey 

Office of Water Quality, 2004). This design provides 14 samples 

in each matrix and 56 samples in all matrices. One additional 

set of four replicates was analyzed at 250 ng/L for ESI+ mode 
Results and Discussion of Method Validation Data 23

analytes providing 60 samples in all matrices. Additional bias 

and variability estimates in reagent water at various concentrations are summarized in the MDL determination results (tables 

18, 19, 20, and 21), and at 250 ng/L in 47 different stream matrices in the “Paired-Sample Stream Study” section of the report.

The samples used for the matrix-effects study and 

their basic characteristics are shown in in table 21). The 

two stream samples from Illinois and California had similar 

pHs of about 8 but different specific conductivities, 537 and 

1,100 microsiemens per centimeter (μS/cm), respectively. 

The groundwater sample had a pH of about 7.6 and a specific 

conductivity of 697 μS/cm. The environmental samples have 

higher pH and specific conductivities than reagent water. 

The specific conductance is a measure of the ability of water 

to conduct an electrical current, and is a good measure of 

the ionic content of water which might affect the ionization efficiency of analytes in the MS electrospray interface. 

The pH of the sample—if very different than the conditions 

used for optimization of ionization conditions—might also 

affect the ionization efficiency of analytes during the elution 

window where the pH and (or) ionic strength of the mobile 

phase are altered. These environmental samples are typical of 

the water matrices intended to be used with the DAI LC-MS/

MS method. Both stream sites are part of the large river sites 

in the USGS National Water Quality Assessment (NAWQA) 

Program national fixed-site network and receive runoff from 

mixed sources (Rowe and others, 2013). Bias and variability 

of the pesticide compounds in other water matrices with very 

different characteristics, such as wastewater effluent, should 

be verified with matrix spikes.

The samples were prepared in 40-mL and 20-mL analytical 

vials and analyzed by LC-MS/MS in one analytical batch in each 

ESI mode. Concentrations of pesticide compounds determined 

in the unspiked environmental sample shown in tables 22 and 23

were subtracted from spike sample results to calculate recovery.

Recovery (R) was calculated according to equation 3:

a b

c

− R= ×100

(3)

where

a is the concentration of analyte in the spiked 

sample, in nanograms per liter;

b is the concentration of analyte in the unspiked 

environmental sample, in nanograms per 

liter; and

c is the expected concentration, in nanograms 

per liter.

The expected concentration (c) of the spike sample was 

calculated according to equation 4:

d × e

f

c =

(4)

where

d is the concentration of analyte in spike 

mixture, in nanograms per milliliter;

e is the volume of spike added, in milliliters; 

and

f is the sample volume, in liters.

Qualification of Matrix-Spike Sample Results

The large range of responses and calculated MDLs 

resulted in spike levels less than the MDL for some analytes 

in this study design with fixed concentration levels. Quantitative results for analytes at concentrations less than the MDL 

have larger uncertainty than at higher concentrations, so 

these would not be considered representative of the bias and 

variability of the analytes in general. Consequently, if the 

calculated MDL was less than the spike fortification level, 

the recovery results for that level were qualified by highlighting in the tables and excluded from the data summaries 

discussed below.

Another factor affecting the reliability of the recovery calculation was the concentration of the environmental 

sample relative to the fortification level. If the environmental 

sample concentration is greater than the spike concentration, there can be large errors in the calculated recoveries. 

Background environmental concentrations more than 3 times 

greater than the spike amount resulted in spike recoveries 

either much higher or lower than typical for schedule 2033 

pesticides (Martin and Eberle, 2011). For the pesticide compounds in this method, graphs of recovery versus background 

concentration showed that recoveries were exceptionally high 

or low for some analytes when the environmental concentrations were equal to or greater than the spike amount. This is 

shown in examples of the recovery of atrazine and atrazine 

degradates as a function of environmental sample concentration in field-matrix spikes (fig. 5). Consequently, when the 

background environmental concentration was greater than the 

nominal spike level, the recovery results for that level were 

highlighted in the data tables.

ESI+ Mode Analytes

Mean recoveries of pesticide compounds in different 

water matrices and concentrations determined by the ESI+ 

mode are shown in table 22. The recoveries as a function of 

concentration in the different matrices for each pesticide compound are shown in supporting figure S5.

Salient features of the mean recoveries of pesticide 

compounds at different concentrations in different matrices are 

shown by the recovery of metolachlor in figure 6. There was 

no major change in recovery at different concentration levels, 

similar to the linear trend of response factors with concentrations discussed previously. In addition, there was no obvious 

difference in recovery in the different matrices. The background concentration of metolachlor in the Mississippi River 

sample was 17 ng/L, which is above the lowest spike level of 

10 ng/L. In this case, the calculated spike recoveries at this 

level were 94 percent and did not appear to be affected by the 

background concentration.
24 Determination of Pesticides and Pesticide Degradates in Filtered Water

A

C

E

B

D

F

2-Hydroxy-4-isopropylamino-6-amino-s-triazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

Deethylatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

2-Hydroxyatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

Deisopropylatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

Atrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

Didealkylatrazine

0

100

200

300

400

500

600

700

800

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration
Results and Discussion of Method Validation Data 25

Figure 5 above and prevous page. Recovery of atrazine and 

degradates as a function of background environmental sample 

concentration determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

in 146 field-matrix-spike samples collected during the National 

Water Quality Assessment (NAWQA) Program field study from 

May to September 2012. Environmental samples, spike samples, 

and spike replicate samples were collected at 48 stream sites 2 

to 6 times; most sites had spike samples collected 3 times and a 

spike replicate 1 time. Recovery of 100 percent shown as solid 

black line, 70 percent and 130 percent as dashed lines. Spike 

level/environmental sample of 1 shown as vertical dashed line. 

Results with nominal spike level/environmental concentration 

<1 shown as open symbols.

A

G

B

2-Hydroxy-6-ethylamino-4-amino-s-triazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent 0.01 0.1 0.04 1 0.2 0.5 10 532 100 20 40 1,000 300 10,000

Spike level/Environmental concentration

Concentration, in nanograms per liter

Reagent water

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Groundwater well 1N/14W-8K1S

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter
26 Determination of Pesticides and Pesticide Degradates in Filtered Water

Mean recoveries of most analytes in the ESI+ mode (181 

of 185) were within data-quality objectives of 100±30 percent 

at spike concentrations above MDLs in all four matrices 

(table 22). The exceptions were 1H-1,2,4-triazole, asulam, 

didealkylatrazine, and phosmet. Mean recoveries at medium and 

high concentrations (100, 500, and 5,075 ng/L combined; n=10) 

in reagent water were from 89 to 119 percent (median 104 percent), with relative standard deviation (RSD) less than 22 percent for 169 analytes with MDLs above the spike level. More 

than half (96) of these analytes had mean recoveries between 95 

and 105 percent, indicating low bias for many of the analytes in 

the DAI LC-MS/MS method. Mean recoveries in other matrices 

were similar, although there were some analytes with more bias 

in some matrices. Analytes with slightly higher recovery in different matrices included cyanazine and imidacloprid in Mississippi River water and oxamyl oxime, imidacloprid, tebuthiuron 

TP 109 (OH), and 2-hydroxy-6-ethylamino-4-amino-s-triazine 

in Santa Ana River water. The carbamate asulam had low 

recoveries in all three environmental matrices but not in reagent 

water and thus were qualified with the permanent estimated 

remark. There was no obvious difference in recovery by type of 

pesticide (analytical method group).

Mean recoveries of the 17 surrogates in reagent water 

were from 90 to 109 percent, with RSD less than 20 percent. 

The RSDs were less than 12 percent for most of the surrogates 

in reagent water, except for alachlor-d13, cis-permethrin-13C6

, 

and deethylatrazine-d6. Recoveries of surrogates in groundwater and Mississippi River water were similar, ranging from 

95 to 100 percent and had RSDs less than 10 percent, except 

Figure 6 above and prevous page. Recovery of metolachlor determined by direct aqueous-injection liquid chromatographytandem mass spectrometry (DAI LC-MS/MS) in positive electrospray ionization (ESI+) mode in samples spiked at different 

concentrations (10, 100, 500, and 5,000 nanograms per liter [ng/L]) in four different matrices. Samples are shown as open symbols 

if the fortification concentration was less than the method detection level (MDL) or the environmental concentration. Dashed lines 

show study objectives of 100±30 percent recovery. Dotted vertical line shows calculated MDL. Background concentrations of 

metolachlor were 17 ng/L in Mississippi River below Grafton, Illinois, samples.

for cis-permethrin-13C6, and deetylatrazine-d6. The mean surrogate recoveries were lower and more variable in the Santa 

Ana River water, from 75 to 88 percent and RSD less than 

35 percent, except for cis-permethrin-13C6 (47 percent) and 

deetylatrazine-d6 (66 percent). A matrix interference caused 

high response for the internal standard (atrazine-d5) used for 

the surrogates that resulted in biased results for all the surrogates in these samples but did not appear to have affected any 

of the pesticide compounds.

Mean recoveries at the lowest spike level (10 ng/L) 

had more bias and variability, especially when MDLs were 

near the spike level. Mean recoveries at low concentrations 

(10 ng/L, n=4) in reagent water were from 52 (phthalazinone) 

to 158 percent (imazethapyr), with a median of 96 percent and 

RSD less than 67 percent for 155 analytes without qualification. However, most of the analytes (137) had mean recoveries within data-quality objectives of 100±30 percent at the 

lowest concentration (10 ng/L). Molinate and cyanazine had 

mean recoveries of 0 and 2 percent, respectively, at this low 

level that was slightly above their calculated MDLs. Mean 

recoveries of disulfoton sulfone and disulfoton oxon sulfone in 

reagent water were high at about 220 percent, which might be 

related to degradation of disulfoton in the samples.

There were 18 pesticide compounds 

(3,4-dichlorophenylurea, carboxy molinate, chlorsulfuron, dichlorvos, didealkylatrazine, halosulfuronmethyl, hexazinone TP G, hydroxyphthalazinone, 

imazamox, methomyl oxime, O-ethyl-S-propyl phosphorothioate, tebuthiuron TP 106, terbacil, 1H-1,2,4-triazole, 

C D

Mississippi River below Grafton, Illinois

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter

Santa Ana River below Prado Dam, California

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter
Results and Discussion of Method Validation Data 27

2-(1-hydroxyethyl)-6-methylaniline [HEMA], asulam, 

famoxadone, and naled) with calculated MDLs (10 to 72 

ng/L) above the lowest (10 ng/L) spike-fortification level and 

3 analytes (EPTC, oxyfluorfen, and parathion-methyl) with 

MDLs above 100 ng/L (103 to 250 ng/L). Mean recoveries for these pesticide compounds (shown in bold highlight) 

were generally outside the expected data-quality objectives, 

with mean recoveries from 0 to 220 percent and RSD as 

much as 200 percent.

There were 18 pesticide compounds (1H-1,2,4-triazole, 

2-hydroxy-6-ethylamino-4-amino-s-triazine, 2-hydroxyatrazine, 

atrazine, bromacil, carbendazim, carboxy molinate, deethylatrazine, deisopropylatrazine, didealkylatrazine, diuron, 

hexazinone, hydroxysimazine, imidacloprid, metolachlor, 

N-(3,4-dichlorophenyl)-N-methylurea, simazine, and 

sulfometuron-methyl) that had concentrations in the environmental samples greater than the lowest spike level (10 ng/L). 

Recoveries for the analytes at these fortification levels were 

generally outside the data-quality objectives, with recoveries 

from –137 to 222 percent and RSD as high as 340 percent. 

However, when the concentration of the environmental sample 

was within 1 to 2 times the fortification levels, some analytes 

(for example, hydroxysimazine, hexazinone, 2-hydroxy6-ethylamino-4-amino-s-triazine in Santa Ana River water, 

metolachlor in Mississippi River water) had recoveries within 

the data-quality objectives. 

ESI− Mode Analytes

Recovery of pesticides and degradates in different 

water matrices and concentrations determined by the ESI− 

mode are shown in table 23. The recoveries as a function 

of concentration in the different matrices for each pesticide 

compound are shown in supporting figure S6.

Salient features of the recovery of pesticide compounds 

determined in the ESI− mode by concentration and matrix 

is shown by the recovery of 2,4-D in figure 7. There was no 

obvious difference in the recovery in the different matrices. 

Recoveries of analytes in the ESI− mode were more variable 

in general than the ESI+ mode because many of the analytes 

have calculated MDLs higher than the lowest spike level. 

The calculated MDL of 2,4-D was 31 ng/L, and recoveries in 

reagent-water samples at the 10-ng/L spike level were from 

0 to 76 percent, with a mean of 37 percent and RSD of 116 

percent (recoveries were 0, 0, 73, and 76 percent for each 

sample). Variability increased as concentration of the spike 

decreased. In the groundwater sample, the background concentration of 2,4-D was 4 ng/L, and spike recoveries were –44, 0, 

23, and 30 percent (mean of 2 percent, RSD of 1,509 percent). 

The stream samples had background concentrations of 23 and 

25 ng/L, and calculated recoveries at 100 ng/L (Mississippi 

River) and at 500 ng/L (Santa Ana River) were high (133 and 

138 percent recovery, respectively). 

Mean recoveries of most analytes in the ESI− mode (42 

of 44) were within data-quality objectives of 100±30 percent 

at spike concentrations above MDLs in all four matrices 

(table 23). The exceptions were fipronil sulfonate and 

metribuzin DK. Mean recoveries of analytes in the ESI− mode 

in reagent-water samples were from 93 to 136 percent (median 

112 percent), with RSDs from 6 to 43 percent (39 analytes). 

cis-Cyhalothric acid and fipronil sulfonate had recoveries slightly higher in reagent water (134 and 136 percent), 

whereas dimethenamid SAA had an RSD of 37 percent in 

reagent-water samples. Metribuzin DK had very high recovery 

(353 percent) in reagent-water samples at the 100-ng/L level, 

the same concentration as the calculated MDL.

Mean recoveries of analytes in the ESI− mode in the 

other matrices were from 84 to 131 percent, with RSDs from 

12 to 47 percent (39 analytes), similar to that in reagent 

water. Mean recoveries of most analytes (n=38) were within 

data-quality objectives of 100±30 percent at all spike levels, 

if the spike was above the calculated MDL. The mean recoveries of isoxaflutole acid RPA 203328 was more variable, 

with RSDs of recovery of 35 percent. Metribuzin DK had 

very high mean recovery (201 percent) in environmental 

samples at the 100 ng/L level; the same level as the calculated MDL. Mean recoveries of the fungicide famoxadone 

were low in both the groundwater and Mississippi River 

samples, so the results for this analyte are reported with an 

estimated validation quality code.

Mean recoveries of the seven surrogates in reagent water 

samples (n=14) were from 99 to 109 percent, with RSD from 

4 to 18 percent. The RSDs of five surrogates that have nominal 

spike concentrations of 1,000 to 10,0000 ng/L were less than 

8 percent, whereas 2,4-D-d3 and diuron-d6, with relatively low 

nominal spike concentrations of 330 ng/L, had higher RSD of 

18 and 15 percent. Mean recoveries of each surrogate in all the 

environmental samples (n=42) were similar to reagent water, 

from 100 to 111 percent, with RSDs from 4 to 19 percent. 

There was no meaningful difference in recovery of the surrogates in any of the environmental matrices compared to reagent 

water (supporting figure S2).

Most of the acetanilides had calculated MDLs above 

the lowest spike level. Average recoveries at medium- and 

high-spike concentrations were within 100±30 percent. In the 

samples from Mississippi River, the background concentration of metolachlor SA was 476 ng/L, yet average recoveries 

at 100- and 500-ng/L spike level were within 100±30 percent. 

Pesticide compounds that have calculated MDLs less than the 

lowest spike level were bentazon, dechlorofipronil, desulfinylfipronil, desulfinylfipronil amide, fipronil, fipronil amide, 

fipronil sulfide, fipronil sulfone, flubendiamide, isoxaflutole 

acid RPA203328, oryzalin, and sulfentrazone.

Performance of Laboratory Quality Control 

Samples During Implementation of Method

This method was implemented in the NWQL in June 

2012 during a field comparison study of stream samples collected by the NAWQA Program. During that time, laboratory 

quality-control samples included third-party check standards, 

laboratory blank, and laboratory spike samples.
28 Determination of Pesticides and Pesticide Degradates in Filtered Water

Figure 7. Recovery of 2,4-D determined by direct aqueous-injection liquid chromatography-tandem mass spectrometry (DAI LC-MS/

MS) in negative electrospray ionization (ESI−) mode in samples spiked at different concentrations (10, 100, 500, and 5,000 nanograms 

per liter [ng/L]) in four different sample matrices. Samples are shown as open symbols if the fortification concentration was less than 

the method detection level (MDL) or the environmental concentration. Dashed lines show study objectives of 100±30 percent recovery. 

Dotted vertical line shows calculated MDL. Background concentrations of 2,4-D were 4, 25, and 23 ng/L in groundwater well 1N/14W8K1S, Mississippi River below Grafton, Illinois, and Santa Ana River below Prado Dam, California, samples.

C

A

D

B

Reagent water

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter

Groundwater well 1N/14W-8K1S

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter

Mississippi River below Grafton, Illinois

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter

Santa Ana River below Prado Dam, California

0

20

40

60

80

100

120

140

160

180

200

Recovery, in percent

1 10 2 3 4 5 7 100 604020 1,000 300 500 4,00010,000

Concentration, in nanograms per liter
Results and Discussion of Method Validation Data 29

Third-Party Check Standards

There was no standard or group of standards commercially available that included all the analytes in the DAI 

LC-MS/MS method that could be used for a TPC. Spike 

mixtures for NWQL laboratory schedules 2033 and 2060 

have 85 analytes that are common with the DAI LC-MS/MS 

method, including 11 analytes that are in both spike mixes. 

These were used as third-party check (TPC) standards during 

implementation of the method because they were prepared 

from completely independent concentrated standard mixtures. They also provide information about the differences in 

environmental samples and field-matrix-spike recoveries in 

comparison of samples analyzed by the different methods.

The TPC standard mixtures are analyzed with each batch 

of samples in the LC-MS/MS method. Results of TPC recovery from 11 batches of samples analyzed in the ESI+ mode 

between September 2012 and February 2013 are summarized 

in table 24. Recoveries ranged from 44 to 179 percent, with 

a median of 99 percent for all results. Mean recovery of each 

analyte in the 2033 and 2060 spike mixes was between 66 and 

151 percent for all analytes without an estimated “E” validation quality code, and the RSD of each analyte was from 7 

to 24 percent. Most of these analytes without an estimated 

validation quality code (76 of 89) had mean recoveries that 

were between 80 and 120 percent. Ten analytes that were in 

both 2033 and 2060 spike mixes had comparable recoveries 

in schedule 2437, illustrating excellent comparability for three 

independent standard solutions.

A few analytes had mean recoveries outside the 

100±30-percent data-quality objectives, including organophosphate (dichlorvos, dicrotophos, fenamiphos) and carbamate 

(aldicarb sulfoxide) analytes. Degradation in one of the standard 

mixtures might be the cause of these differences because analytes in these chemical classes can be unstable during storage. 

Analytes reported with an estimated validation quality code had 

more variable recoveries (RSD from 11 to 65 percent). Benomyl, a component of the schedule 2060 spike mixture, rapidly 

degrades to carbendazim in water, as indicated by its quantitative recovery as carbendazim in the TPC standard analyzed by 

the DAI LC-MS/MS method. 

Results of TPC recovery from 11 batches of samples 

analyzed in the ESI− mode between September 2012 and 

February 2013 are summarized in table 25. Recoveries 

ranged from 52 to 121 percent, with a mean of 97 percent 

and median of 98 percent for all results. Mean recovery of 

each of the 11 analytes (without validation quality code) in 

the TPC standards was between 84 and 102 percent. The 

RSDs of these analytes were from 6 to 22 percent. Dicamba 

and chlorthal-monomethyl, both qualified with “E” remark in 

this method, had more variable recoveries, with RSD of 28 

and 35 percent.

Although the TPC standards only included about one-half 

of the analytes in the DAI LC-MS/MS method, the analytes 

are from a broad range of chemical classes representative of 

all the analytes in the DAI LC-MS/MS method. These TPC 

results show that the standards used in the DAI LC-MS/MS 

method provide comparable results to the existing methods 

for those analytes for which a TPC was available. Differences 

in environmental samples and field-matrix-spike recovery 

in comparison of samples analyzed by the different methods 

should be less than the bias shown in tables 24 and 25 based 

on calibration standard differences alone. Conversely, larger 

differences would be due to other factors, such as matrix 

effects or differences in calibration, but not the analytical standards. Results for dichlorvos might be biased high in comparisons between the different methods.

Laboratory Reagent Blank Samples

Laboratory reagent blank samples are prepared with each 

batch of samples analyzed and are used to monitor contamination from laboratory procedures, which is most likely to 

occur when the sample is transferred to the analytical vial 

and when injected by the autosampler during data acquisition. The addition of surrogate solution can also be a source 

of contamination if the solution contains analytes. For this 

method, the standard solutions of the IS diazinon-d10 have low 

concentrations of the diazinon degradate pyrimidinol, so the 

reporting level for this analyte is generally limited by the level 

of the pyrimidinol from the surrogate (about 5 ng/L). Laboratory blanks determined during implementation of the method 

in 2012 and 2013 are summarized in table 26. Detections of 

pesticide compounds in laboratory blanks were infrequent. 

There were 212 analytes with no detections in any lab blanks 

determined during the implementation studies (17 blanks for 

ESI+ mode, 18 blanks for ESI− mode). There were 16 analytes 

with detections in the lab blanks in the ESI+ mode and 2 analytes with detections in the ESI− mode. Detection frequencies 

were greater than 15 percent, from 18 to 35 percent (more than 

3 of the 17 blanks) for fenbutatin oxide, piperonyl butoxide, 

and pyrimidinol in the ESI+ mode and for fipronil sulfone in 

the ESI− mode. The 95th percentile of concentration for most 

pesticide compounds in the lab blanks was less than the LRL.

These results show that contamination from laboratory 

procedures is unlikely for most of the pesticides in the method. 

The four pesticide compounds with detection frequencies above 

18 percent should be carefully monitored, and if blank detections continue at a comparable frequency, they must be qualified 

with a “v” code for detections in environmental samples. 

Laboratory Reagent Spike Samples

Laboratory reagent spike samples are prepared with each 

batch of samples analyzed and are used to monitor performance of the analytes in a relatively matrix-free sample—in 

addition to the CCV samples. The laboratory reagent spikes 

are added to sample collection containers and are directly 

comparable to samples and field-matrix spikes which are collected in the same containers, unlike CCV samples that are 

added to 2-mL analytical vials. Laboratory spikes determined 

during implementation of the method from June 2012 to 

March 2013 are summarized in table 27.
30 Determination of Pesticides and Pesticide Degradates in Filtered Water

The mean spike recoveries in laboratory reagent spike 

samples for analytes in the ESI+ mode were from 66 to 

114 percent, with an overall mean of 92 percent. The RSDs 

were from 7 to 31 percent. Mean spike recoveries in laboratory reagent spike samples for analytes in the ESI− mode were 

from 74 to 119 percent, with an overall mean of 93 percent. 

The RSDs were less than 30 percent in the ESI− mode, except 

for a few analytes (acetochlor SA, alachlor SA, 4-hydroxychlorothalonil, and metribuzin DADK), with RSD between 

31 and 59 percent. Overall results were very good, with mean 

recoveries for most analytes between 80 and 120 percent and 

a few analytes (indoxacarb, lactofen, tribufos, cis-permethrin, 

trans-permethrin, chlorimuron-ethyl, orthosulfamuron, prosulfuron, and fipronil sulfone) having slightly lower mean 

recoveries between 66 and 80 percent. Analytes qualified with 

the estimated remark shown in table 27 had recoveries outside 

the objectives of 100±30 percent and RSDs greater than 30 

percent. Exceptions were the pesticide compounds within the 

objectives in the reagent-water-spike samples but with low 

recoveries or high bias in other matrices (asulam, 3-hydroxycarbofuran, 1H-1,2,4-triazole, butralin, flumetsulam, dicamba, 

and chlorosulfonamide acid).

The nonparametric statistical equivalents of the mean 

recovery and RSD—median and F-pseudosigma (Helsel and 

Hirsch, 2002)—are also shown in table 27. The mean and 

median recoveries are generally equivalent. The RSDs and 

F-pseudosigma also are similar, except in a few cases where 

the RSD is much larger than the F-pseudosigma. For example, 

hydroxytebuthiuron in the ESI+ mode had 0 percent recovery 

in both samples in one batch. This resulted in an RSD of 30 

percent, whereas the F-pseudosigma was 14 percent because 

the nonparametric calculation is resistant to a few outliers 

compared to the parametric summary. Both statistical summaries are available from the NWQL QC sample-data application 

(http://nwqlqc.cr.usgs.gov/).

Application to Environmental Samples—PairedSample Stream Study

A comparison of the new DAI LC-MS/MS analytical 

method (schedule 2437) with two existing NWQL analytical methods (schedules 2033 and 2060) for determination of 

pesticides was conducted in collaboration with the NAWQA 

Program. Environmental samples, field-matrix spikes, and 

field blanks collected from a variety of NAWQA stream sites 

from May to September 2012, as well as routine laboratory 

QC samples, were analyzed by all three methods. Samples 

analyzed by the new method were compared to the existing 

pesticide methods by determination of the common analytes in 

existing method spike mixtures used as third-party check standards and in environmental samples and field-matrix spikes 

collected during a NAWQA field study. The results provide a 

characterization of the performance of the new method during 

implementation in routine production, including bias and variability of analytes in schedule 2437 over a range of environmental matrices and concentrations and in comparison to 

schedules 2033 and 2060; potential for bias and contamination 

using the new field filtration procedure for schedule 2437; and 

an opportunity to assess efficiency, costs, implementation, and 

logistical issues of schedule 2437 from both field and laboratory perspectives.

Field-Matrix Spikes

Study Design

Environmental samples, spike samples, and spike replicate samples were collected at 48 NAWQA stream sites two 

to six times during May through September, 2012; most sites 

had spike samples collected three times and a spike replicate 

one time. During this study, there were a total of 146 spike 

samples and 50 spike replicates, resulting in 196 spike 

samples that used the schedule 2437 spike mixture. In addition, four spike samples and one spike replicate were incorrectly spiked with the schedule 2033 spike mixture, resulting 

in an expected spike concentration of 4,747 ng/L, and these 

are summarized separately.

Recovery of analytes in the spike samples was calculated 

using equation 3. Spike-recovery calculation can have large 

bias and variability if the environmental sample concentration is close to or greater than the spike concentration. This 

is illustrated in examples of calculated spike recovery for 

analytes with concentrations of pesticide compounds in the 

environmental samples larger than the spike level of 250 ng/L 

in supporting figure S7. Graphs of spike recoveries relative to 

environmental sample concentration for all analytes are shown 

in supporting figure S8. Recoveries for samples with the analytes for which the environmental sample concentrations were 

larger than the spike level (shown as open symbols in supporting figure S8) generally have larger bias and are not within 

the data-quality objectives. Consequently, sample results with 

spike concentration/environmental sample concentrations >1 

were excluded from the data summary discussed here. There 

were 36 analytes with background environmental sample concentrations higher than the spike level, although most of these 

had from 1 to 8 results that were excluded (<5 percent of the 

146 spike samples). There were seven pesticide compounds 

(acetochlor OA, metolachlor, metolachlor OA, metolachlor 

SA, 2,4-D, atrazine, and didealkylatrazine) that had from 9 to 

46 results excluded (supporting table S8).

Spike Recovery in Stream Samples

The results of the calculated spike recovery for the spike 

samples are shown in table 28. Example graphs of atrazine 

and atrazine degradates are shown in figure 8 (graphs of 

matrix-spike recovery by site for all analytes are shown in 

supporting figure S7). Most analytes had low environmental 

sample concentrations, so results for all 146 spike samples 

for each analyte were included in the data analysis. There 

were 36 analytes with high environmental sample concentrations that were excluded in some samples, so the number 

of spike samples included in the data analysis was between 
Results and Discussion of Method Validation Data 31

A

B

2-Hydroxy-4-isopropylamino-6-amino-s-triazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID

2-Hydroxy-6-ethylamino-4-amino-s-triazine

0

20

40

80

60

100

120

140

160

240

220

200

180

280

260

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID

Figure 8 (above and following pages). Recovery of atrazine and degradates determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) in 146 field-matrix-spike samples collected during the National 

Water Quality Assessment (NAWQA) Program field study from May to September 2012. Recovery of 100 percent shown as solid 

black line, 70 percent and 130 percent shown as dashed lines; results reported with “E” validation quality code because of 

interference shown in light gray filled symbols, results with nominal spike level/environmental concentration <1 shown as open 

symbols. (ID, identifier)
32 Determination of Pesticides and Pesticide Degradates in Filtered Water

C

D

2-Hydroxyatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID

Atrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID
Results and Discussion of Method Validation Data 33

E

F

Deethylatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500

394340085524601

Site ID

Deisopropylatrazine

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID
34 Determination of Pesticides and Pesticide Degradates in Filtered Water

Figure 8. Recovery of atrazine and degradates determined by direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in 146 field-matrix-spike samples collected during the National Water Quality Assessment (NAWQA) 

Program field study from May to September 2012. Recovery of 100 percent shown as solid black line, 70 percent and 130 percent 

shown as dashed lines; results reported with “E” validation quality code because of interference shown in light gray filled symbols, 

results with nominal spike level/environmental concentration <1 shown as open symbols. (ID, identifier)—Continued

G

H

Didealkylatrazine

0

100

200

300

400

500

600

700

800

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID

Deethylatrazine-d6 (surrogate)

0

20

40

60

80

100

120

140

160

180

200

FMS recovery, in percent

01184000013561900140330001463500016540000208950002091500021742500231850002335870023380000246976203374100034662080407205004186500052887050542050005420680054655000549050005572000055861000627950006610000068000000680550007022000072636200728895507373420073740000738149508057410081166500817880008364000084750000916350010350340110740001130350012510500130927471420130014211720

322023090544500394340085524601

Site ID
Results and Discussion of Method Validation Data 35

100 (metolachlor SA) and 146. Two pesticide compounds 

(famoxadone and 1H-1,2,4-triazole) had fewer than 146 

matrix-spike sample results because of missing values 

reported—generally because of interferences. Mean recoveries of most pesticide compounds (192 of 207 pesticide compounds not qualified, labeled “A” in tables) in field-matrix 

spikes were within the data-quality objective of 100±30 

percent, with a median recovery of 93 percent and RSD of 

29 percent, although for some stream matrices recoveries 

were outside these objectives. 

For analytes with a validation quality code “A,” 

there were four analytes with mean recoveries higher 

than 130 percent (diketonitrile isoxaflutole, sulfentrazone, 

2-hydroxy-6-ethylamino-4-amino-s-triazine [HDIA], and 

didealkylatrazine). Didealkylatrazine and HDIA elute in the 

first few minutes of the chromatogram and have wide and 

non-Gaussian chromatographic peak shapes, so they are likely 

influenced by co-eluting stream matrix components that elute 

in the early part of the chromatogram. Matrix suppression is 

the likely cause of mean recoveries less than 70 percent for 

11 pesticide compounds (oxamyl, indoxacarb, malaoxon, 

terbufos oxon, chlorimuron-ethyl, hydroxymonodemethyl 

fluometuron, hydroxyfluometuron, halosulfuron-methyl, 

nicosulfuron, orthosulfamuron, and hexazinone TP E) in the 

field-matrix-spike samples. These compounds typically have 

recoveries within 100±30 percent in reagent-water samples. 

Recoveries were more variable (30 to 54 percent) in the 

field-matrix-spike samples than study objectives for about 

23 pesticide compounds (highlighted in bold in table 28), 

indicating some matrix effects for these analytes.

Some sites had sample matrices that resulted in low 

recoveries of some analytes covering a wide range of chemical classes. For example, recoveries of carbaryl, carbofuran, 

kresoxim-methyl, trifloxystrobin, indoxacarb, isoxaflutole, 

lactofen, propargite, dimethoate oxon, profenofos, hydroxytebuthiuron, and malathion were between 0 and 50 percent 

in matrix-spike samples from Iowa River at Wapello, Iowa; 

Des Moines River at Keosauqua, Iowa; and Platte River at 

Louisville, Nebr. (supporting figure S7). These sites were 

characterized by some of the highest pH values (8.4 to 9.6) of 

all stream sites, whereas other water-quality parameters such 

as specific conductivity, major ions, and dissolved organic 

carbon were not atypical at these sites (supporting tables S9 

and S10). Hydrolysis is more rapid for these pesticides at basic 

pH, which likely resulted in their degradation after they were 

spiked at the field site and stored at 4 °C until analysis (about 

5–10 days). For example, the half-life of carbaryl is 1.8 days at 

pH 8 and 10.5 days at pH 7 (Howard, 1991). This degradation 

of carbaryl over time is in contrast to the surrogate carbaryld7, which had recoveries within expected limits in these same 

samples—the surrogate is added to the samples prior to analysis so there is less time for hydrolysis of the surrogate. Recovery of carbaryl in Iowa River at Wapello, Iowa samples with 

pH values between 8.8 and 9.2 were 3 to 59 percent, whereas 

the carbaryl-d7

 recovery was from 54 to 102 percent. This is an 

example of a sample matrix effect that results in biased results, 

but the matrix effect is not related to ionization suppression in the ion source of the LC-MS/MS instrument. Similar 

results for degradation of carbamates in high pH samples were 

observed during development of EPA method 538, also a DAI 

LC-MS/MS method for pesticides (Shoemaker, 2011).

One of the disadvantages of a method with a large number of analytes having wide range of chemical characteristics 

is that it is difficult to find sample preservation techniques that 

stabilize all analytes without adversely affecting some. Future 

improvements to the method should explore better sample 

preservation techniques such as pH adjustment. 

Mean recoveries of most surrogates in the stream samples 

were generally within the objectives of 100±30 percent. Mean 

recoveries of each surrogate in the stream samples were 

from 77 to 111 percent, with RSD between 6 and 23 percent 

(table 28). Recoveries of carbaryl-d7

, malathion-d10, and 

cis-permethrin-13C6 were more variable than the other surrogates, with recoveries from 10 to 175 percent, and RSDs 

greater than 15 percent (17 to 23 percent). The larger variability of these surrogates indicates they are more susceptible to 

interference from the sample matrix.

Another 22 analytes shown in table 28 are listed separately and reported with the estimated validation quality 

code because of difficulty in meeting data-quality objectives 

during method validation. Also shown are spike recovery 

results for five analytes that were analyzed by the ESI− mode 

but had better performance in the ESI+ mode and one analyte 

that was analyzed by the ESI+ mode but had better performance in the ESI− mode. Recovery results for 10 analytes 

deleted from the LC-MS/MS method are also shown (analytes with the “D” validation quality code at the bottom of 

table 28), illustrating the high bias and variability in these 

stream samples.

There were samples from five sites that incorrectly used 

the schedule 2033 spike mixture to prepare the matrix spikes. 

This mixture is a higher concentration than the schedule 

2437 mixture, so the expected spike concentration of the 

20-mL samples was 4,747 ng/L. The results of the calculated spike recovery for these samples are shown in table 29. 

There were 60 analytes in the 2033 spike mix also in the 

2437 analytical method. The average recoveries of most 

analytes were within the study objectives of 100±30 percent. 

These results show overall good consistency between the two 

methods, even though the spike mixtures were prepared from 

different concentrated standard solutions. A few organophosphate analytes (chlorpyrifos oxon, fenamiphos, malaoxon) 

had lower recovery, although recoveries of these analytes 

in the same matrices using the schedule 2437 spike mix are 

acceptable. This might indicate degradation of these pesticide compounds in the 2033 spike mixture ampoule. 

Relative Percent Difference of Spike Recovery

Differences in recoveries of the pesticide compounds 

in spike and spike-replicate samples provide information on 

reproducibility of the analysis in a particular sample matrix. 

There were 50 spike replicate samples collected from 45 sites 
36 Determination of Pesticides and Pesticide Degradates in Filtered Water

(3 sites had two pairs of replicate spikes, 1 site had three 

pairs; 1 site that used the 2033 spike mix was excluded). 

The difference in recovery was calculated as the relative 

percent differences (RPD) and summarized in table 30. Spike 

samples with background environmental concentrations of 

the pesticide compounds larger than the nominal spike level 

of 250 ng/L were analyzed separately. The median RPD of 

the pesticide compounds with a validation quality indicator 

of “A” and low background environmental concentration 

was from 4 to 20 percent for 169 of the ESI+ analytes and 

from 4 to 28 percent for 38 of the ESI− analytes. Most of the 

pesticide compounds had a median RPD of less than 15 percent (190 out of 207 of these analytes). Pesticide compounds 

with a validation quality indicator code of “E” had somewhat 

larger RPD, from 4 to 39 percent.

Field Blank Samples

Field blank samples were analyzed from 43 sites during the NAWQA field comparison study. There were few 

detections of pesticide compounds in field blanks, with only 

15 detections found in more than 9,000 determinations (229 

pesticide compounds in 43 samples). Twelve analytes were 

found in at least one of the 43 field blank samples analyzed, 

with three analytes (pyrimidinol, metolachlor, and methoxyfenozide) detected in two blank samples (table 31). Concentrations were from 1 to 20 ng/L and close to the LRLs, except for 

pyrimidinol (a diazinon degradate) where concentrations were 

69 and 113 ng/L. 

These results indicate relatively low tendency for bias 

from contamination for analytes in the DAI LC-MS/MS 

method from all field collection and processing and laboratory analysis steps. Pyrimidinol was an exception, found in 

two blanks (5 percent detection frequency) at relatively high 

concentrations. This analyte appears to be derived from degradation of the diazinon-d10 internal standard because it has 

been consistently found in laboratory blanks. Environmental 

sample results might need to be censored at an appropriate 

LRL based on summaries of concentrations found in field and 

laboratory blanks.

The field blank samples with analytes detected were 

reanalyzed after storage in the freezer for about 3 months to 

confirm the detections. Six of the analytes found at concentration from 1 to 8 ng/L were not confirmed in the re-analysis. 

A likely explanation is that the initial contamination was not 

in the sample collected at the field site, but introduced at the 

laboratory during processing and analysis. However, another 

explanation is that the analytes degraded during storage in 

the environmental matrix because the holding time studies 

were done in reagent water. Careful monitoring of laboratory 

blanks and more experience with the method after implementation in the laboratory might help identify and eliminate the 

sources of laboratory contamination as a contribution to the 

field blank contamination.

Environmental Sample Results

The new DAI LC-MS/MS method expanded the number of pesticide compounds (229) determined in the samples 

compared to previous methods (about 100 compounds in 

three different analytical methods). These analytes were rated 

high-priority for monitoring and studies because of measured 

or predicted detection frequency, concentration in streams and 

groundwater, and agricultural and nonagricultural pesticideuse estimates. The results of the initial field comparison study 

show that a relatively large number of these high-priority 

pesticide compounds were found at many sites. Mixtures of 

3 to 56 pesticide compounds were found in different streams 

and times, with a median of 26 pesticide compounds for all 

samples. Mixtures of more than 14 pesticide compounds were 

found in 75 percent of the samples (25th percentile was 14 

detections). The number of detections and summary of concentrations of the pesticide compounds are shown in table 32. 

Seventeen pesticide compounds were found frequently, 

with detection frequencies from 52 to 86 percent (shown in 

bold in table 32). The pesticide compounds found most frequently included herbicides heavily used in agriculture, primarily corn and soybean production: atrazine and five degradates 

(2-hydroxy-4-isopropylamino-6-amino-s-triazine, 2-hydroxyatrazine, deethylatrazine, deisopropylatrazine, and didealkylatrazine), metolachlor and three degradates (dechlorometolachlor, 

hydroxymetolachlor, and metolachlor SA), and acetochlor 

SA+alachlor SA mixture. The other pesticide compounds were 

those used in both agricultural and nonagricultural uses: the 

fungicides azoxystrobin and carbendazim and herbicides diuron and its degradate (N-(3,4-dichlorophenyl)-N-methylurea), 

prometon, and the simazine degradate hydroxysimazine. The 

fungicide carbendazim is a new analyte not previously included 

in USGS water-quality studies. About 44 percent of the pesticide compounds were not found in any of the samples.

The concentrations of pesticide compounds that were 

found in the stream samples ranged from undetected to 

17,899 ng/L. There were eight pesticide compounds with the 

median concentration greater than 10 ng/L (shown in bold in 

table 32): acetochlor SA + alachlor SA mixture, metolachlor 

SA, diuron, 2-hydroxyatrazine, atrazine, deethylatrazine, 

deisopropylatrazine, and didealkylatrazine.

Interlaboratory Comparison of Methods

An interlaboratory comparison study of stream sample 

concentrations was conducted with the DAI LC-MS/MS 

method (schedule 2437) compared to SPE GC-MS SIM 

method (schedule 2033) and a SPE LC-MS SIM method 

(schedule 2060). The comparison included linear regression 

analysis to evaluate potential bias between concentrations 

determined by the different methods and an evaluation and 

signed rank test to determine whether there were differences in 

the number of detections found by the different methods.
Results and Discussion of Method Validation Data 37

Linear Regression of Concentrations

For analytes with detections in more than five samples, 

the agreement between the two methods was evaluated from 

a linear regression analysis of a graph of the concentrations obtained from each method (Helsel and Hirsch, 2002; 

Odetokun and others, 2010). A t-test was conducted to determine whether the linear correlation coefficient was different from 0, and therefore whether a linear correlation exists 

between the two methods. In addition, if the 95th percentile 

of the slope was within 15 percent of unity (0.85 to 1.15), the 

methods were considered similar for the specific analytes. 

These older methods (SPE GC-MS and SPE LC-MS) involve 

sample preparation steps that probably introduce more variability than is common for inorganic analytes, where a slope 

within 10 percent is considered similar (Garbarino and others, 2006).

Graphs of the correlation between concentrations of 

selected pesticides obtained from schedules 2437 and 2033 are 

shown in figure 9 and for schedules 2437 and 2060 in figure 10. 

The graphs show generally good correlations of concentrations determined in the stream samples over 3 to 4 orders of 

magnitude for pesticides with frequent detections (acetochlor, 

metolachlor, atrazine, deethylatrazine, simazine, and metalaxyl 

in schedules 2437 and 2033; 2-hydroxyatrazine, deethylatrazine, atrazine, and 2,4-D in schedules 2437 and 2060). The 

linear regression analysis parameters are summarized in tables 

33 and 34. The linear correlation coefficients were evaluated 

for pesticide compounds with more than five detections in both 

methods. These results show that the t-statistic for the linear 

correlation coefficients (slopes) are significant (p<0.001) for 

most of the analytes, and a strong linear correlation exists. 

The correlation coefficients for imidacloprid in 2437–2060 

methods and fipronil sulfide in 2437–2033 methods were not 

significant, and the graphs show a general lack of correlation 

of the concentrations (not shown). An explanation for fipronil 

sulfide lack of correlation is that the concentrations are below 

the reporting levels of both methods where there is more error 

in the calculated concentration. Imidacloprid is reported as a 

highly variable analyte in the 2060 method with large error in 

the calculated concentrations.

The correlation coefficient for the slope was equal 

to 1±0.15 within the 95-percent confidence limits for 4 of 

the 20 compounds in the 2437–2033 comparison (atrazine, 

metribuzin, prometon, and simazine), indicating that the 

concentrations were similar and there was low bias between 

the concentrations. Similarly, the correlation coefficients 

were equal to 1±0.15 for three of the 15 compounds in the 

2437–2060 comparison (metalaxyl, 2-hydroxyatrazine, and 

atrazine). The correlation coefficients for the slope were 

greater than 1 for the other 16 compounds in the 2437–2033 

comparison and generally less than 1 for the other 12 compounds in the 2437–2060 comparison. For example, the slope 

for acetochlor in the 2437–2033 comparisons was 1.58. This 

estimate predicts that the concentrations determined by the 

2437 method will be 58 percent larger than those determined 

in the 2033 method. The reasons for these differences might 

be the result of differences in calibration model (curve), 

extraction and processing steps, and matrix effects. The 

2437 method used spiking solution standards from the 2033 

methods as third-party check standards, and the recovery 

of acetochlor in those standards was 85 percent (table 24). 

Similarly, the recovery of acetochlor in stream matrix spikes 

was 96 percent (table 28). These differences would account 

for 19 percent larger concentrations in the 2033 concentration compared to the 2437 concentration. Other factors, such 

as matrix enhanced response for the GC-MS method and 

use of weighting in the 2437 calibration model compared to 

no weighting in the 2033 method might be responsible for 

the remaining differences. Additional explanation related to 

matrix-spike recovery in paired samples determined by the 

2033 method are described in the field comparison study.

Detection Frequency in Stream Samples

The consistency of detection frequency of the different 

methods was compared by counting the number of detections 

(at any concentration) and nondetections in the paired-stream 

samples reported by each method. Graphs of the concentrations found by the different methods (figs. 9 and 10) also show 

agreement or differences in detections, with different symbols 

for detections in the methods. The consistency in the number of 

detections and nondetections in the stream samples is summarized in table 35 for the comparison of schedule 2437 and 2033. 

Consistent nondetections are paired samples where the 

pesticide compound was not detected by both methods and 

consistent detections are paired samples where the pesticide 

compound was detected by both methods. Inconsistent detections in one method are paired samples where the pesticide 

compound was not detected by one method and was detected 

in the other. Agreement between results reported for both 

methods as detections or as nondetections was from 66 to 

100 percent. For example, the agreement between methods 

for metalaxyl was 76 percent, with both methods reporting 85 

nondetections and both methods reporting 29 consistent detections. The lack of agreement for metalaxyl was 24 percent, 

with three detections reported as nondetection by schedule 

2437 when a detection was reported by schedule 2033, and 33 

detections reported as nondetection by schedule 2033 when a 

detection was reported by schedule 2437 (fig. 9).

The sign test was used to test whether these differences 

in the number of detections and nondetections found in the 

paired-stream samples were significant (table 36). Some 

analytes had significantly larger number of detections in the 

2033 method (acetochlor, carbaryl, chlorpyrifos, deethylatrazine, metribuzin, simazine, desulfinylfipronil, desulfinylfipronil amide, and fipronil sulfide), whereas other analytes had 

significantly larger number of detections reported with the 

2437 method (metalaxyl, dicrotophos, dimethoate, tebuthiuron, and hexazinone).

Most of the differences in number of detections were 

close to or below the LRL of the method, as expected by 

the definition of MDL and LRL. For example, the detections of acetochlor by the 2033 method that were reported as 
38 Determination of Pesticides and Pesticide Degradates in Filtered Water

A

C

E

B

D

F

Schedule 2437 concentration, in nanograms per liter

Acetochlor

1 10 2 3 4 5 7 100 60403020 1,000 600400200

1

10

6

4

2

100

60

40

20

1,000

600

400

200

Schedule 2033 concentration, in ng/L

Atrazine

1

10

5

3

100

50

30

1,000

500

300

10,000

5,000

3,000

Schedule 2033 concentration, in ng/L

1 10 2 3 4 5 7 100 20 40 60 1,000 300 500 10,000 4,000

Schedule 2437 concentration, in nanograms per liter

Deethylatrazine

Schedule 2437 concentration, in nanograms per liter

1 10 2 3 4 5 7 100 60403020 1,000 600400200

1

10

6

4

2

100

60

40

20

1,000

600

400

200

Schedule 2033 concentration, in ng/L

Metolachlor

1

10

5

3

100

50

30

1,000

500

300

10,000

5,000

3,000

Schedule 2033 concentration, in ng/L

1 10 2 3 4 5 7 100 20 40 60 1,000 300 500 10,000 4,000

Schedule 2437 concentration, in nanograms per liter

Metalaxyl

Schedule 2437 concentration, in nanograms per liter

1 10 2 3 4 5 76 8 100 20 4030 7050

1

5

3

2

20

30

7

100

10

50

70

Schedule 2033 concentration, in ng/L

Simazine

Schedule 2437 concentration, in nanograms per liter

1 10 2 3 4 5 7 100 60403020 1,000 600400200

1

10

6

4

2

100

60

40

20

1,000

600

400

200

Schedule 2033 concentration, in ng/L
Results and Discussion of Method Validation Data 39

Figure 10. Comparison of selected pesticides determined by direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) (schedule 2437) and liquid chromatography-mass spectrometry (LC-MS) (schedule 2060) in 150 samples 

from 48 stream sites collected during National Water Quality Assessment (NAWQA) Program field comparison study from May to 

September 2012. Pesticide compounds detected in both methods shown as filled circles, analyte not detected in either schedule as 

open circles, analyte detected in 2437 and non-detect in 2060 as open diamonds, and analyte detected in 2060 and non-detect in 2437 

as crosses. Laboratory reporting levels in nanograms per liter (ng/L) are shown as dotted lines.

Figure 9 (previous page). Comparison of selected pesticides determined by direct aqueous-injection liquid chromatography-tandem 

mass spectrometry (DAI LC-MS/MS) (schedule 2437) and gas chromatography-mass spectrometry (GC-MS ) (schedule 2033) in 150 

samples from 48 stream sites collected during National Water Quality Assessment (NAWQA) Program field comparison study from May to 

September 2012. Pesticide compounds detected in both methods shown as filled circles, analyte not detected in either schedule as open 

circles, analyte detected in 2437 and non-detect in 2033 as open diamonds, and analyte detected in 2033 and non-detect in 2437 as crosses. 

Laboratory reporting levels in nanograms per liter (ng/L) are shown as dotted lines.

A

C

B

D

2-Hydroxyatrazine

Schedule 2060 concentration, in ng/L

Schedule 2437 concentration, in nanograms per liter

1 10 2 3 4 5 7 100 60403020 1,000 600400200

1

10

6

4

2

100

60

40

20

1,000

600

400

200

2,4-D

10

60

40

20

100

1,000

600

400

200

3,000

5,000

2,000

Schedule 2060concentration, in ng/L

10 20 30 50 70 100 1,000 200 400 600 2,000 4,000

Schedule 2437 concentration, in nanograms per liter

Atrazine

1

10

5

3

100

50

30

1,000

500

300

10,000

5,000

3,000

Schedule 2060 concentration, in ng/L

1 10 2 3 4 5 7 100 20 40 60 1,000 300 500 10,000 4,000

Schedule 2437 concentration, in nanograms per liter

Deethylatrazine

1

10

5

3

100

50

30

1,000

500

300

10,000

5,000

3,000

Schedule 2060 concentration, in ng/L

1 10 2 3 4 5 7 100 20 40 60 1,000 300 500 10,000 4,000

Schedule 2437 concentration, in nanograms per liter
40 Determination of Pesticides and Pesticide Degradates in Filtered Water

nondetections by the 2437 method were reported as concentrations with a median of 10 ng/L (range from 6 to 24 ng/L), 

similar to the LRL of 10 ng/L (table 37). Similarly, 90 percent 

of the 42 detections of simazine in the 2033 method that were 

reported as nondetections by the 2437 method were less than 

11 ng/L, whereas the LRL of the 2437 method was 10 ng/L. 

On the other hand, 90 percent of the detections of metalaxyl 

by the 2437 method that were reported as nondetections by 

the 2033 method were less than 4 ng/L whereas the reporting 

level was 14 ng/L (table 38). 

Similarly, the agreement in the number of detections in 

the stream samples is summarized in table 39 for the comparison of schedule 2437 and 2060. Agreement between results 

reported for both methods as concentrations or as nondetections was from 56 to 100 percent. For example, the agreement 

in results in paired-stream samples for diuron was 72 percent, 

with both methods reporting 86 consistent detections and both 

methods reporting 21 nondetections. The lack of agreement 

for diuron was 28 percent, with 4 results reported as nondetection by schedule 2437 when a concentration was reported by 

schedule 2060, and 37 reported as nondetection by schedule 

2060 when a concentration was reported by schedule 2437. 

The sign test was used to test whether there were 

significant differences in the number of detections found in 

the paired-stream samples (table 40). Most of the analytes 

had a significantly larger number of detections in the 2437 

method compared to the 2060 method, and most of these 

detections were lower than the LRL of the 2060 method 

(tables 41 and 42).

Stability Studies

Stability of the analytes stored at different temperatures 

and in potential preservative or dechlorination reagents was 

measured in different studies. The relevant stability issues 

for this LC-MS/MS method for pesticides (schedule 2437) 

include stability in filtered water similar to those expected 

from time of collection to analysis (refrigeration and ice cooler 

at 4 °C) and storage in a freezer to allow flexibility in sample 

scheduling. The stability in citric acid (pH 3.8) as a reagent 

to decrease microbial activity (Pepich and others, 2005) was 

studied, especially for uses in studies where overnight shipping to the NWQL was unachievable, and stability in ascorbic 

acid used to reduce free chlorine for studies that collected 

samples from sites that might have residual chlorine from 

water treatment plants (Winslow and others, 2001).

Stability Study Design

Stability of the analytes spiked into reagent water was 

measured at one concentration under different temperatures 

and treatments. This design provides information about 

stability only under the simplest conditions, with minimal 

microbial activity, stable pH, and low concentrations of dissolved organic carbon and trace metals (pH and conductivity 

of the reagent water are shown in table 21). A large study of 

the effects of various sample matrices is beyond the scope of 

the validation study. However, this study design will identify 

analytes that are unstable even in reagent water or with the 

various preservative treatments.

The recovery of all analytes spiked at a mid-level concentration (250 ng/L) into reagent water contained in a 20-mL vial 

was measured after storage at various times for as many as 

28 days. Four replicates were prepared for each time interval. 

Since the LC-MS/MS method includes both pesticide and 

transformation products (degradates), spike solutions containing only pesticides or pesticide degradates were prepared to 

conduct the stability studies separately so the transformation 

product stability can be evaluated independently from the 

pesticide. At the end of each storage time, subsamples were 

removed from each vial and analyzed by LC-MS/MS, resulting in five analytical batches.

The four different treatments for pesticides and degradates are shown in table 43. Note that there was no day zero 

for the –10 °C treatment because the samples took about a day 

to freeze. Long-term storage of the samples in the freezer was 

evaluated by re-analysis of a set of samples from day 3 stored 

in the freezer for 133 days (about 4 months).

Storage stability samples were prepared by adding 

100 μL of the spike mixtures to 20 mL of reagent water 

or treated water in a 40-mL amber vial to exclude light. 

The treated water samples contained a buffer used in EPA 

method 527 (reagent water acidified to pH 3.8 with potassium dihydrogen citrate, 9.4 g/L) or a dechlorination reagent 

(reagent water with ascorbic acid, 0.1 g/L). After adding the 

spike mixture, the vials were capped and the samples were 

stored at 4 °C or –10 °C until analysis. At the end of storage, 

subsamples were transferred to analytical vials and prepared 

for analysis by adding the internal standard and methanol. 

Frozen samples were allowed to thaw at room temperature 

for about 4 h before subsampling.

All treatments for a particular storage time were analyzed 

in one analytical batch. Thus, differences over time might reflect 

batch-to-batch variability as well as any degradation or stabilityrelated changes that occurred during storage. In particular, the 

samples for ESI+ analytes day 3 were from a repeat experiment, 

and because these results appear to be higher than other samples, 

they could give misleading indication of trends over time.

Maximum Holding Times

Stability results are discussed in terms of recovery rather 

than concentration. This allows easy comparison of the overall 

data-quality objectives of recovery within 100±30 percent. 

Stability can then be evaluated as an additional source of bias 

yielding results lower than 70 percent. 

Maximum holding times were estimated using two 

approaches—a statistical calculation specified by the ASTM 

and a practical approach based on the data-quality objectives 

of the validation experiments. The ASTM standard D4841-88 

defines the holding time as the time the predicted concentration falls below the lower two-sided 99-percent confidence 

interval of the analyte concentration found at zero time 
Results and Discussion of Method Validation Data 41

(American Society for Testing and Materials, 1993). The dataquality objectives of the validation experiments include a bias 

specification within 30 percent of expected concentration or 

recovery, so the practical approach applied here was changes 

in recovery lower than 30 percent after storage would indicate 

the time when degradation of the analyte causes significant 

bias in recovery. 

The 99-percent confidence interval used in the ASTM 

specification was calculated from 29 CCV standards analyzed 

during the holding time experiments. The CCVs were analyzed in five separate batches over the 28-day stability study 

and are expected to provide a good estimate of measurement 

variability. The tolerable range of variation (99-percent confidence interval) was calculated according to equation 5:

d t s

n

= ± 

×

(5)

where

d is the range of tolerable variation from the 

initial mean recovery, in percent;

t is the Student’s t (based on number of 

replicates);

s is the standard deviation of replicates, in 

percent; and 

n is the number of replicates at each time.

For this study, n=29 (replicate CCVs) and the Student’s 

t for a two-tailed 99-percent confidence interval is 2.763. The 

lower limit of stability was then calculated as 100 percent 

minus the tolerable range of variation. The practical approach 

defined the lower limit of stability as 100 percent minus 

30 percent (70 percent).

Stability Study Results

Results of the stability experiments by treatment for the 

two analyte groups (pesticides and degradates) are discussed 

in the following sections. Storage at 4 °C and –10 °C with 

no preservative are discussed in detail, whereas stability results with ascorbic acid and pH 3.8 are provided in 

supplementary tables. The pesticide group is discussed first, 

followed by pesticide degradates. Analytes determined by 

ESI+ mode are discussed before the ESI− analytes. Graphs 

of the recoveries as a function of storage time in the different treatments for each pesticide compound are shown in 

supporting information figures S9, S10, S11, S12, S13, and 

S14. Variability of study results is represented by the RSD 

in tables 44–62.

Tolerable Range of Variation

The lower limits of stability calculated from the CCVs 

for analytes determined in the ESI+ mode are summarized 

in table 44. Most analytes determined by ESI+ mode had 

relatively narrow tolerable ranges of variation from the 

mean concentration (99-percent confidence intervals) less 

than 20 percent (mean, 17 percent; median, 17 percent; 

75th percentile, 20 percent). The corresponding lower limits 

of recovery expected from analytical variability for most 

analytes were from 80 to 91 percent and were higher than the 

practical data-quality objectives of recovery of 70 percent. A 

few analytes had larger confidence intervals, including didealkylatrazine (42 percent), 2-hydroxy-6-ethylamino-4-aminos-triazine (31 percent), and 4-chlorobenzylmethyl sulfoxide 

(36 percent). The analytes with validation quality code “E” 

generally had a larger range of confidence intervals, from 13 

to 78 percent.

The lower limits of stability calculated from the CCVs 

for analytes determined in the ESI− mode are summarized in 

table 45. Most analytes (37 of 45) determined by ESI− mode 

had tolerable ranges of variation from the mean concentration (99-percent confidence intervals) of 40 percent or less 

(mean 30 percent; median 25 percent; 75th percentile 31 percent). The corresponding lower limits of recovery expected 

from analytical variability alone for most analytes were from 

63 to 88 percent. Seven analytes (flubendiamide, oryzalin, 

diflufenzopyr, sulfentrazone, chlorothal-monomethyl, chlorosulfonamide acid, and hexazinone TP D) had larger analytical variability of the CCV samples, so the corresponding 

lower limits of recovery are very low (25 to 52 percent) and 

might not be useful for estimating holding times.

Stability at 4 °C Pesticides ESI+ Mode Analytes

The variability of recoveries, represented by the RSD, 

of pesticides analyzed in ESI+ mode generally were less 

than 15 percent for any day, with median of recovery variability from 4 to 9 percent for the five study holding times 

are shown in table 46 (for the 90 pesticides with validation 

quality code of “A”). These results indicate that relative 

measurement error for any storage time was low so that 

the concentration trends over time were less likely to be 

obscured by variability in results. Some of the qualified 

analytes, with a validation quality code of “E” had variability larger than 15 percent, for example oxyfluorfen on day 

7 (47 percent) and parathion methyl on day 14 (78 percent). 

Concentration trends over time for these pesticides will be 

more difficult to determine.

Changes in recovery of pesticides determined by ESI+ 

mode after 28 days of storage at 4 °C were relatively low 

(table 46). Acetanilide, carbamate, fungicide, sulfonylurea, 

and triazine pesticides were within the ASTM tolerable range 

of variation with few exceptions. Triallate (79 percent) and 

kresoxim-methyl (84 percent) exceeded the ASTM limit, but 

these were above the practical limit of 70 percent. Miscellaneous, organophosphate, and pyrethroid pesticides had 

slightly larger changes in recovery after 28 days, with many 

(27) analytes below the ASTM limit after this time. Three 

organophosphates (disulfoton, phorate, and terbufos) had 

recoveries lower than 70 percent (54 to 66 percent) after 

28 days. In addition, five pesticides (etoxazole, indoxacarb, 

lactofen, cis-permethrin, and trans-permethrin).were less 

than the practical limit of 70 percent after 28 days, but they 

were also below 70 percent on day 0 and other times, so 
42 Determination of Pesticides and Pesticide Degradates in Filtered Water

any changes in variability might be obscured by the analytical variability. Famoxadone, naled, and phosmet are qualified (E) for various reasons but also appeared to degrade 

and have recoveries less than 70 percent after storage for 

28 days. Overall, all 102 pesticides analyzed in ESI+ mode 

were 70 percent or above after 14 days of storage, and 92 

pesticides were above 70 percent after 28 days of storage. 

Flumiclorac-pentyl and bifenazate exceeded practical recovery limits after storage for 7 and 14 days, so were considered 

unsuitable for the method and candidates for deletion after 

the validation studies.

Few of the targeted degradates were found in the 

samples spiked with pesticides only and stored at 4 °C, 

indicating stability of most of the 44 pesticides that have a 

degradate measured in the ESI+ mode. Fenamiphos sulfoxide, the diazinon degradate pyrimidinol, and the ethoprop 

degradate O-ethyl-O-methyl-S-propyl phosphorothioate 

were found at concentrations representing about 5 to 8 

percent of the nominal concentration of the pesticide and 

remained at that level during storage. This might reflect 

some degradate present in the mixed standard rather than 

formation during storage. Disulfoton sulfoxide, however, 

increased from 8 percent on day 0 to 19 percent on day 

28 of the spike concentration of disulfoton added to the 

sample on day 0 and probably represents transformation 

of disulfoton during storage. Similarly, bifenazate diazine 

was found at concentrations representing 16 to70 percent 

of bifenazate concentration, although both these analytes 

had very high measurement variability.

Stability at 4 °C Pesticides ESI− Mode Analytes

The variability of pesticides analyzed in ESI− mode 

stored at 4 °C for as many as 28 days were generally less 

than 15 percent for most pesticides, although on some days 

variability was as high as 28 percent for some pesticides. The 

overall variability was from 6 to 33 percent, with the sulfonylureas diflufenzopyr and sulfentrazone generally having 

larger variability, as shown in table 47. There were relatively 

small changes in recovery of the pesticides over the 28-day 

storage time. All pesticides were within both the ATSM tolerable range of variation and the practical limit of greater than 

70 percent recovery after 28 days except for flubendiamide. 

Recovery of flubendiamide decreased to 68 percent by day 

28. This pesticide had a very low ASTM limit (25 percent) 

because of the variability of CCVs used to calculate the 

standard deviation. This probably represents some degradation of flubendiamide in the CCVs during data acquisition 

of the sample batch. There were few degradates found in 

the samples spiked with pesticides only and stored at 4 °C, 

indicating stability of most pesticides (at least those that 

have a degradate determined by this method). Diketonitrile 

isoxaflutole was the only degradate found in the pesticideonly spike samples, at 5 and 7 percent of the spike level at 

day 14 and 28.

Stability at 4 °C Degradates ESI+ Mode Analytes

The variability of recoveries of degradates analyzed in 

ESI+ mode generally were less than 20 percent for any day, 

with median recovery variability from 5 to 7 percent for the 

five sampling days (table 48). Chlorpyrifos oxon on day 3 and 

methomyl oxime and hydroxy monomethyl fluometuron on 

day 28 were more variable (25–50 percent). 7-Hydroxycarbofuran had high measurement variability (24 to 70 percent) on 

some days.

Changes in recovery of degradates determined by ESI+ 

mode after 28 days of storage at 4 °C were relatively low. 

Overall, 82 of 84 degradates with validation quality code 

of “A” were above 70 percent recovery after 14 days of 

storage, and 74 degradates were above 70 percent recovery 

after 28 days of storage. Many degradates were within the 

ASTM tolerable range of variation after 14 days of storage but exceeded the limit after 28 days. However, only 

10 degradates exceeded the practical limit of 70 percent 

(phorate oxon, terbufos oxon, 4-hydroxy-tert-fluometuron, 

hydroxy mono demethyl fluometuron, hydroxyfluometuron, 

tebuthiuron TP 106, 2-hydroxy-4-isopropylamino-6-aminos-triazine, 2-hydroxy-6-ethylamino-4-amino-s-triazine, 

didealkylatrazine, and hexazinone TP C), and all were within 

the practical limit after 14 days of storage except for phorate 

oxon and terbufos oxon, and for diazinon oxon after 7 days. 

Methomyl oxime and desiodoflubendiamide were less than 

70 percent on day 0 but were above 80 percent after that 

from 3 to 28 days.

Stability at 4 °C Degradates ESI− Mode Analytes

The variability of recoveries of degradates analyzed in 

ESI− mode were less than 20 percent for day 3 through 28 for 

about half of the analytes (table 49). Many of the analytes had 

relatively high recovery (greater than 130 percent) and high 

analytical variability, from 20 to 30 percent on different days. 

Despite this high recovery, the overall changes in recovery 

after 28 days of storage were relatively low. All degradates 

determined in ESI− mode were within the ASTM tolerable 

range of variation after 28 days of storage. The ASTM limit 

was lower than the practical limit of 70 percent for some analytes (4-(hydroxymethyl)pendimethalin, cis-cyhalothric acid, 

metribuzin DK) because of the larger variability on a particular day. However, the recoveries on day 28 for these analytes 

were still above the practical limit of 70 percent.

Stability at –10 °C Pesticides ESI+ Mode Analytes

The variability of recoveries of pesticides analyzed in 

ESI+ mode stored at –10 °C were mostly less than 15 percent 

for any time period, with the median of recovery variability for 91 pesticides from 5 to 9 percent for the five sample 

storage times (table 50). As in the case for storage at 4 °C the 

relative analytical variability for any storage time was low so 

that the concentration trends over time are distinct. However, 

recoveries for many analytes were relatively high on day 3.
Results and Discussion of Method Validation Data 43

Changes in recovery of pesticides determined by ESI+ 

mode after 28 days of storage at –10 °C were relatively low 

(table 50) and are similar to the results of storage at 4 °C. 

Overall, most (101 of 102) pesticides analyzed in ESI+ mode 

were greater than 70 percent recovery after 14 days of storage, and 94 pesticides were within 70 percent after 28 days 

of storage. Acetanilide, carbamate, fungicide, sulfonylurea, 

and triazine pesticides were within the ASTM tolerable range 

of variation with few exceptions. Triallate (83 percent), 

linuron (88 percent), and siduron (88 percent) exceeded the 

ASTM limit but were within the practical limit of above 

70 percent recovery. Miscellaneous, organophosphate, and 

pyrethroid pesticides had the larger changes in recovery after 

28 days, with many analytes below the ASTM limit after 

this time. Only eight pesticides were less than the practical 

limit of above 70 percent recovery (etoxazole, indoxacarb, 

lactofen, pyridaben, phosmet, oxyfluorfen, cis-permethrin, 

and trans-permethrin). Notably phorate and terbufos had 

higher recoveries (74 and 86 percent) after 28 days of storage 

at –10 °C compared to 4 °C (59 and 54 percent). Similarly, 

flumiclorac-pentyl and bifenazate had higher recoveries (82 

and 80 percent) after 14 days of storage at –10 °C compared to 

4 °C (21 and 46 percent). Recoveries of pesticides in samples 

stored at –10 °C for 133 days (about 4 months) were similar 

to those at 28 days, with eight pesticides with recoveries less 

than 70 percent (etoxazole, lactofen, pyridaben, disulfoton, 

tribufos, phosmet, cis-permethrin, and trans-permethrin).

The few degradates found in the samples spiked with pesticides only and stored at –10 °C were the same ones as found 

in samples stored at 4 °C. Fenamiphos sulfoxide, pyrimidinol, 

and O-ethyl-O-methyl-S-propyl phosphorothioate were found 

at concentrations representing about 5 to 8 percent of the 

spiked concentration of the pesticide (250 ng/L) and remained 

at that level during storage. It is also possible these degradates 

were the result of contamination, carryover, or some other 

artifact and not due to degradation of the pesticide because the 

concentrations of the degradate did not increase over time, and 

the corresponding pesticide did not decrease. Disulfoton sulfoxide increased from 9 to 20 percent over the 28-day storage, 

very similar to the samples stored at 4 °C, corresponding to 

the decrease in disulfoton. This pesticide compound increased 

during storage at –10 °C for 133 days, as well as three other 

organophosphate degradates (fenamiphos sulfoxide, phorate 

sulfoxide, and terbufos sulfoxide) (supporting table S12).

Stability at –10 °C Pesticides ESI− Mode Analytes

Storage temperature had no measurable effect on recovery of pesticides determined by ESI− mode. Changes in 

recovery of pesticides determined by ESI− mode after 28 

days of storage at –10 °C were relatively low (table 51) and 

are similar to the results of storage at 4 °C. As in the case of 

samples stored at 4 °C, all pesticides were within both the 

ATSM tolerable range of variation and the practical limit of 

70 percent recovery after 28 days except for flubendiamide. 

However, many recoveries were high (>130 percent) on the 

first two sample storage times, so the fact that recoveries 

were above 70 percent after 14 and 28 days could also reflect 

some degradation that was obscured by the high recoveries. 

Flubendiamide recovery was below 70 percent (69 percent) 

after 28 days, but 95 percent after 133 days, perhaps a result of 

batch-to-batch analytical variability of this analyte rather than 

degradation during storage. Similar to the samples stored at 

4 °C, diketonitrile isoxaflutole increased to 13 percent of the 

spiked concentration during the 28-day storage from degradation of isoxaflutole. Isoxaflutole, a pesticide determined in 

the ESI+ mode, recoveries correspondingly decreased by 21 

percent over the same time. This was also the only ESI− mode 

degradate found in the samples spiked with pesticides only 

during storage at –10 °C for 133 days (supporting table S12).

Stability at –10 °C Degradates ESI+ Mode Analytes

There were relatively small changes in recovery of degradates determined by ESI+ mode after 28 days of storage at 

–10 °C (table 52). Many acetanilide, carbamate, fungicide, 

miscellaneous, and organophosphate degradates were within the 

ASTM tolerable range after 28 days. There were a few exceptions, including 4-hydroxymolinate, carboxy molinate, and 

desiodo flubendiamide (77, 77, and 78 percent, respectively), 

but these were above the practical limit of 70 percent recovery. 

Recoveries were less than 70 percent for only two degradates 

(diazinon oxon and disulfoton oxon) after 133 days of storage 

at –10 °C. Many sulfonylurea and triazine degradates exceeded 

the ASTM tolerable range at 28 days, and seven were less than 

70 percent (4-hydroxy-tert-fluometuron, 2-hydroxy-4-isopropylamino-6-amino-s-triazine, 2-hydroxy-6-ethylamino-4-amino-striazine, didealkylatrazine, 4-hydroxyhexazinone A, hexazinone 

TP C, and hydroxysimazine). However, these lower recoveries 

in the samples stored for 28 days might have been the result of 

bias with that particular analytical batch rather than degradation during storage because the samples stored for 133 days had 

recoveries above 70 percent. Compared with storage at 4 °C, 

storage at –10 °C substantially improved the recovery of phorate 

oxon and terbufos oxon, from 39 and 7 percent to 102 and 82 

percent recovery after 28 days.

Stability at –10 °C Degradates ESI− Mode Analytes

There were relatively small changes in recovery of 

degradates determined by ESI− mode after 28 days of storage 

(table 53). Most degradates were within the ASTM tolerable range after 28 days and above the practical limit of 70 

percent recovery. A few degradates had recoveries less than 

70 percent after 28 (4-(hydroxymethyl)pendimethalin) and 133 

(acetochlor SA and cis-cyhalothric acid) days of storage, but 

the variability in each batch for these compounds (RSD >30 

percent) might have obscured any trends in storage stability. 

Pesticide Stability Using Dechlorination Reagent 

(Ascorbic Acid) ESI+ Mode Analytes

The addition of the dechlorination reagent ascorbic acid 

resulted in lower recoveries for about 18 percent of the ESI+ 

pesticides (table 54). Seven pesticides (indoxacarb, lactofen, 
44 Determination of Pesticides and Pesticide Degradates in Filtered Water

propargite, pyridaben, tribufos, cis-permethrin, and transpermethrin) had recoveries lower than 70 percent on day 0 but 

exhibited no substantial change in concentration over time. 

This is likely the result of matrix suppression caused by the 

ascorbic acid. Another 11 pesticides (etoxazole, pymetrozine, 

diazinon, terbufos, chlorimuron-ethyl, chlorsulfuron, orthosulfamuron, prosulfuron, and the analytes with validation quality 

code “E” [bifenthrin, fenbutatin oxide, and flumiclorac-pentyl]) 

had large changes in recovery over time such that by day 28 

recoveries were less than 70 percent. This indicates degradation of the pesticide was caused by the ascorbic acid because 

these analytes were stable in samples stored with no reagent. 

The ascorbic acid in general resulted in higher variability and 

slightly higher recoveries compared to reagent water with no 

ascorbic acid. For example mean recoveries of asulam were 

from 130 to 161 percent in ascorbic acid compared to 71 to 108 

in reagent water alone.

The few degradates found in the samples spiked with 

pesticides only and stored with the dechlorination reagent 

ascorbic acid at 4 °C were the same ones as found in samples 

without acid and stored at 4 °C. O-ethyl-O-methyl-S-propyl 

phosphorothioate was found at concentrations representing 

about 5 to 8 percent of the nominal concentration of the pesticide and remained at that level during storage. Fenamiphos 

sulfoxide, pyrimidinol (a diazinon degradate), and disulfoton 

sulfoxide stored with ascorbic acid increased from 5 percent 

to 12 to 38 percent over the 28-day storage, more than the 

samples stored in reagent water alone. In addition, phorate 

sulfoxide and terbufos sulfoxide increased to 7 and 6 percent 

over the 28-day storage in ascorbic acid.

Pesticide Stability Using Dechlorination Reagent 

(Ascorbic Acid) ESI− Mode Analytes

The addition of the dechlorination reagent ascorbic acid 

had no substantial effects on recoveries during storage in comparison to storage at 4 °C for pesticides determined by ESI− 

mode (table 55). Recovery of diflufenzopyr (60 percent) after 

28 days was below the practical limit, but overall results were 

too variable to evaluate a definite trend. Recovery of flubendiamide (65 percent) after 28 days was also below the practical 

limit, but this was similar to stability results for samples stored 

with no reagent. 

Pesticide Degradate Stability Using Dechlorination 

Reagent (Ascorbic Acid) ESI+ Mode Analytes

The addition of the dechlorination reagent ascorbic acid 

resulted in no change in recovery after 14 days and small 

changes in recovery after 28 days of storage for pesticide degradates determined in the ESI+ mode (table 56). Most analytes 

were within the ASTM limits after 28 days, and 75 of the 84 

degradates were above the practical limit of 70 percent recovery. However, nine analytes (hydroxydiazinon, phorate oxon, 

terbufos oxon, 4-hydroxy-tert-fluometuron, 2-hydroxy-4-isopropylamino-6-amino-s-triazine, didealkylatrazine, hexazinone 

TP C, 1H-1,2,4-triazole, and diazinon oxon) were lower than 

70 percent recovery after 28 days and some had large changes 

in recovery over time. This was clearly evident for the diazinon degradates: diazinon oxon, which decreased to 0 percent 

recovery after only 7 days of storage, and hydroxydiazinon, 

which decreased to 6 percent recovery after 28 days. Pyrimidinol initially increased from degradation of hydroxydiazinon 

or diazinon oxon to about 151 percent recovery and then later 

decreased. The other organophosphate degradates phorate oxon 

and terbufos oxon degraded during storage at 4 °C with no 

reagent, so the loss in this experiment is not due to the ascorbic 

acid. In addition, 2-hydroxy-6-ethylamino-4-amino-s-triazine 

recoveries changed from 177 percent on day 0 to 97 percent on 

day 28, which might be an example of degradation obscured by 

the enhanced recovery.

Pesticide Degradate Stability Using Dechlorination 

Reagent (Ascorbic Acid) ESI− Mode Analytes

The addition of the dechlorination reagent ascorbic 

acid had no substantial effects on recoveries during storage for most pesticide degradates determined by ESI− mode 

(table 57). There was no substantial change in recovery over 

the 28-day storage time, although two analytes (cis-cyhalothric acid and hexazinone TP E) did not meet the 70 percent 

threshold for one and three of the time points, respectively. 

Variability was large on day 0 caused by one sample with low 

recoveries for most analytes.

Pesticide Stability Using Antimicrobial Reagent (Citric 

Acid) ESI+ Mode Analytes

The addition of the antimicrobial reagent citric acid to 

adjust the sample pH to 3.8 used in EPA method 527 resulted 

in changes in recovery for a small number of pesticides analyzed in the ESI+ mode (table 58). Many of the pesticides 

(76 of 106), mainly acetanilide, carbamate, fungicide, sulfonylurea, and triazine pesticides, had recoveries within the 

ASTM limits over the 28-day storage with citric acid. Only a 

few pesticide recoveries from these groups were less than the 

70-percent-recovery practical limit after this time including 

the sulfonylureas chlorsulfuron, orthosulfamuron, prosulfuron, 

and the triazine cyanazine. However, many (15 of 43) miscellaneous and organophosphate pesticides had recoveries below 

the ASTM limit at three or more sampling times, probably due 

to matrix suppression caused by the acid. A few pesticides had 

large decreases in concentration over the storage time that was 

due to degradation caused by the acid, including etoxazole, 

pymetrozine, diazinon, orthosulfamuron, and prosulfuron. 

Bifenazate, terbufos and flumiclorac-pentyl also degraded over 

time in the acid, but these pesticide compounds also degraded in 

untreated water.

Few of the targeted degradates were found in the samples 

spiked with the pesticides only and stored with the citric acid. 

The organophosphate pesticide degradates O-ethyl-O-methylS-propyl phosphorothioate, fenamiphos sulfoxide, and terbufos sulfoxide were found at concentrations representing about 

5 to 8 percent of the nominal concentration of the pesticide 
Problematic Compounds 45

and remained at that level during storage. Pyrimidinol and 

disulfoton sulfoxide increased from 5 percent to 38 and 

19 percent over the 28-day storage, corresponding to decreases 

in the pesticide diazinon and disulfoton, similar to the samples 

stored in ascorbic acid.

Pesticide Stability Using Antimicrobial Reagent (Citric 

Acid) ESI− Mode Analytes

The addition of the antimicrobial reagent citric acid to 

adjust the sample pH to 3.8 used in EPA method 527 resulted in 

large changes in recovery for a number of pesticides analyzed 

in the ESI− mode (table 59). Many of the acids had high recoveries (mean recoveries from 106 to 517 percent), although there 

was no trend in recovery over time. The change in sample pH 

seems to have caused electrospray ionization enhancement in 

the MS/MS instrument for these acidic pesticides. Other effects 

of the citric acid were large variability for the sulfonylureas. 

Chlorsulfuron recoveries also were low (49 to 58 percent) and 

less than ASTM limits by day 14, similar to the samples treated 

with ascorbic acid and unlike those without reagent added. One 

degradate was found in the samples spiked with the pesticides 

only and stored with the citric acid. Diketonitrile isoxaflutole 

recoveries increased to 11 and 13 percent after 14 and 28 

days of storage respectively, similar to samples stored without 

reagent added.

Pesticide Degradate Stability Using Antimicrobial Reagent 

(Citric Acid) ESI+ Mode Analytes

The addition of antimicrobial reagent citric acid resulted 

in no change in recovery after 14 days and small changes in 

recovery after 28 days of storage for most pesticide degradates 

determined in the ESI+ mode (table 60). Most analytes were 

within the ASTM limits after 28 days, and 73 of the 84 degradates 

were above the 70-percent-recovery practical limit. However, 11 

degradates, mainly organophosphates and triazines (methomyl 

oxime, hydroxydiazinon, phorate oxon, terbufos oxon, phthalazinone, 4-hydroxy-tert-fluometuron, 2-hydroxy-6-ethylamino4-amino-s-triazine, deisopropylatrazine, didealkylatrazine, 

1H-1,2,4-triazole, and diazinon oxon) were lower than 70 percent 

recovery after 28 days. Some degradates, including hydroxydiazinon, diazinon oxon, phorate sulfoxide, and terbufos sulfoxide, 

were degraded in the citric acid and had large decreases over the 

time of the study. The diazinon degradate pyrimidinol increased 

from degradation of hydroxydiazinon or diazinon oxon to 146 

percent recovery by day 14 and then decreased.

Pesticide Degradate Stability Using Antimicrobial Reagent 

(Citric Acid) ESI− Mode Analytes

The addition of the antimicrobial reagent citric acid 

to adjust the sample pH to 3.8 used in EPA method 527 

resulted in large changes in recovery for a number of pesticide degradates analyzed in the ESI− mode (table 61). Many 

of the acetanilide degradates had high recoveries (mean 

recoveries as much as 367 percent), although there was no 

trend in recovery over time. We assume this was the result of 

ionization enhancement. The variability of pesticide degradate recovery was also large, from 15 to 38 percent for many 

analytes. The hexazinone degradate hexazinone TP E had 

low recoveries (0 to 28 percent) at all times, most likely due 

to ionization suppression because there was no change in 

recovery over time.

Summary and Recommendations Based on Stability Study 

Results

The stability studies demonstrated that the largest number 

of the pesticide compounds (227 of 229) were stable in reagent 

water after 14 days of storage at 4 °C, so that was selected as 

the practical holding time for routine sample processing. Upon 

arrival at the laboratory, samples are stored in a refrigerator 

and analyzed within 14 days of sample collection. The use 

of ascorbic acid as a dechlorination reagent resulted in lower 

recoveries of some analytes due to degradation and ion suppression, so use of this reagent to prevent reactions of pesticide 

compounds with free chlorine needs to be weighed against the 

negative effects of the reagent. The use of antimicrobial reagent 

citric acid to adjust the sample pH to about 4 also resulted in 

lower recoveries of some analytes, so it should not be used as a 

routine sample preservative. Storing the samples at –10 °C for 

28 to 133 days resulted in recoveries less than 70 percent for 10 

of the 229 pesticide compounds and formation of four organophosphate degradates. If samples cannot be analyzed within the 

14-day holding time, storing at –10 °C would be a reasonable 

alternative for most of the pesticide compounds but should 

not be used for routine operation. If the samples are stored for 

longer than the holding time, laboratory reagent spikes should 

be prepared and stored with the samples to verify the results of 

the holding-time experiments described here, and appropriate 

data qualifiers should be assigned to sample results for analytes 

that exceed control limits.

Problematic Compounds

For a number of pesticide compounds (four in ESI+, six 

in ESI−), there was only one major product ion, so the qualifier MRM selected was precursor-to-precursor type (shown 

in bold in table 6). These pesticide compounds generally had 

higher MDLs (above 50 ng/L) than other compounds, and 

identification in a sample requires careful evaluation by the 

analyst and comparison with calibration standards because 

these MRMs tend to have more background interferences.

Acetochlor SA and alachlor SA are not separated using the 

chromatography conditions of the DAI LC-MS/MS method, 

and they have the same quantifier (314 → 80) and qualifier 

ions (314 → 121) MRMs (table 6). Additional unique but low 

abundance qualifier MRMs are used to identify these pesticide 

compounds, but the analyst needs to carefully compare ion 

ratios of samples with calibration standards for identification.

Siduron is the only pesticide compound that has cis- and 

trans-isomers separated using the chromatography conditions 
46 Determination of Pesticides and Pesticide Degradates in Filtered Water

of the DAI LC-MS/MS method and require integration as one 

peak. This requires manual integration by the analyst because 

the automated integrator software identifies each peak as a 

separate compound. cis-Permethrin and trans-permethrin are 

also separated using the chromatography conditions of the 

DAI LC-MS/MS method, but these are integrated and reported 

as separate compounds.

Pymetrozine is an amine with pKa of 4.06 and therefore 

is appreciably ionic at low pH. Consistent recoveries within 

data-quality objectives were obtained for pymetrozine in 

all three matrices (table 22) but were more variable (RSD = 

40 percent) in the field-matrix-spike samples analyzed during 

the field study (table 28). This may be due to the inherent 

trade-off related to control of pH at 3–5 in samples with wider 

ranges of pH. Analysts might need to qualify results based on 

variability in QC samples during an analytical run related to 

changes in sample pH.

Asulam and chlorosulfonamide acid both have a chromatographic peak shape that changes, sometimes appearing 

as one peak, other times as two peaks. Asulam peak shape 

changes with concentration, whereas the peak shape of chlorosulfonamide acid is affected by matrix and age of the chromatographic column; both of these analytes require careful 

review and manual integration by the analyst.

Analytes that elute in the first few minutes of the chromatogram are prone to matrix-related electrospray ionization interference. For example, the triazine degradate didealkylatrazine 

(retention time 2.0 min) had mean recoveries from 180 to 231 

percent in all the matrices (tables 22 and 28). Other analytes that 

elute early and have interferences that cause problems with identification at low concentrations are 1H-1,2,4-triazole, 2-Hydroxy6-ethylamino-4-amino-s-triazine, and metribuzin DK. 

Summary and Conclusions

A liquid chromatography-tandem mass spectrometry 

(LC-MS/MS) method was developed for determination of 

229 pesticides compounds (113 pesticides and 116 pesticide 

degradates) in filtered water samples from stream and groundwater sites. The method involves direct injection of a 100-μL 

sample onto the LC-MS/MS with filtration as the only sample 

preparation. Samples are analyzed with two injections, one 

in ESI positive mode and one in ESI negative mode, using 

dynamic MRM conditions, with two MRM transitions for 

each analyte. Isotope-labeled pesticide compounds are used 

as internal standards for quantitation to minimize effects of 

water-sample matrix components that cause ion suppression 

or enhancement. Information about the pesticide compounds 

included in the new method is provided in table 1, and a summary of results from selected validation studies is provided in 

table 62.

In the current method, DAI eliminates the need for 

sample extraction and lengthy sample preparation steps, which 

in turn eliminates analyte losses due to inefficient sample 

extractions or evaporation steps. Because the DAI LC-MS/MS 

method requires a relatively small sample volume (100 μL), 

smaller sample volumes need to be collected and filtered than 

required for previous pesticide analytical methods. In addition, 

the specificity of the MS/MS technique allows use of disposable polypropylene sample collection equipment because 

plasticizer interferences in polypropylene that are a problem 

with single quadrupole MS instruments are not a problem 

with MS/MS. Together, these new requirements for sample 

processing offer substantial savings in sample collection and 

processing supplies, equipment, and time.

The pesticide compounds in the new method were 

considered high priority (tier 1) for method development and 

future monitoring studies in the USGS because of potential 

for occurrence and persistence in streams and groundwater, 

and toxicity to humans and aquatic organisms. The highpriority pesticide compounds included 78 new compounds 

not previously monitored in USGS analytical methods for 

determination of pesticides in water (based on their unique 

NWIS method code) as well as many pesticide compounds 

determined infrequently or in small research studies. In addition, 96 pesticide compounds are included in commonly used 

USGS analytical methods A unique feature of the new method 

is the relatively large number of pesticide degradates included 

(122 of 240), which provides the ability to more thoroughly 

study the fate of pesticides in the environment, as well as 

evaluate the toxicity and exposure effects.

The analytical method was first developed by selection 

and optimization of the MRM conditions for each pesticide 

compound. The MS/MS was operated in both positive and 

negative electrospray ionization modes for different groups of 

pesticide compounds. For most compounds in the ESI+ mode 

(171 of 185), the protonated molecular ion [M+H]+ was most 

abundant and was selected as precursor ion. For 10 pesticide 

compounds, the ammonia adducts [M+NH4

]+ were most 

abundant and were selected as precursor ion, but there were no 

differences in performance for these compounds compared to 

the compounds with protonated ion precursor (table 62). For 

most pesticide compounds in the ESI− mode (38 of 44), the 

deprotonated molecular ion [M-H]- was most abundant and 

was selected as precursor ion. For six pesticide compounds in 

the ESI− mode, there was only one major product ion, so the 

qualifier MRM selected was precursor-to-precursor type.

The method detection levels determined in reagent 

water range from 1 to 250 ng/L, with a median of 3 ng/L, for 

pesticide compounds determined in ESI+ mode, and from 2 to 

250 ng/L, with a median of 40 ng/L, for pesticide compounds 

determined in the ESI− mode. These MDLs are comparable to 

or less than the MDLs of NWQL’s existing analytical methods 

for pesticides.

Mean recoveries of most analytes in the ESI+ mode 

were within data-quality objectives of 100±30 percent at all 

spike levels and matrices. The spiked sample recoveries in the 

three different matrices tested during a validation study were 

comparable to those in laboratory reagent water, indicating 

the absence of major matrix effects that caused ion suppression (or enhancement) of many of the pesticide compounds 
Summary and Conclusions 47

included in the method. A few pesticide compounds, such as 

1H-1,2,4-triazole, 2-hydroxy-6-ethylamino-4-amino-s-triazine, 

methamidophos, didealkylatrazine, and chlorosulfonamide 

acid, elute in the first few min of the chromatographic run and 

have larger bias and variability because of interference from 

highly polar matrix components. Mean recoveries at medium 

and high concentrations (100, 500, and 5,075 ng/L combined; 

n=10) in reagent water were from 89 to 119 percent (median 

104 percent), with RSD less than 22 percent for 169 analytes 

with MDLs above the spike level. More than half (96) of these 

analytes had recoveries between 95 and 105 percent, indicating low bias for many of the analytes in the DAI LC-MS/MS 

method. There was no obvious difference in recovery by type 

of pesticide (analytical method group). Mean recoveries in 

other matrices were similar, although there were some analytes 

with more bias in some matrices. The carbamates asulam and 

oxamyl oxime had low recoveries in all three environmental 

matrices but not in reagent water and thus were qualified with 

the permanent estimated remark (E). Recoveries at the lowest 

spike level (10 ng/L) had more bias and variability, especially 

when MDLs were near the spike level. Average recoveries of most analytes were within data-quality objectives of 

100±30 percent at spike levels above the calculated MDL. 

Recoveries of analytes in the ESI− mode were more variable 

in general than the ESI+ mode because many of the analytes 

have calculated MDLs higher than the lowest spike level.

Analysis of TPC standards demonstrate that the standards 

used in the DAI LC-MS/MS method provide comparable 

results to the existing methods for most analytes. Differences 

in environmental samples and field-matrix-spike recovery 

in comparison to samples analyzed by the different methods 

should be less than 20 percent based on calibration standard 

differences. Conversely, larger differences would be due to 

other factors such as matrix effects or differences in calibration but not the analytical standards. Detections of pesticide 

compounds in laboratory blanks were infrequent. There were 

16 analytes with detections in the laboratory blanks in the 

ESI+ mode and two analytes with detections in the ESI− 

mode. There were four pesticide compounds (fenbutatin oxide, 

piperonyl butoxide, and pyrimidinol in the ESI+ mode and 

fipronil sulfone in the ESI− mode) with 3 to 6 detections in 17 

laboratory blank samples (18 to 36 percent).

A comparison of the new DAI LC-MS/MS analytical method with two existing NWQL analytical methods 

(schedules 2033 and 2060) for determination of pesticides 

was conducted in collaboration with the NAWQA Program. 

Environmental samples, field-matrix spikes, and field blanks 

collected from a variety of NAWQA stream sites from June 

to September 2012, as well as routine laboratory QC samples, 

were analyzed by all three methods. The results provided 

a characterization of the performance of the new method 

during implementation in routine production, including bias 

and variability of analytes in schedule 2437 over a range of 

environmental matrices and concentrations and in comparison 

to schedules 2033 and 2060, potential for bias and contamination using the new field filtration procedure for schedule 2437, 

and an opportunity to assess efficiency, costs, implementation, 

and logistical issues of schedule 2437 from both field and 

laboratory perspectives. Overall, the agreement in matrix spike 

recoveries, detection frequencies, and environmental concentrations was very good. At concentrations near the reporting 

levels, there were larger differences in detection frequencies 

and environmental concentrations. Significant differences in 

environmental concentrations were found between methods 

for some compounds. Recovery and other factors result in different environmental concentrations, and the differences vary 

by compound. These differences need to be understood and 

characterized prior to any data analysis when comparing data 

from different methods.

Agreement between different methods in number of 

detections found at concentrations near the LRL varies by pesticide compound. Different reporting levels in different methods result in fewer detections in one method. For example, 

LRLs are much lower in the LC-MS/MS method compared 

to the previous schedule 2060 for many pesticide compounds, 

so detection frequencies at low concentrations are larger with 

the LC-MS/MS method. Some pesticide compounds that have 

similar LRLs in reagent water have fewer detections in the 

LC-MS/MS method because matrix components can cause a 

decrease in one or both of the MRM signals or an increase in 

background noise. 

Stability of the analytes added to laboratory reagent water 

and stored at different temperatures and in potential preservatives was measured in different studies. The stability studies 

demonstrated that the largest number of the pesticide compounds were stable in reagent water after 14 days of storage 

at 4 °C, so that was selected as the practical holding time for 

routine sample processing. Upon arrival at the laboratory, samples are stored in a refrigerator and analyzed within 14 days of 

sample collection.

Recoveries of pesticides after 28 days of storage 

at 4 °C were relatively consistent. Only nine pesticides 

(etoxazole, indoxacarb, lactofen, disulfoton, phorate, terbufos, cis-permethrin, trans-permethrin, and flubendiamide) 

exceeded the practical limit of above 70 percent recovery 

after 28 days of storage, and all were within the practical limit after 14 days of storage. Similarly, 10 degradates 

exceeded the practical limit of 70 percent (phorate oxon, 

terbufos oxon, 4-hydroxy-tert-fluometuron, hydroxy mono 

demethyl fluometuron, hydroxyfluometuron, tebuthiuron 

TP 106, 2-hydroxy-4-isopropylamino-6-amino-s-triazine, 

2-hydroxy-6-ethylamino-4-amino-s-triazine, didealkylatrazine, 

and hexazinone TP C), and all were within the practical limit 

after 14 days of storage except for phorate oxon and terbufos 

oxon. Few degradates were found in the samples spiked with 

pesticides only and stored at 4 °C, indicating stability of most 

of the 44 pesticides that have one or more degradate measured 

in the ESI+ mode.

Changes in mean recovery of pesticides after 28 days of 

storage at –10 °C were relatively low and are similar to the 

results of storage at 4 °C. A few analytes (phorate, terbufos, 

phorate oxon, terbufos oxon, flumiclorac-pentyl, bifenazate) 
48 Determination of Pesticides and Pesticide Degradates in Filtered Water

had higher recoveries after storage at –10 °C compared to 4 °C. 

Storing the samples at –10 °C for 28 to 133 days resulted in 

recoveries of less than 70 percent for 10 of the 229 pesticide 

compounds and formation of four organophosphate degradates.

The addition of the dechlorination reagent ascorbic acid 

resulted in lower recoveries for some analytes, caused by both 

matrix suppression and degradation of the compound during storage. The ascorbic acid in general resulted in higher 

variability and slightly higher recoveries compared to reagent 

water with no ascorbic acid.

The addition of the antimicrobial reagent citric acid to 

adjust the sample pH to 3.8 used in EPA method 527 resulted 

in small changes in recovery for a number of pesticides analyzed in the ESI+ mode. A few pesticides had large decreases 

in concentration over the storage time that was due to degradation caused by the acid. The addition of the antimicrobial 

reagent citric acid to adjust the sample pH to 3.8 used in EPA 

method 527 resulted in large changes in recovery for a number 

of pesticide degradates analyzed in the ESI− mode, so it 

should not be used as a routine antimicrobial preservative.

References Cited

Alder, L., Greulich, K., Kempe, G., and Vieth, B., 2006, 

Residue analysis of 500 high priority pesticides—Better 

by GC-MS or LC-MS/MS?: Mass Spectrometry Reviews, 

v. 25, no. 6, p. 838–865.

American Society for Testing and Materials, 1993, Standard 

practice for estimation of holding time for water samples 

containing organic and inorganic constituents, D4841-88, 

Annual Book of ASTM Standards, Section 11, Water and 

Environmental Technology, book 11.01 Water (II), sec. B: 

Philadelphia, Pa., American Society for Testing and Materials, p. 8–12.

Anisuzzaman, A.K.M., Storehalder, T., Williams, D.C., Ogg, 

N., Kilbourne, T.D., JohnSamuel, J., and Cottrell, C.E., 

2008, Effect of alcohols on the stability of iprodione in 

solution: Journal of Agricultural and Food Chemistry, v. 56, 

no. 2, p. 502–506.

Backe, W.J., and Field, J.A., 2012, Is SPE necessary for environmental analysis? A quantitative comparison of matrix 

effects from large-volume injection and solid-phase extraction based methods: Environmental Science and Technology, v. 46, no. 12, p. 6750–6758.

Bester, K., 2008, Quantification with HPLC-MS/MS for environmental issues—Quality assurance and quality assessment: Analytical and Bioanalytical Chemistry, v. 391, no. 1, 

p. 15–20.

Childress, C.J., Foreman, W.T., Connor, B.F., and Maloney, 

T.J., 1999, New reporting procedures based on long-term

 method detection levels and some considerations for 

interpretations of water-quality data provided the U.S. 

Geological Survey National Water Quality Laboratory: U.S. 

Geological Survey Open-File Report 99–133, 19 p.

Díaz, L., Llorca-Pórcel, J., and Valor, I., 2008, Ultra trace 

determination of 31 pesticides in water samples by direct 

injection-rapid resolution liquid chromatography-electrospray tandem mass spectrometry: Analytica Chimica Acta, 

v. 624, no. 1, p. 90–96.

Eichelberger, J.W., Munch, J.W., and Shoemaker, J.A., 1995, 

Method 525.2. Determination of organic compounds in 

drinking water by liquid-solid extraction and capillary column gas chromatography/mass spectrometry—Methods for 

the determination of organic compounds in drinking water, 

suppl. III: U.S. Environmental Protection Agency, Office of 

Research and Development, National Exposure Research 

Laboratory EPA/600/R-95/131, 60 p.

European Commission, 2012, Method validation and quality 

control procedures for pesticide residues analysis in food 

and feed: European Commission Directorate General for 

Health and Consumers, SANCO/12495/2011, p. 41.

Fillâtre, Y., Rondeau, D., Jadas-Hécart, A., and Communal, 

P.Y., 2010, Advantages of the scheduled selected reaction 

monitoring algorithm in liquid chromatography/electrospray 

ionization tandem mass spectrometry multi-residue analysis 

of 242 pesticides—A comparative approach with classical 

selected reaction monitoring mode: Rapid Communications 

in Mass Spectrometry, v. 24, no. 16, p. 2453–2461.

Fuhrman, J.D., and Allan, J.M., 2003, Determination of acetanilide degradates in ground and surface waters by direct 

aqueous injection LC/MS/MS, in Ferrer, I., and Thurman, 

E.M., eds., Liquid Chromatography/Mass Spectrometry, 

Ms/Ms and Time-of-Flight Ms, book 850, sec. B: Washington, D.C., American Chemical Society ACS Symposium 

Series 850, p. 256–272.

Furey, A., Moriarty, M., Bane, V., Kinsella, B., and Lehane, 

M., 2013, Ion suppression—A critical review on causes, 

evaluation, prevention and applications: Talanta, v. 115, 

p. 104–122.

Furlong, E.T., Anderson, B.D., Werner, S.L., Soliven, P.P., 

Coffey, L.J., and Burkhardt, M.R., 2001, Methods of 

analysis by the U.S. Geological Survey National Water 

Quality Laboratory—Determination of pesticides in water 

by graphitized carbon-based solid-phase extraction and 

high-performance liquid chromatography/mass spectrometry: U.S. Geological Survey Water-Resources Investigations 

Report 01-4134, Method O-2060-01, 73 p.

Garbarino, J.R., Kanagy, L.K., and Cree, M.E., 2006, Determination of elements in natural-water, biota, sediment, and 

soil samples using collison/reaction cell inductively coupled 

plasma—mass spectrometry: U.S. Geological Survey Techniques and Methods, book 5, sec. B, chap. 1, p. 88.
References Cited 49

Greulich, K., and Alder, L., 2008, Fast multiresidue screening of 300 pesticides in water for human consumption 

by LC-MS/MS: Analytical and Bioanalytical Chemistry, 

v. 391, no. 1, p. 183–197.

Hahn, G.J., and Meeker, W.Q., 1991, Statistical intervals—A 

guide for practitioners: New York, Wiley, 392 p.

Helsel, D.R., and Hirsch, R.M., 1992, Statistical methods in 

water resources: New York, Elsevier, 522 p.

Helsel, D.R., and Hirsch, R.M., 2002, Statistical methods in 

water resources: U.S. Geological Survey Techniques of 

Water-Resources Investigations, book 4, chap. A3, available 

at http://water.usgs.gov/pubs/twri/twri4a3/.

Howard, P.H., ed., 1991, Handbook of environmental fate and 

exposure data for organic chemicals—Pesticides, v. III: 

Chelsea, Mich., Lewis Publishers, 684 p.

Huang, S.B., Mayer, T.J., and Yokley, R.A., 2008, Direct 

aqueous injection LC-ESI/MS/MS analysis of water for 11 

chloro- and thiomethyltriazines and metolachlor and its ethanesulfonic and oxanilic acid degradates: Journal of Agricultural and Food Chemistry, v. 56, no. 8, p. 2595–2602.

Le Grand, R., Dulaurent, S., Gaulier, J.M., Saint-Marcoux, F., 

Moesch, C., and Lachâtre, G., 2012, Simultaneous determination of five synthetic pyrethroid metabolites in urine by 

liquid chromatography-tandem mass spectrometry—Application to 39 persons without known exposure to pyrethroids: Toxicology Letters, v. 210, no. 2, p. 248–253.

Lehotay, S.J., De Kok, A., Hiemstra, M., and Van Bodegraven, 

P., 2005, Validation of a fast and easy method for the 

determination of residues from 229 pesticides in fruits and 

vegetables using gas and liquid chromatography and mass 

spectrometric detection: Journal of AOAC International, 

v. 88, no. 2, p. 595–614.

Lehotay, S.J., Mastovska, K., Amirav, A., Fialkov, A.B., 

Martos, P.A., Kok, A., and Fernández-Alba, A.R., 2008, 

Identification and confirmation of chemical residues in food 

by chromatography-mass spectrometry and other techniques: TrAC Trends in Analytical Chemistry, v. 27, no. 11, 

p. 1070–1090.

Lovell, D.P., 2013, Biological importance and statistical 

significance: Journal of Agricultural and Food Chemistry, 

v. 61, no. 35, p. 8340–8348.

Malcolm Pirnie Inc., 1999, Evaluation of the fate and transport of 

methanol in the environment: Malcolm Pirnie, Inc., accessed 

August 4, 2014, at http://www.methanol.org/Environment/

Resources/Environment/MP-Methanol-Fate.aspx.

Maloney, T.J., ed., 2005, Quality management system, U.S. 

Geological Survey National Water Quality Laboratory: U.S. 

Geological Survey Open-File Report 2005–1263, version 

1.3, chapters and appendixes variously paged.

Martin, J.D., and Eberle, M., 2011, Adjustment of pesticide 

concentrations for temporal changes in analytical recovery, 

1992–2010: U.S. Geological Survey Data Series 630, 11 p.

Miller, J.C., and Miller, J.N., 1993, Statistics for analytical 

chemistry (3d ed.): New York, Ellis Horwood Limited, 233 p.

Murray, K.K., Boyd, R.K., Eberlin, M.N., John Langley, G., 

Li, L., and Naito, Y., 2013, Definitions of terms relating to 

mass spectrometry (IUPAC Recommendations 2013): Pure 

and Applied Chemistry, v. 85, no. 7, p. 1515–1609.

Norman, J.E., Kuivila, K.M., and Nowell, L.H., 2012, Prioritizing pesticide compounds for analytical methods development: U.S. Geological Survey Scientific Investigations 

Report 2012–5045, 74 p.

O’Boyle, N.M., 2012, Towards a universal SMILES representation—A standard method to generate canonical SMILES based 

on the InChI: Journal of Cheminformatics, v. 4, no. 22, p. 14.

Odetokun, M.S., Montesano, M.A., Weerasekera, G., Whitehead, R.D., Jr, Needham, L.L., and Barr, D.B., 2010, 

Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate 

sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry: 

Journal of Chromatography B, v. 878, no. 27, p. 2567–2574.

Pepich, B.V., Prakash, B., Domino, M.M., Dattilio, T.A., 

Munch, D.J., and Price, E.K., 2005, Development of U.S. 

EPA method 527 for the analysis of selected pesticides and 

flame retardants in the UCMR survey: Environmental Science and Technology, v. 39, no. 13, p. 4996–5004.

Pozo, O.J., Sancho, J.V., Ibáñez, M., Hernández, F., and Niessen, W.M.A., 2006, Confirmation of organic micropollutants 

detected in environmental samples by liquid chromatography 

tandem mass spectrometry—Achievements and pitfalls: TrAC 

Trends in Analytical Chemistry, v. 25, no. 10, p. 1030–1042.

Radtke, D.B., Horowitz, A.J., and Sandstrom, M.W., 2004 

with updates through 2009, Filtered samples: U.S. Geological Survey Techniques of Water-Resources Investigations, 

book 5, chap. A5, section 5.2, accessed October 14, 2009, 

at http://water.usgs.gov/owq/FieldManual/chapter5/html/

Ch5_contents.html.

Rowe, G.L., Gilliom, R.J., and Woodside, M.D., 2013, Tracking and forecasting the Nation’s water quality—Priorities 

and strategies for 2013–2023: U.S. Geological Survey Fact 

Sheet 2013–3008, 6 p.

Sandstrom, M.W., Stroppel, M.E., Foreman, W.T., and Schroeder, M.P., 2001, Methods of analysis by the U.S. Geological 

Survey National Water Quality Laboratory—Determination 

of moderate-use pesticides in water by C-18 solid-phase 

extraction and capillary-column gas chromatography/mass 

spectrometry: U.S. Geological Survey Water-Resources 

Investigations Report 01-4098, Method O-2002-01, 70 p.
50 Determination of Pesticides and Pesticide Degradates in Filtered Water

Sandstrom, M.W., and Lewis, J.A., 2009, Instructions for field 

use of spike solutions for organic-analyte samples: U.S. 

Geological Survey Techniques of Water-Resources Investigations, book 9, chap. A5, sec. 3.2, accessed October 14, 

2009, at http://pubs.water.usgs.gov/twri9A5/.

SAS Institute Inc., 2010, Using JMP 9: Cary, N.C., SAS Institute Inc., 540 p.

Schlusener, M.P., and Bester, K., 2005, Determination of 

steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants 

utilising high-performance liquid chromatography/tandem 

mass spectrometry with electrospray and atmospheric pressure chemical ionisation: Rapid Communications in Mass 

Spectrometry, v. 19, no. 22, p. 3269–3278.

Shoemaker, J.A., and Bassett, M.V., 2005, Method 535. Measurement of chloroacetanilide and other acetamide herbicide 

degradates in drinking water by solid phase extraction and 

liquid chromatography/tandem mass spectrometry (LC/

MS/MS), ver. 1.1: Cincinnati, Ohio, U.S. Environmental 

Protection Agency, Office of Research and Development, 

National Exposure Research Laboratory, EPA Document 

600/R-05/053, 61 p.

Shoemaker, J.A., 2009, Method 538. Determination of 

selected organic contaminants in drinking water by direct 

aqueous injection- liquid chromatography/tandem mass 

spectrometry (DAI-LC/MS/MS), ver. 1: Cincinnati, Ohio, 

U.S. Environmental Protection Agency, Office of Research 

and Development, National Exposure Research Laboratory, 

EPA Document 600/R-09/149, 40 p.

Shoemaker, J.A., 2011, Development and multi-laboratory 

verification of U.S. EPA method 538 for the analysis of 

drinking water contaminants by direct aqueous injectionLC/MS/MS: Analytical Methods, v. 3, no. 7, p. 1628–1636.

Sinclair, C.J., Boxall, A.B., Parsons, S.A., and Thomas, M.R., 

2006, Prioritization of pesticide environmental transformation products in drinking water supplies: Environmental 

Science and Technology, v. 40, no. 23, p. 7283–7289.

Smith, G.A., Pepich, B.V., and Munch, D.J., 2011, Method 

536. Determination of triazine pesticides and their degradates in drinking water by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/

MS): Cincinnati, Ohio, U.S. Environmental Protection 

Agency, Office of Research and Development, National 

Exposure Research Laboratory, EPA 815-B-10-001, 36 p.

The Commission of the European Communities, 2002, 

2002/657/EC—Commission Decision of 12 August 2002 

implementing Council Directive 96/23/EC concerning the 

performance of analytical methods and the interpretation of 

results: Brussels, Official Journal of the European Communities, p. 36.

U.S. Geological Survey, 2006, Collection of water samples 

(ver. 2.0): U.S. Geological Survey Techniques of WaterResources Investigations, book 9, chap. A4, accessed 

August 4, 2014, at http://pubs.water.usgs.gov/twri9A4/.

U.S. Geological Survey, variously dated, National field manual 

for the collection of water-quality data: U.S. Geological Survey Techniques of Water-Resources Investigations, book 9, 

chaps. A1–A10, available at http://pubs.water.usgs.gov/

twri9A.

U.S. Geological Survey Office of Water Quality, 2004, 

Revised policy for the approval of U.S. Geological Survey 

Water-Quality Analytical Methods: Office Of Water Quality 

Technical Memorandum 04.01, accessed March 14, 2014, at 

http://water.usgs.gov/admin/memo/QW/qw04.01.html.

Weininger, D., 1988, SMILES, a chemical language and information system .1. Introduction to methodology and encoding rules: Journal of Chemical Information and Computer 

Sciences, v. 28, no. 1, p. 31–36.

Wilde, F.D., Sandstrom, M.W., and Skrobialski, S.C., 2014, 

Section 5.2.2.B. Syringe-filter procedure for processing samples for analysis of organic compounds by DAI 

LC-MS/MS: U.S. Geological Survey Techniques of WaterResources Investigations, book 9, chap. A5, accessed July 1, 

2014, at http://water.usgs.gov/owq/FieldManual/chapter5/

html/Ch5_contents.html.

Winslow, S.D., Prakash, B., Domino, M.M., Pepich, B.V., and 

Munch, D.J., 2001, Considerations necessary in gathering 

occurrence data for selected unstable compounds in the 

USEPA Unregulated Contaminant Candidate List in USEPA 

Method 526: Environmental Science and Technology, v. 35, 

no. 9, p. 1851–1858.

Zaugg, S.D., Sandstrom, M.W., Smith, S.G., and Fehlberg, 

K.M., 1995, Methods of analysis by the U.S. Geological 

Survey National Water Quality Laboratory—Determination of pesticides in water by C-18 solid-phase extraction 

and capillary-column gas chromatography/mass spectrometry with selected-ion monitoring: U.S. Geological Survey 

Open-File Report 95–181, Method O-1126-95, 49 p.

Zwiener, C., and Frimmel, F.H., 2004a, LC-MS analysis in the 

aquatic environment and in water treatment technology—A 

critical review. Part II. Applications for emerging contaminants and related pollutants, microorganisms and humic 

acids: Analytical and Bioanalytical Chemistry, v. 378, no. 4, 

p. 862–874.

Zwiener, C., and Frimmel, F.H., 2004b, LC-MS analysis in 

the aquatic environment and in water treatment—A critical 

review. Part I. Instrumentation and general aspects of analysis and detection: Analytical and Bioanalytical Chemistry, 

v. 378, no. 4, p. 851–861.
Glossary 51

Glossary

analytical reference standard Samples of 

pure active ingredients or technical grade 

active ingredients of pesticides, and regulated 

metabolites, degradates, and related compounds, obtained from the EPA National Pesticide Standard Repository or other supplier.

bias Systematic error inherent in the method 

or measurement system. The error can be 

positive (for example, from contamination or 

ionization enhancement) or negative (from 

analyte loss or ionization suppression).

calibration (CAL) standard A solution prepared from the primary dilution standard solution and internal standard solution. The CAL 

solutions are used to calibrate the instrument 

response relative to analyte concentration.

concentrated standard solution A concentrated solution containing the individual 

analyte prepared in the laboratory using reference materials or purchased from a commercial vendor. 

instrument blank (IBLNK) A sample of 

reagent water processed through the instrument analysis steps in the analytical process 

used to measure bias from the instrument 

analysis. Results for these samples analyzed 

by the DAI LC-MS/MS pesticide method are 

stored in the NWQL LIMS system as “instrument blank (IBLNK)” samples.

instrument detection level (IDL) standard A 

low-concentration calibration standard 

analyzed during the analytical run used to 

measure LC-MS/MS response during and at 

the end of the analytical batch. Results for 

these samples analyzed by the DAI LC-MS/

MS pesticide method are stored in the NWQL 

LIMS system as “instrument detection level 

(IDL)” samples.

internal standard (IS) solution A solution 

of isotope-labeled pesticides compounds 

used as internal standards for quantitation of 

method analytes. Internal standard recoveries 

are reported and used to monitor the method 

performance of a group of analytes in each 

sample, similar to surrogates.

internal standard (IS) An analyte added to 

the sample and used to measure the response 

of the pesticide compound relative to the 

internal standard—measured as chromatographic peak area for the quantitation MRM. 

Isotope-labeled pesticides or other chemicals 

similar to the pesticide compounds but not 

expected to be in the environmental sample 

are selected as internal standards.

laboratory reagent blank (PBLNK) A 

reagent-water sample processed through the 

sample preparation and analysis steps of the 

analytical process used to measure bias of the 

analytical method. Results for these samples 

analyzed by the DAI LC-MS/MS pesticide 

method are stored in the NWQL LIMS system 

as “preparation blank (PBLNK)” samples.

laboratory reagent spike (PSPK) A reagentwater sample spiked with the spiking solution 

and used to measure intra-laboratory bias and 

variability of the analytical method. The spiking solution is added in the laboratory prior 

to sample analysis. Results for these samples 

analyzed by the DAI LC-MS/MS pesticide 

method are stored in the NWQL LIMS system 

as “preparation spike (PSPK)” samples. The 

EPA uses the term “laboratory fortified blank” 

for this type of sample.

laboratory reporting level (LRL) The LRL 

is set at twice the MDL. The LRL is used to 

control false negative error—the risk of a 

false negative (not detecting an analyte when 

present) is less than 1 percent at the LRL 

(Childress and others, 1999).

matrix spike (MSPK) An environmental 

sample (stream or groundwater) spiked with 

the spiking solution and used to measure bias 

and variability due to sample matrix interferences. The spiking solution can be added at 

the collection site (field-matrix spike) or at the 

laboratory (laboratory matrix spike) and, if 

spiked at the collection site, is used to measure 

degradation during shipping and storage as 

well as matrix interferences. Results for these 

samples analyzed by the DAI LC-MS/MS pesticide method are stored in the NWQL LIMS 

system as “matrix-spike (MSPK)” samples.

method detection level (MDL) The MDL is 

the minimum concentration of an analyte that 

can be identified, measured, and reported with 

99-percent confidence that the analyte concentration is greater than zero. At the MDL, the 
52 Determination of Pesticides and Pesticide Degradates in Filtered Water

risk of a false positive (reporting a detection 

when no analyte is present) is predicted to be 

less than 1 percent. The MDL is determined 

from replicate analysis of low-concentration 

spike samples and, if done over an extended 

time period to obtain a more realistic measurement of the standard deviation, was 

termed the long-term MDL (Childress and 

others, 1999). The EPA uses the term “method 

detection limit.”

mixed intermediate standard solution A 

solution containing a group of pesticide 

compounds based on pesticide use (herbicide, insecticide, fungicide), chemical class 

(acetanilide, triazine, acid), and whether it is 

a pesticide or degradate. The mixed solution 

is prepared from the individual concentrated 

standard solutions.

nominal concentration The amount of a 

pesticide compound that is expected to be 

present in a sample at the time the sample is 

prepared by spiking. Nominal is used as a 

synonym for “expected” in some contexts.

primary dilution standard solution A solution containing all the pesticide compounds 

prepared from all the mixed intermediate standard solutions.

reagent water Deionized water prepared by 

distillation, ion exchange, and filtration. The 

deionized water was then pumped through 

charcoal prefilters and a dual wavelength 

ultraviolet oxidizer lamp for removal of trace 

organic compounds to produce high-purity 

organic-free reagent water.

spiking solution A solution for preparing 

reagent spikes and matrix spikes. The spiking 

solution is prepared by dilution of the primary 

dilution standard solution.

surrogate An analyte added to the sample 

and used to measure the performance of the 

method in every sample. Isotope-labeled 

pesticides or other chemicals similar to the 

pesticide compounds but not expected to be in 

the environmental sample are selected as surrogates. The surrogate is chemically similar 

to the analyte so that losses in the analytical 

procedure are expected to be the same for 

both compounds.

tandem mass spectrometry or mass spectrometry/mass spectrometry (MS/MS) The 

acquisition and study of the spectra of the 

product ions or precursor ions of m/z selected 

ions (Murray and others, 2013).

third-party check (TPC) standards An analytical standard prepared from an independent 

source—different from that used to prepare 

the calibration and spike standards. The 

third-party check standard is used to verify 

that the calibration standards are accurate 

and have not changed as a result of degradation or errors in preparation. Results for these 

samples analyzed by the DAI LC-MS/MS 

pesticide method are stored in the NWQL 

LIMS system as “third-party check (TPC)” 

samples.

triple quadrupole mass spectrometer A tandem mass spectrometer comprising two transmission quadrupole mass spectrometers in 

series with a (nonselecting) radio frequencyonly quadrupole (or other multipole) between 

them to act as a collision cell (Murray and 

others, 2013).

variability Random error in independent 

measurements as the result of repeated application of the process or analytical method 

under specific conditions.
Appendix 1. Supporting Figures and Tables 53

Appendix 1. Supporting Figures and Tables

Figure S7. Recovery of pesticides determined by direct 

aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in field-matrix-spike samples by 

site in 200 field-matrix-spike samples that were prepared for the 

National Water Quality Assessment (NAWQA) Program field study 

from May to September 2012.

Figure S8. Pesticide compound recovery as a function of 

background environmental sample concentration determined by 

direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in 146 field-matrix-spike samples 

that were prepared for the National Water Quality Assessment 

(NAWQA) Program field study from May to September 2012.

Figure S9. Recovery of pesticides in reagent water after 

storage for as many as 28 days with different treatments (none, 

4 °C [degrees Celsius]; none, –10 °C; citric acid pH 3.8, 4 °C; 

ascorbic acid, 4 °C) determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) in 

positive electrospray ionization (ESI+) mode.

Figure S10. Recovery of pesticide degradates in reagent water 

after storage for as many as 28 days with different treatments 

(none, 4 °C [degrees Celsius]; none, –10 °C; citric acid pH 3.8, 

4 °C; ascorbic acid, 4 °C) determined by direct aqueous-injection 

liquid chromatography-tandem mass spectrometry (DAI LC-MS/

MS) in positive electrospray ionization (ESI+) mode.

Figure S11. Pesticide degradates found in reagent water 

fortified with pesticides after storage for as many as 28 days 

with different treatments (none, 4 °C [degrees Celsius]; none, 

–10 °C; citric acid pH 3.8, 4 °C; ascorbic acid, 4 °C) determined 

by direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in positive electrospray ionization 

(ESI+) mode.

Figure S12. Recovery of pesticides in reagent water after 

storage for as many as 28 days with different treatments (none, 

4 °C [degrees Celsius]; none, –10 °C; citric acid pH 3.8, 4 °C; 

ascorbic acid, 4 °C) determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) in 

negative electrospray ionization (ESI−) mode.

Supporting Figures

Figure S1. The qualifier ion response ratio of pesticide 

compounds determined by liquid chromatography-tandem mass 

spectrometry (LC-MS/MS) in positive electrospray ionization 

(ESI+) mode fortified in different matrices (Santa Ana Prado Dam, 

Mississippi River, Well 1N/14W-8K1S) at 10, 100, 500, and 5,075 

nanograms per liter (ng/L) and in reagent water (calibration [CAL] 

and continuing calibration verification [CCV] standards) at 1, 2.5, 5, 

10, 25, 50, 100, 500, 5,000, and 10,000 ng/L.

Figure S2. The qualifier ion response ratio of pesticide 

compounds determined by liquid chromatography-tandem mass 

spectrometry (LC-MS/MS) in negative electrospray ionization 

(ESI−) mode fortified in different matrices (Santa Ana Prado Dam, 

Mississippi River, Well 1N/14W-8K1S) at 10, 100, 500, and 5,075 

nanograms per liter (ng/L) and in reagent water (calibration [CAL] 

and continuing calibration verification [CCV] standards) at 1, 2.5, 5, 

10, 25, 50, 100, 500, 5,000, and 10,000 ng/L.

Figure S3. Graphs of response factor (peak area/nominal 

concentration) and parameters of linear fit of response factor 

by nominal concentration for direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

method positive electrospray ionization (ESI+) mode analytes.

Figure S4. Graphs of response factor (peak area/nominal 

concentration) and parameters of linear fit of response factor 

by nominal concentration for direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

method negative electrospray ionization (ESI−) mode analytes.

Figure S5. Recovery of pesticide analytes determined by liquid 

chromatography-tandem mass spectrometry (LC-MS/MS) in 

positive electrospray ionization (ESI+) mode fortified at different 

concentrations (10, 100, 500, and 5,000 nanograms per liter [ng/L]) 

in four different matrices.

Figure S6. Recovery of pesticide analytes determined by liquid 

chromatography tandem mass spectrometry (LC-MS/MS) in 

negative electrospray ionization (ESI−) mode fortified at different 

concentrations (10, 100, 500, and 5,000 nanograms per liter [ng/L]) 

in four different matrices.

Supporting figure PDF files and supporting table Microsoft Excel files are available at http://dx.doi.org/10.3133/tm5B11.
54 Determination of Pesticides and Pesticide Degradates in Filtered Water

Figure S13. Recovery of pesticide degradates in reagent water 

after storage for as many as 28 days with different treatments 

(none, 4 °C [degrees Celsius]; none, –10 °C; citric acid pH 3.8, 4 °C; 

ascorbic acid, 4 °C) determined by direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) in 

negative electrospray ionization (ESI−) mode.

Figure S14. Pesticide degradates found in reagent water 

fortified with pesticides after storage for as many as 28 days 

with different treatments (none, 4 °C [degrees Celsius]; none, 

–10 °C; citric acid pH 3.8, 4 °C; ascorbic acid, 4 °C) determined 

by direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in negative electrospray ionization 

(ESI−) mode.

Supporting Tables

Table S1. Groups of pesticide compounds used to prepare 

intermediate standard solutions of pesticide compounds for 

direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) method.

Table S2. Preparation of primary dilution standard solutions in 

methanol used to make calibration standards for direct aqueousinjection liquid chromatography-tandem mass spectrometry (DAI 

LC-MS/MS) method.

Table S3. Examples of National Water Information System 

(NWIS) qualifier codes used by analyst with direct aqueousinjection liquid chromatography-tandem mass spectrometry (DAI 

LC-MS/MS) method (laboratory schedule 2437) to qualify samples 

or analytes not meeting quality-control criteria.

Table S4. Summary of quality-control criteria for determination 

of pesticides by direct aqueous-injection liquid chromatographytandem mass spectrometry (DAI LC-MS/MS) (laboratory schedule 

2437).

Table S5. Pesticide compounds identified as high priority for 

method development but not included in direct aqueous-injection 

liquid chromatography-tandem mass spectrometry (DAI LC-MS/

MS) method for various reasons.

Table S6. Summary of response factor (peak area/nominal 

concentration) and parameters of linear fit of response factor 

by nominal concentration for direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

method positive electrospray ionization (ESI+) mode analytes.

Table S7. Summary of response factor (peak area/nominal 

concentration) and parameters of linear fit of response factor 

by nominal concentration for direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

method negative electrospray ionization (ESI−) mode analytes.

Table S8. Recovery of pesticides determined by direct aqueousinjection liquid chromatography-tandem mass spectrometry 

(DAI LC-MS/MS) method (laboratory schedule 2437) in fieldmatrix-spike samples collected during National Water Quality 

Assessment (NAWQA) Program field comparison study from 

May to September 2012 excluded from data summaries because 

environmental concentration was greater than spike level.

Table S9. Summary of water quality parameters measured in 

streams when direct aqueous-injection liquid chromatographytandem mass spectrometry (DAI LC-MS/MS) method (schedule 

2437) samples were collected during National Water Quality 

Assessment (NAWQA) Program field comparison study from May 

to September 2012.

Table S10. Summary of ancillary water quality parameters 

for each stream site when direct aqueous-injection liquid 

chromatography-tandem mass spectrometry (DAI LC-MS/MS) 

method (schedule 2437) samples were collected during National 

Water Quality Assessment (NAWQA) Program field comparison 

study from May to September 2012.

Table S11. Degradates found in reagent water samples spiked 

with pesticides spiked at 250 nanograms per liter (ng/L) and stored 

at 4 °C (degrees Celsius) for as many as 28 days and analyzed by 

direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in positive electrospray ionization 

(ESI+) and negative electrospray ionization (ESI−) modes.

Table S12. Degradates found in reagent water samples spiked 

with pesticides spiked at 250 nanograms per liter (ng/L) and stored 

at –10 °C (degrees Celsius) for as many as 133 days and analyzed 

by direct aqueous-injection liquid chromatography-tandem mass 

spectrometry (DAI LC-MS/MS) in positive electrospray ionization 

(ESI+) and negative electrospray ionization (ESI−) modes.
Publishing support provided by: 

Denver Publishing Service Center

For more information concerning this publication, contact:

Chief, USGS National Water Quality Laboratory

Box 25585, Mail Stop 407

Denver, CO 80225-0585

(303) 236-2000

Or visit the National Water Quality Laboratory Web site at:

http://nwql.usgs.gov/
Sandstrom and others—Determination of Pesticides in Filtered Water by DAI LC-MS/MS—Techniques and Methods 5–B11

ISSN 2328-7055 (online)

http://dx.doi.org/10.3133/tm5B11