536 -1

EPA Document #: 815-B-07-002 

METHOD 536 DETERMINATION OF TRIAZINE PESTICIDES AND THEIR 

DEGRADATES IN DRINKING WATER BY LIQUID 

CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM 

MASS SPECTROMETRY (LC/ESI-MS/MS) 

Version 1.0 

October 2007 

G.A. Smith and B.V. Pepich (Shaw Environmental, Inc.) 

D. J. Munch (U.S. EPA, Office of Ground Water and Drinking Water) 

TECHNICAL SUPPORT CENTER 

OFFICE OF GROUND WATER AND DRINKING WATER 

U. S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OHIO 45268 
536 -2

METHOD 536 

DETERMINATION OF TRIAZINE PESTICIDES AND THEIR DEGRADATES IN DRINKING 

WATER BY LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM 

MASS SPECTROMETRY (LC/ESI-MS/MS)

1. SCOPE AND APPLICATION

1.1 This is a liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESIMS/MS) method for the determination of triazine pesticides and their degradation products in 

finished drinking waters. This method requires the use of MS/MS in Multiple Reaction 

Monitoring (MRM) mode to enhance selectivity. Precision and accuracy data have been 

generated for the detection of the triazine pesticides and their degradates in reagent water and 

finished drinking water from both groundwater and surface water sources. The single 

laboratory Lowest Concentration Minimum Reporting Level (LCMRL) has also been 

determined in reagent water.1

 The following compounds can be determined using this 

method: 

Analyte

Chemical Abstract Services 

Registry Number (CASRN)

Atrazine 1912-24-9 

Atrazine-desethyl 6190-65-4 

Atrazine-desethyl-desisopropyl 3397-62-4 

Atrazine-desisopropyl 1007-28-9 

Cyanazine 21725-46-2 

Propazine 139-40-2 

Simazine 122-34-9 

1.2 The MRM mass spectrometry conditions described in this method were developed using a 

conventional LC/MS/MS system. 

1.3 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets the Data 

Quality Objectives (DQOs), which are based on the intended use of this method. The single 

laboratory LCMRL is the lowest true concentration for which the future recovery is predicted 

to fall between 50-150% recovery with 99% confidence. Single laboratory LCMRLs for the 

analytes in this method ranged from 0.020 – 0.14 μg/L, and are listed in Table 4. The 

procedure used to determine the LCMRL is described elsewhere.1

1.4

1.5

Laboratories using this method are not required to determine an LCMRL, but they will need 

to demonstrate that their laboratory Minimum Reporting Level (MRL) for this method meets 

the requirements described in Section 9.2.4. 

Detection limit (DL) is defined as the statistically calculated minimum concentration that can 

be measured with 99% confidence that the reported value is greater than zero.2 The DL is 

dependent on sample matrix, fortification concentration, and instrument performance. 
536 -3

Determining the DL for analytes in this method is optional (Sect. 9.2.6). DLs for method 

analytes fortified into reagent water ranged from 0.010 – 0.043 μg/L. These values are also 

provided in Table 4. 

1.6 This method is intended for use by analysts skilled in the operation of LC/MS/MS 

 instrumentation and the interpretation of the associated data. 

1.7 METHOD FLEXIBILITY – In recognition of technological advances in analytical systems 

and techniques, the laboratory is permitted to modify the separation technique, LC column, 

mobile phase composition, LC conditions and MS conditions (Sect. 6.10, 9.4, 10.2.2, 10.2.3, 

10.2.4, and 12.1). Changes may not be made to sample collection and preservation (Sect. 

8) or to the quality control requirements (Sect. 9). Method modifications should be 

considered only to improve method performance. Modifications that are introduced in the 

interest of reducing cost or sample processing time, but result in poorer method performance, 

may not be used. The analytes must be resolved chromatographically in order to avoid 

the effects of matrix suppression or enhancement and to provide analyte specificity. In 

all cases where method modifications are proposed, the analyst must perform the procedures 

outlined in the Initial Demonstration of Capability (IDC, Sect. 9.2), verify that all QC 

acceptance criteria in this method (Tables 9 and 10) are met, and verify method performance 

on a real sample matrix (Sect. 9.4). 

2. SUMMARY OF METHOD

2.1 Samples are pH adjusted and dechlorinated with ammonium acetate and protected from 

microbial degradation using sodium omadine during sample collection. Prior to analysis, 

isotopically enriched analytes [Atrazine-d5(ethyl-d5), Atrazine-desethyl-d7, Atrazinedesethyl-desisopropyl(13C3), Atrazine-desisopropyl-d5(ethyl-d5), Cyanazine-d5(N-ethyl-d5), 

Propazine-d14(di-isopropyl-d14), and Simazine-d10(diethyl-d10)] are added to the samples 

as internal standards. An aliquot of the sample is injected without cleanup or concentration 

onto a chromatographic column (C18). The analytes are separated using an ammonium 

acetate-buffered binary mobile phase and subsequently detected by positive electrospray 

ionization tandem mass spectrometry. The concentration of each identified component is 

determined using the internal standard technique.

3. DEFINITIONS

3.1 ANALYSIS BATCH – A sequence of samples, which are analyzed within a 30-hour period 

and include no more than 20 field samples. Each Analysis Batch must also include all 

required QC samples, which do not contribute to the maximum field sample total of 20. The 

required QC samples include: 

Laboratory Reagent Blank (LRB), 

 Continuing Calibration Check Standards (CCCs), 

Laboratory Fortified Sample Matrix (LFSM), and 

Laboratory Fortified Sample Matrix Duplicate or Laboratory Duplicate (LFSMD or LD). 

3.2 CALIBRATION STANDARD (CAL) – A solution of the target analytes prepared from the 

primary dilution standard solution(s) or stock standard solution(s) and the internal standards. 

The CAL solutions are used to calibrate the instrument response with respect to analyte 

concentration. 
536 -4

3.3 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing the 

method analytes and internal standards which is analyzed periodically to verify the accuracy 

of the existing calibration. 

3.4 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be 

identified, measured and reported with 99% confidence that the analyte concentration is 

greater than zero. This is a statistical determination (Sect. 9.2.6), and accurate quantitation is 

not expected at this level.2

3.5 INTERNAL STANDARD (IS) – A pure compound added to all standard solutions and field 

samples in a known amount. It is used to measure the relative response of the method 

analyte. The internal standard must be a compound that is not a sample component. 

3.6 ION SUPRESSION/ENHANCEMENT – An observable loss or increase in target analyte 

response in complex (field) samples as compared to the response obtained in standard 

solutions. 

3.7 LABORATORY DUPLICATES (LDs) – Two sample aliquots (LD1 and LD2), taken in the 

laboratory from a single sample bottle, and analyzed separately with identical procedures. 

Analyses of LD1 and LD2 indicate precision associated specifically with the laboratory 

procedures by removing variation contributed from sample collection, preservation and 

storage procedures. 

3.8 LABORATORY FORTIFIED BLANK (LFB) – An aliquot of reagent water or other blank 

matrix to which a known quantity of the method analytes is added. The LFB is analyzed 

exactly like a sample including the preservation procedures in Section 8. Its purpose is to 

determine whether the methodology is in control, and whether the laboratory is capable of 

making accurate measurements. 

3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of a field sample to 

which a known quantity of the method analytes is added. The LFSM is processed and 

analyzed exactly like a sample, and its purpose is to determine whether the sample matrix 

contributes bias to the analytical results. The background concentration of the analytes in the 

sample matrix must be determined in a separate aliquot and the measured value in the LFSM 

corrected for background concentrations. 

3.10 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A second 

aliquot of the field sample used to prepare the LFSM which is fortified and analyzed 

identically to the LFSM. The LFSMD is used instead of the Laboratory Duplicate to assess 

method precision and accuracy when the occurrence of the target analytes is infrequent. 

3.11 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or other blank 

matrix that is treated exactly as a sample including exposure to all storage containers, buffers, 

preservatives and internal standards. The LRB is used to determine if the method analytes or 

other interferences are present in the laboratory environment, the reagents, or the apparatus. 
536 -5

3.12 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The single 

laboratory LCMRL is the lowest true concentration for which the future recovery is predicted 

to fall between 50-150% recovery with 99% confidence.1

3.13 MATERIAL SAFETY DATA SHEETS (MSDS) – These sheets contain written information 

provided by vendors concerning a chemical’s toxicity, health hazards, physical properties, 

fire, and reactivity data including storage, spill, and handling precautions. 

3.14 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be 

reported by a laboratory as a quantified value for the target analyte in a sample following 

analysis. This concentration must meet the criteria defined in Section 9.2.4 and must be no 

lower than the concentration of the lowest calibration standard for the target analyte. 

3.15 MULTIPLE REACTION MONITORING (MRM) – A mass spectrometric technique in 

which a precursor ion is first isolated then subsequently fragmented into product ion(s). 

Quantitation is accomplished by monitoring a specific product ion. As described in Section 

10.2.2, MS parameters must be optimized for each precursor ion (Sect. 3.16) and product ion 

(Sect. 3.18). 

3.16 PRECURSOR ION – For the purpose of this method, the precursor ion is the protonated 

molecule ([M+H]+

) of the target analyte ([M]). In MS/MS, the precursor ion is mass selected 

and fragmented by collisionally activated dissociation to produce distinctive product ions of 

smaller m/z. 

3.17 PRIMARY DILUTION STANDARD SOLUTION (PDS) – A solution containing the 

method analytes prepared in the laboratory from stock standard solutions and diluted as 

needed to prepare calibration standards and other analyte solutions. 

3.18 PRODUCT ION – For the purpose of this method, a product ion is one of the fragment ions 

produced in MS/MS by collisionally activated dissociation of the precursor ion. 

3.19 QUALITY CONTROL SAMPLE (QCS) – A solution containing the method analytes at a 

known concentration which is obtained from a source external to the laboratory and different 

from the source of calibration standards. It is used to check laboratory performance with 

externally prepared test materials. 

3.20 REAGENT WATER (RW) – Purified water which does not contain any measurable quantity 

of the target analytes or interfering compounds at or above 1/3 the MRL for the target 

analytes. 

3.21 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing one or more 

of the method analytes that is prepared in the laboratory using assayed reference materials or 

purchased from a reputable commercial source, so that the concentration and purity of 

analytes are traceable to certificates of analysis. 

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware thoroughly and rinse with 

reagent water. 
536 -6

4.2 Method interferences may be caused by contaminants in solvents, reagents (including reagent 

water), sample bottles and caps, and other sample processing hardware. These interferences 

may lead to discrete artifacts and/or elevated baselines in the chromatograms. All laboratory 

reagents and equipment must be routinely demonstrated to be free from interferences (less 

than 1/3 the MRL for the target analytes) under the conditions of the analysis. This may be 

accomplished by analyzing LRBs as described in Section 3.11. Subtracting blank values 

from sample results is not permitted.

4.3 Equipment used for sample collection and storage has the potential to introduce 

interferences. The potential for interferences from these devices must be investigated during 

the Initial Demonstration of Capability (Sect. 9.2) by preparing and analyzing a Laboratory 

Reagent Blank (LRB). This procedure should be repeated each time a new brand or lot of 

equipment is used to ensure that background contamination does not interfere with the 

identification and quantitation of the analytes. 

4.4 Matrix interferences may be caused by contaminants that are present in the sample. The 

extent of matrix interferences will vary considerably from source to source depending upon 

the nature of the water. 

4.4.1 ION SUPPRESSION/ENHANCEMENT – Field samples containing moderate-to-high 

levels of organic carbon or dissolved solids may cause suppression or enhancement of 

instrumental response for one or more of the analytes. Suppression and enhancement 

effects are introduced during the ionization process in the electrospray source when a coeluting contaminant influences the ionization of the analyte of interest. MS/MS methods 

are not immune to suppression or enhancement effects because the fragmentation process 

occurs after ionization. Isotopically enriched internal standards can help mitigate the 

effect of matrix-induced suppression or enhancement, but good performance is best 

achieved by optimizing the chromatographic separation. 

4.4.2 INTERFERING COMMON IONS – While MS/MS detection offers increased selectivity 

relative to MS mode detection, interference can still occur in complex samples if 

common ions are present in co-eluting components. Due to a lack of LC/MS/MS 

reference spectral libraries, optimized chromatographic separation is required for mass 

resolution of the analytes of interest in the method. 

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely 

defined. Each chemical should be treated as a potential health hazard, and exposure to these 

chemicals should be minimized. Each laboratory is responsible for maintaining an awareness 

of OSHA regulations regarding safe handling of chemicals used in this method. A reference 

file of MSDSs should be made available to all personnel involved in the chemical analysis. 

5.2 Pure standard materials and stock standard solutions of the method compounds should be 

handled with suitable protection for skin, eyes, etc. 3-5
536 -7

6. EQUIPMENT AND SUPPLIES (References to specific brands or catalog numbers are included 

as examples only and do not imply endorsement of the product. This does not preclude the use of 

other vendors or suppliers.) 

6.1 SAMPLE CONTAINERS – Pre-cleaned, amber vials (40 mL or larger) fitted with 

PTFE/silicone septa and polypropylene screw caps (Fisher Cat. No. 02-912-377 or 

equivalent). 

6.2 VIALS – Amber 2-mL glass autosampler vials with PTFE/silicone septa for storing standards 

(Fisher Cat. No. 03-375-20B or equivalent). 

6.3 MICRO SYRINGES – Suggested sizes include 10, 25, 50, 100, 250, and 500 μL. 

6.4 VOLUMETRIC FLASKS – Class A, suggested sizes include 5, 10, 50, 100, 250, and 500 

mL for preparation of reagents and standards. 

6.5 VOLUMETRIC PIPETTES – Class A, suggested sizes include 1, 4, 5, 10, 20, and 25 mL. 

6.6 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g. 

6.7 DESOLVATION GAS – High purity compressed gas (e.g., nitrogen or zero-air) used for 

desolvation in the mass spectrometer. The specific type of gas, purity and pressure 

requirements will depend on the instrument manufacturers’ specifications. 

6.8 COLLISION GAS – High purity compressed gas (e.g., nitrogen or argon) used in the 

collision cell of the mass spectrometer. The specific type of gas, purity and pressure 

requirements will depend on the instrument manufacturer’s specifications. 

6.9 DISPOSABLE PASTEUR PIPETS – 9” borosilicate glass, used to transfer samples to 

autosampler vials (Fisher Cat. No. 13-678-20C or equivalent). 

6.10 LIQUID CHROMATOGRAPHY ELECTROSPRAY TANDEM MASS SPECTROMETRY 

SYSTEM (LC/ESI-MS/MS) 

6.10.1 LC COLUMN – LC column, Waters Xterra® RP18, 2.1 x 150 mm, 3.5 μm dp (Waters 

Part No. 186000410 or equivalent). Any column that provides adequate resolution, peak 

shape, capacity (Sect. 10.2.3 and 10.2.4), accuracy and precision (Sect. 9), and does not 

result in suppression or enhancement of analyte response (Sect. 13.4.2) may be used. 

6.10.2 LC SYSTEM –The LC system (Waters 2695 or equivalent) must provide consistent 

sample injection volumes and be capable of performing binary linear gradients at a 

constant flow rate. 

6.10.3 MASS SPECTROMETER –The MS (Micromass QuattroMicro or equivalent) must be 

capable of providing electrospray ionization with positive ion detection. The system 

must be capable of performing MS/MS to produce unique product ions (Sect. 3.18) for 

the target analytes within specified retention time windows. Data presented in Section 

17, Tables 4 - 8 were collected using an ESI-MS/MS system. 
536 -8

6.10.4 DATA SYSTEM – An interfaced data system is required to acquire, store, and output MS 

data. The computer software should have the capability of processing stored LC/MS/MS 

data by recognizing a chromatographic peak within a given retention time window. The 

software must allow integration of the ion abundance of any specific ion between 

specified times or scan number limits. The software must be able to construct a linear 

regression or quadratic calibration curve, and calculate analyte concentrations using the 

internal standard technique. 

7. REAGENTS AND STANDARDS

7.1 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used. Unless 

otherwise indicated, it is intended that all reagents will conform to the specifications of the 

Committee on Analytical Reagents of the American Chemical Society (ACS), where such 

specifications are available. Other grades may be used, as long as the reagent is of 

sufficiently high purity to permit its use without lessening the quality of the determination. 

7.1.1 METHANOL – (CH3OH, CAS# 67-56-1) – High purity, demonstrated to be free of 

analytes and interferences (Fisher GC Resolv grade or equivalent). 

7.1.2 AMMONIUM ACETATE – (NH4C2H3O2, CAS# 631-61-8) – High purity, demonstrated 

to be free of analytes and interferences (Fisher Brand HPLC grade or equivalent). 

7.1.3 5 mM AMMONIUM ACETATE MOBILE PHASE – To prepare 1 L of mobile phase, 

add 0.3854 g ammonium acetate to 1 L of reagent water (final pH ~ 6.5). This solution is 

prone to volatility losses and should be replaced every 48 hours. 

7.1.4 2.0 M AMMONIUM ACETATE/REAGENT WATER CONCENTRATED STOCK – 

Add 38.5 g of ammonium acetate to a 250-mL volumetric flask. Dilute to volume with 

reagent water. An aliquot of this concentrated stock solution is diluted 1:100 in all 

calibration standards and samples to yield a final concentration of 20 mM (1.5 g/L). 

7.1.5 SODIUM OMADINE® (Sodium 2-pyridinethiol-1-oxide, CAS# 3811-73-2) – 

(C5H4NOSNa) – 40 wt.% in water (Omadine is a registered trademark of Arch 

Chemicals, Inc.). 

7.1.6 SODIUM OMADINE CONCENTRATED STOCK SOLUTION (32 g/L) – Add 20 mL 

of the 40 wt.% sodium omadine in water to a 250-mL volumetric flask. Dilute to volume 

with reagent water. The concentrated stock solution is diluted 1:500 in all calibration 

standards and samples to yield a final concentration of 64 mg/L. 

7.2 STANDARD SOLUTIONS – When a compound’s purity is assayed to be 96 percent or 

greater, the weight can be used without correction to calculate the concentration of the stock 

standard. Solution concentrations listed in this section were used to develop this method 

and are included only as an example. Stock standard solutions are estimated to be stable 

for six months. Any fortified or dilute solutions made from the stock standards are stable for 

at least 60 days provided they are stored at a temperature < 6 o

C and the stock standard 

solutions have not exceeded their six month stability period. Although estimated stability 

times for standard solutions are given, laboratories should use standard QC practices to 

determine when their standards need to be replaced.
536 -9

7.2.1 INTERNAL STANDARD SOLUTIONS – This method uses a separate isotopically 

enriched internal standard for each target analyte, specifically Atrazine-d5(ethyl-d5) 

(CAS# 163165-75-1), Atrazine-desethyl-d7, Atrazine-desethyl-desisopropyl(13C3), 

Atrazine-desisopropyl-d5(ethyl-d5), Cyanazine-d5(N-ethyl-d5), Propazine-d14(diisopropyl-d14), and Simazine-d10(diethyl-d10) (CAS# 220621-39-6). It should be noted 

that all of the internal standards except atrazine-desethyl-desisopropyl (13C3) are 

commercially available; the atrazine-desethyl-desisopropyl (13C3) internal standard used 

during method development was custom synthesized by Cambridge Laboratories. 

7.2.1.1 INTERNAL STANDARD STOCK STANDARDS (ISSS) (500 μg/mL, except as 

noted) – Prepare the stock standards individually by weighing 2.5 mg of the solid 

standards into tared 5-mL volumetric flasks and diluting to volume with methanol. 

Atrazine-desethyl-desisopropyl (13C3) and Simazine-d10 (diethyl-d10) exhibit 

limited solubility in methanol and must be prepared as < 100 μg/mL stock solutions. 

To prepare the 100 μg/mL stock solutions, weigh 2.5 mg of the solid standards into 

tared 25-mL volumetric flasks and dilute to volume with methanol. Sonication may 

be required to achieve full dissolution of the standards. 

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (PDS) (1.0 

μg/mL) – Prepare the internal standard PDS by adding enough of each ISSS to a 

volumetric flask partially filled with methanol to make the final concentrations near 

1.0 μg/mL when filled to the mark with methanol. During method development, 

addition of 5 μL of the internal standard PDS to each 1.0-mL sample produced a 

final concentration of 5 μg/L. Analysts are permitted to use other PDS 

concentrations and volumes provided all field samples and CAL standards contain 

the same amount of internal standard, the concentration of the internal standard 

added provides adequate signal to maintain precision, and the volume added has a 

negligible effect on the final concentration. 

7.2.2 ANALYTE STANDARD SOLUTIONS – Obtain the analytes listed in the table in 

Section 1.1 as neat standards. Prepare the Analyte Stock and Primary Dilution Standards 

as described below. 

7.2.2.1 ANALYTE STOCK STANDARD SOLUTION (500 μg/mL, except as noted) – 

Prepare the stock standards individually by weighing 2.5 mg of the solid standards 

into tared 5-mL volumetric flasks and diluting to volume with methanol. Atrazinedesethyl-desisopropyl and Simazine exhibit limited solubility in methanol and must 

be prepared as < 100 μg/mL stock solutions. To prepare the 100 μg/mL stock 

solutions, weigh 2.5 mg of the solid standards into tared 25-mL volumetric flasks 

and dilute to volume with methanol. Sonication may be required to achieve full 

dissolution of the standards. 

7.2.2.2 ANALYTE PRIMARY DILUTION SOLUTION (PDS) (2.5 – 5 μg/mL) – The 

analyte PDS is prepared by dilution of the Analyte Stock Standard solutions and is 

used to prepare the calibration standards and to fortify the LFBs, LFSMs and 

LFSMDs with the target analytes. 
536 -10

7.2.3 CALIBRATION (CAL) STANDARDS – Prepare a procedural calibration curve from 

dilutions of the analyte PDS in reagent water. Transfer the standard to a proper storage 

container and add appropriate amounts of the ammonium acetate and sodium omadine 

concentrated stocks (Sect. 7.1.4 and 7.1.6) as shown in the table below. The target 

analyte concentrations found in Tables 5-7 can be used as a starting point for determining 

the calibration range. An example of the dilutions used to prepare the CALs that were 

used to collect data in Section 17 is shown below. The lowest concentration calibration 

standard must be at or below the MRL, which may depend on system sensitivity. The 

CAL standards may also be used as CCCs. If stored, the aqueous standards must be 

refrigerated in the same manner as the samples. A constant amount of the internal 

standard is added to each prepared Calibration Standard This is accomplished for each 

standard by taking 1.0 mL of the final calibration standard containing the 20 mM 

ammonium acetate and 64 mg/L sodium omadine, and placing it in a 2.0-mL autosampler 

vial and adding the appropriate volume of Internal Standard PDS (Sect. 7.2.1.2). 

CAL 

Level 

Analyte 

PDS 

Conc. 

(μg/mL) 

Analyte 

PDS 

Volume 

(μL) 

Ammonium 

Acetate Stock 

Volume *

(mL) 

Sodium 

Omadine Stock 

Volume *

(mL) 

Final CAL 

Std. Volume 

(mL) 

Final CAL 

Std. Conc. 

(μg/L) 

1 5 5 1.0 0.2 100 0.25 

2 5 10 1.0 0.2 100 0.50 

3 5 20 1.0 0.2 100 1.00 

4 5 20 0.5 0.1 50 2.00 

5 5 25 0.5 0.1 50 2.50 

6 5 10 0.1 0.02 10 5.00 *

Ammonium acetate stock (2.0 M) and sodium omadine stock (32 g/L) are added after the 

standards have been diluted to volume and transferred to storage containers. 

7.2.3.1 This is a procedural calibration technique. If an alternate set of dilutions are used to 

prepare the calibration standards, the analyst must ensure that the dilution factor 

used to prepare the calibration standards remains consistent with the dilution factor 

during sample collection (Sect. 8.1.1). The dilution factor is currently 101.2/100 in 

the table above and in Section 8.1. The analyst must also ensure that all CAL 

standards, QC and field samples contain the preservation reagents at their proper 

concentrations (Sect. 8.1.1). 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE COLLECTION 

 

8.1.1. Prior to shipment to the field, ammonium acetate and sodium omadine must be added to 

each amber bottle fitted with a PTFE-lined screw cap. If using the suggested 40-mL vials 

to collect a 40-mL aliquot, add 400 μL of the ammonium acetate concentrated stock 

(Sect. 7.1.4) and 80 μL of the concentrated sodium omadine stock (Sect. 7.1.6). If other 

collection volumes are used, adjust the amount of preservation reagent so that the final 

concentrations of ammonium acetate and sodium omadine in the sample containers are 

1.5 g/L and 64 mg/L, respectively. Cap the vials to avoid evaporation of the preservation 

reagents. 
536 -11

8.1.2 When sampling from a cold water tap, open the tap and allow the system to flush until the 

water temperature has stabilized (usually approximately 3 to 5 minutes). Collect a 

representative sample from the flowing system using a beaker of appropriate size. Use 

this bulk sample to generate individual samples as needed. Transfer a volume of at least 

40 mLs into each collection container, cap the container, and invert it several times to 

mix the sample with the preservation reagents. Care should be taken not to overfill the 

vial and flush out the preservation reagents. Samples do not need to be headspace free. 

8.1.3 When sampling from an open body of water, fill a beaker with water sampled from a 

representative area. Use this bulk sample to generate individual samples as needed. 

8.2 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment and 

must not exceed 10 °C during the first 48 hours after collection. Samples should be 

confirmed to be at or below 10 °C when they are received at the laboratory. Samples stored 

in the lab must be stored at or below 6 °C until analysis. Samples should not be frozen.

8.3 SAMPLE HOLDING TIMES – Samples should be analyzed as soon as possible. Samples 

that are collected and stored as described in Sections 8.1 and 8.2 may be stored prior to 

analysis for a maximum of 28 days.

9. QUALITY CONTROL

9.1 Quality Control (QC) requirements include the Initial Demonstration of Capability (IDC) and 

ongoing QC requirements that must be met when preparing and analyzing field samples. 

This section describes each QC parameter, its required frequency, and the performance 

criteria that must be met in order to meet EPA quality objectives. The QC criteria discussed 

in the following sections are summarized in Section 17, Tables 9 and 10. These QC 

requirements are considered the minimum acceptable QC criteria. Laboratories are 

encouraged to institute additional QC practices to meet their specific needs. 

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – The IDC must be successfully 

performed prior to analyzing any field samples. Prior to conducting the IDC, the analyst 

must meet the calibration requirements outlined in Sections 10.2 and 10.3. Requirements for 

the initial demonstration of laboratory capability are described in the following sections and 

are summarized in Table 9. 

9.2.1 DEMONSTRATION OF LOW SYSTEM BACKGROUND – Analyze a LRB and 

confirm that the blank is reasonably free of contamination and that the criteria in Section 

9.3.1 are met. 

NOTE: It is good laboratory practice to include a blank in the calibration of any 

instrument. The method should also be checked for carry-over by analyzing a RW blank 

immediately following the highest CAL standard. If this RW sample does not meet the 

criteria outline in Section 9.3.1, then carry-over is present and should be identified and 

eliminated. 

9.2.2 DEMONSTRATION OF PRECISION – Prepare and analyze 7 replicate LFBs. These 

samples should be fortified near the midrange of the initial calibration curve. 
Ammonium acetate and sodium omadine must be added to the samples as described in 

Section 8.1. The percent relative standard deviation (%RSD) of the concentrations of the 

replicate analyses must be ≤ 20% for all target analytes. 

100

Concentrat Average ion

Concentrat Measured ofDeviation Standard ions = RSD % ×

9.2.3 DEMONSTRATION OF ACCURACY – Using the same sets of replicate data generated 

for Section 9.2.2, calculate the average percent recovery. The average percent recovery 

of the replicate analyses must be within + 30% of the true value. 

100

Fortified Concentration

Concentrat Measured Average ion Recovery % = ×

9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target 

concentration for the MRL based on the intended use of the method. The lowest 

calibration standard used to establish the initial calibration (as well as the low-level 

Continuing Calibration Check) must be at or below the concentration of the MRL. 

Establishing the MRL concentration too low may cause repeated failure of ongoing QC 

requirements. Confirm or validate the MRL following the procedure outlined below. 

9.2.4.1 Fortify and analyze seven replicate LFBs at or below the proposed MRL 

concentration. All samples must be fortified and processed using the sample 

collection protocols described in Section 8.1. Calculate the mean (Mean) and 

standard deviation (S) for these replicates. Determine the Half Range for the 

Prediction Interval of Results (HRPIR) using the equation below 

HRPIR = 3.963S

where S is the standard deviation, and 3.963 is a constant value for seven replicates.1

9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Results (PIR = 

Mean + HRPIR) meet the upper and lower recovery limits as shown below. 

The Upper PIR Limit must be ≤ 150 percent recovery. 

Mean HR

FortifiedConcentration

PIR +

× ≤ 100 150%

The Lower PIR Limit must be ≥ 50 percent recovery. 

 Mean HR

FortifiedConcentration

PIR − × ≥ 100 50%

536 -12
536 -13

9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the criteria 

described above. If these criteria are not met, the MRL has been set too low and 

must be determined again at a higher concentration. 

9.2.5 QUALITY CONTROL SAMPLE (QCS) – Analyze a mid-level Quality Control Sample 

(Sect. 9.3.7) to confirm the accuracy of the calibration curve fit. 

9.2.6 DETECTION LIMIT DETERMINATION (optional) – While DL determination is not a 

specific requirement of this method, it may be required by various regulatory bodies 

associated with compliance monitoring. It is the responsibility of the laboratory to 

determine if DL determination is required based upon the intended use of the data.

Analyses for this procedure should be done over at least 3 days. Prepare at least 7 

replicate LFBs. Ammonium acetate and sodium omadine must be added to the samples 

as described in Section 8.1. Use the solutions described in Section 7.2 to fortify at a 

concentration estimated to be near the DL. This concentration may be estimated by 

selecting a concentration at 2-5 times the noise level. Analyze the seven replicates 

through all steps of Section 11. 

 NOTE: If an MRL confirmation data set meets these requirements, a DL may be 

calculated from the MRL confirmation data, and no additional analyses are necessary. 

Calculate the DL using the following equation: 

DL = St(n-1,1-α = 0.99) 

 where 

 t(n-1,1-α = 0.99) = Student's t value for the 99% confidence level with n-1 degrees of 

freedom (for seven replicate determinations, the Student’s t value 

is 3.143 at a 99% confidence level), 

n = number of replicates, and 

S = standard deviation of replicate analyses. 

NOTE: Do not subtract blank values when performing DL calculations. 

9.3 ONGOING QC REQUIREMENTS – This section describes the ongoing QC criteria that 

must be followed when processing and analyzing field samples. Table 10 summarizes these 

requirements. 

9.3.1 LABORATORY REAGENT BLANK (LRB) – A LRB is analyzed during the IDC and is 

required with each Analysis Batch (Sect. 3.1) to confirm that background contaminants 

are not interfering with the identification or quantitation of the target analytes. If within 

the retention time window of any analyte, the LRB produces a peak that would prevent 

the determination of that analyte, determine the source of contamination and eliminate 

the interference before processing samples. The LRB must contain ammonium acetate, 

sodium omadine, and the internal standard at the same concentrations used to fortify all 

field samples and CAL standards. Background from target analytes or contaminants that 

interfere with the measurement of target analytes must be < 1/3 the MRL. If the target 

analytes are detected in the LRB at concentrations equal to or greater than this level, then 
536 -14

all data for the problem analyte(s) must be considered invalid for all samples which 

yielded a positive result. 

 NOTE: Although quantitative data below the MRL may not be accurate enough for data 

reporting, such data are useful in determining the magnitude of background interference. 

Therefore, blank contamination levels may be estimated by extrapolation when the 

concentration is below the MRL. 

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC standards are analyzed at the 

beginning of each Analysis Batch, after every ten field samples and at the end of the 

Analysis Batch. See Section 10.4 for concentration requirements and acceptance criteria. 

 

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – Since this method utilizes procedural 

calibration standards, which are fortified reagent waters, there is no difference between 

the LFB and the continuing calibration check standard. Consequently, the analysis of a 

separate LFB is not required as part of the ongoing QC; however, the acronym LFB is 

used for clarity in the IDC. 

9.3.4 INTERNAL STANDARDS (IS) – The analyst must monitor the peak areas of the 

internal standards in all injections of the Analysis Batch. The IS responses (as indicated 

by peak areas) for any chromatographic run must not deviate by more than ± 50% from 

the average areas measured during the initial calibration for the internal standards. If the 

IS area counts for a sample do not meet this criterion, inject a second aliquot of the 

suspect sample as part of the same or a new Analysis Batch. 

9.3.4.1 If the re-injected aliquot produces an acceptable internal standard response, report 

results for that aliquot. 

9.3.4.2 If the IS area counts of the re-injected aliquot still do not meet the IS criterion, 

check the IS area of the most recent CCC. If the IS criterion is met in the CCC but 

not the sample, report the sample results as suspect/matrix. 

9.3.4.3 If the IS area criterion is not met in both the sample and the CCC, instrument 

maintenance such as sample cone cleaning may be necessary. Perform the 

appropriate instrument maintenance and then re-inject the sample in a subsequent 

analytical batch. 

 

9.3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of a LFSM (Sect. 

3.9) is required in each Analysis Batch. The LFSM is processed and analyzed exactly like 

a sample, and its purpose is to determine whether the sample matrix contributes bias to 

the analytical results. The background concentrations of the analytes in the sample 

matrix must be determined in a separate aliquot and the measured values in the LFSM 

corrected for background concentrations. If a variety of different sample matrices are 

analyzed regularly, for example drinking water from groundwater and surface water 

sources, performance data should be collected for each source. 

9.3.5.1 Within each Analysis Batch, a minimum of one field sample is fortified as an LFSM 

for every 20 samples analyzed. The LFSM is prepared by fortifying a sample with 

an appropriate amount of the analyte PDS (Sect. 7.2.2.2). Select a spiking 
concentration that is greater than or equal to the native background concentration, if 

known. Selecting a duplicate aliquot of a sample that has already been analyzed 

aids in the selection of an appropriate spiking level. If this is not possible, use 

historical data and rotate through low, medium and high calibration concentrations 

when selecting a fortifying concentration. 

9.3.5.2 Calculate the percent recovery (%REC) using the equation: 

( ) 100

C

B-A %REC ×=

 where 

 A = measured concentration in the fortified sample, 

 B = measured concentration in the unfortified sample, and 

 C = fortification concentration. 

9.3.5.3 Recoveries for samples fortified at concentrations near or at the MRL (within a 

factor of two times the MRL concentration) should be within + 50% of the true 

value. Recoveries for samples fortified at all other concentrations should be within +

30% of the true value. If the accuracy for any analyte falls outside the designated 

range, and the laboratory performance for that analyte is shown to be in control in 

the CCCs, the recovery is judged to be matrix biased. The result for that analyte in 

the unfortified sample is labeled suspect/matrix to inform the data user that the 

results are suspect due to matrix effects. 

9.3.5.3.1 Field samples that have native analyte concentrations above the DL but below 

the MRL and are fortified at concentrations at or near the MRL should be 

corrected for the native levels in order to obtain meaningful %REC values. 

This example and the LRB (Sect. 9.3.1) are the only permitted use of analyte 

results below the MRL.

9.3.6 LABORATORY DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX 

DUPLICATE (LD or LFSMD) – Within each Analysis Batch, a minimum of one 

Laboratory Duplicate (LD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD) 

must be analyzed. Laboratory Duplicates check the precision associated with laboratory 

procedures. If target analytes are not routinely observed in field samples, a LFSMD 

should be analyzed rather than a LD. 

9.3.6.1 Calculate the relative percent difference (RPD) for duplicate measurements (LD1

and LD2) using the equation: 

( ) 100

2/LDLD

LDLD

RPD

1 2

1 2 ×

+

− =

9.3.6.2 RPDs for Laboratory Duplicates should be ≤ 30%. Greater variability may be 

observed when Laboratory Duplicates have analyte concentrations that are within a 

factor of 2 x MRL. At these concentrations, Laboratory Duplicates should have 

536 -15
RPDs that are ≤ 50%. If the RPD of the analyte falls outside the designated range, 

and the laboratory performance for the analyte is shown to be in control in the CCC, 

the precision is judged to be matrix influenced. The result from the unfortified 

sample is labeled suspect/matrix to inform the data user that the results are suspect 

due to matrix effects. 

9.3.6.3 If a LFSMD is analyzed instead of a Laboratory Duplicate, calculate the relative 

percent difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the 

equation: 

( ) 100

LFSM /2LFSMD

LFSM LFSMD

RPD ×

+

− =

9.3.6.4 RPDs for duplicate LFSMs should be ≤ 30%. Greater variability may be observed 

when fortified LFSMs have analyte concentrations that are within 2 x MRL. 

LFSMs at these concentrations should have RPDs that are ≤ 50%. If the RPD of the 

analyte falls outside the designated range, and the laboratory performance for the 

analyte is shown to be in control in the CCC, the precision is judged to be matrix 

influenced. The result from the unfortified sample is labeled suspect/matrix to 

inform the data user that the results are suspect due to matrix effects. 

9.3.7 QUALITY CONTROL SAMPLE (QCS) – A QCS is required if an alternate commercial 

source is available for the target analytes. A QCS should be evaluated as part of the IDC 

(Sect. 9.2.5) and each time new PDS solutions are prepared. If standards are prepared 

infrequently, analyze a QCS at least quarterly. The QCS should be fortified near the 

midpoint of the calibration range and analyzed as a CCC. The acceptance criteria for the 

QCS are the same as the mid- and high-level CCCs (Sect. 10.4). If measured analyte 

concentrations are not of acceptable accuracy, check the entire analytical procedure to 

locate and correct the problem. 

9.4 METHOD MODIFICATION QC REQUIREMENTS - The analyst is permitted to modify 

the separation technique, LC column, mobile phase composition, LC conditions and MS 

conditions. 

9.4.1 Each time method modifications are made, the analyst must repeat the procedures of the 

IDC (Sect. 9.2) and verify that all QC criteria can be met in ongoing QC samples (Sect. 

9.3). 

9.4.2 The analyst is also required to evaluate and document method performance for the 

proposed method modifications in real matrices that span the range of waters that the 

laboratory analyzes. This additional step is required because modifications that perform 

acceptably in the IDC, which is conducted in reagent water, can fail ongoing method QC 

requirements in real matrices. This is particularly important for methods subject to 

matrix effects, such as LC/MS-based methods. If, for example, the laboratory analyzes 

finished waters from both surface and groundwater municipalities, this requirement can 

be accomplished by assessing precision and accuracy (Sects. 9.2.2 and 9.2.3) in a surface 

water with moderate to high total organic carbon ( e.g., 2 mg/L or greater) and a hard 

groundwater (e.g., 250 mg/L as CaCO3 equivalent, or greater). 

536 -16
536 -17

9.4.3 The results of Sections 9.4.1 and 9.4.2 must be appropriately documented by the analyst 

and should be independently assessed by the laboratory’s QA officer prior to analyzing 

field samples. 

9.4.3.1 When implementing method modifications, it is the responsibility of the laboratory 

to closely review the results of ongoing QC, and in particular, the results associated 

with the LFSM (Sect. 9.3.5), LFSMD (Sect. 9.3.6), CCCs (Sect. 9.3.2), and the IS 

area counts (Sect. 9.3.4). If repeated failures are noted, the modification must be 

abandoned. 

10 CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of acceptable MS calibration and initial analyte 

calibration are required before performing the IDC (Sect. 9.2) and prior to analyzing field 

samples. After initial calibration is successful, Continuing Calibration Check (CCC) 

standards are required at the beginning and the end of each analysis batch, and after every 

tenth sample (Sect. 10.4). Verification of the MS calibration and the initial calibration 

should be repeated each time a major instrument modification or maintenance is performed. 

10.2 LC/MS/MS CALIBRATION AND OPTIMIZATION 

10.2.1 MASS CALIBRATION – Calibrate the mass spectrometer with the calibration 

compounds and procedures specified by the manufacturer. 

10.2.2 OPTIMIZING MS PARAMETERS – This method requires the utilization of ESI and 

Multiple Reaction Monitoring as defined above (Sect. 3.15). LC/MS instruments have 

several instrumental parameters that must be optimized. Each LC/MS system will have 

different optimal conditions which are influenced by the source geometry and system 

design. Due to the differences in design, the recommendations of the instrument 

manufacturer should be followed when tuning the instrument. In the development of this 

method, instrumental parameters were optimized for the precursor and product ions listed 

in Section 17, Table 3. Literature resources to date describe similar precursor and 

product ions.6,7 Product ions other than those listed may be selected; however, the 

analyst is cautioned to avoid using ions with lower mass and/or common ions that may 

not provide sufficient discrimination between the analytes of interest and co-eluting 

interferences. 

10.2.2.1 Prepare 0.05 – 1.0 μg/mL solutions of the target analytes in the approximate mobile 

phase compositions that correspond to the elution mobile phase compositions. This 

step will need to be repeated and the parameters re-optimized if an analyte is 

prepared in a mobile phase composition that is significantly different from the 

mobile phase composition at the time the analyte elutes from the column. 

10.2.2.2 Optimize the response of the precursor ion (Sect. 3.16) for each target analyte by 

infusing the prepared solutions (Sect. 10.2.2.1) directly into the mass spectrometer 

as recommended by the instrument manufacturer. The LC should be operating with 

a mobile phase composition and flow rate comparable to the elution conditions. 

Vary the MS parameters (source voltages, source and desolvation temperatures, gas 
536 -18

flows, etc.) until optimal analyte responses are determined. The target analytes may 

have different optima, thus requiring some compromise. LC/MS systems are 

subject to drift, which can affect run-to-run precision. Accordingly, the optimal 

conditions may be those that yield good response while minimizing the potential for 

drift. See Section 17, Table 2 for the ESI+ MS conditions used during method 

development. 

10.2.2.3 In all cases, a precursor ion must be isolated (e.g., in a quadrupole or an ion trap) 

prior to fragmentation. Optimize the response of the product ion (Section 3.18) for 

each target by infusing the prepared solutions (Sect. 10.2.2.1) directly into the mass 

spectrometer as recommended by the instrument manufacturer. The LC should be 

operating with a mobile phase composition and flow rate comparable to the elution 

conditions. The MS/MS parameters (collision gas pressure, collision energy, etc.) 

are varied until optimal analyte responses are determined. The triazine compounds 

listed in this method exhibited very similar optimal MS/MS conditions. See Section 

17, Table 3 for the MS/MS conditions used in method development. 

10.2.3 LC INSTRUMENT CONDITIONS – LC operating conditions are described in Section 

17, Table 1. Conditions different from those described (e.g., LC columns and mobile 

phases) may be used if the QC criteria in Sections 9.2 and 9.3 are met and 

chromatographic separation of the target analytes is achieved (resolution > 50%). 

NOTE: Chromatographic separation as defined does not include the isotopically 

enriched internal standards, which are mass separated. Co-elution of the internal 

standards with their analogous target analytes helps mitigate matrix suppression and/or 

enhancement effects.8

10.2.4 ESTABLISH LC/MS RETENTION TIMES AND MRM SEGMENTS – Inject a mid- to 

high-level CAL standard under optimized LC/MS conditions to obtain the retention times 

of each target analyte. Divide the chromatogram into segments that contain one or more 

chromatographic peaks. For maximum sensitivity in subsequent MS/MS analyses, 

minimize the number of MRM (Sect. 3.15) transitions that are simultaneously monitored 

within each segment. 

10.3 INITIAL CALIBRATION – During method development, daily calibrations were performed; 

however, it is permissible to verify the calibration with daily CCCs. Calibration must be 

performed using peak areas and the internal standard technique. Calibration using peak 

heights or external calibration is not permitted. 

10.3.1 CALIBRATION STANDARDS – Prepare a set of at least five CAL standards as 

described in Section 7.2.3. The lowest concentration of the calibration standards must be 

at or below the MRL. The MRL must be confirmed using the procedure outlined in 

Section 9.2.4 after establishing the initial calibration. Additionally, field samples must be 

quantified using a calibration curve that spans the same concentration range used to 

collect the IDC data (Sect. 9.2). 

10.3.2 CALIBRATION – The LC/MS system is calibrated using peak areas and the internal 

standard technique. Concentrations may be calculated using the data system software to 

generate a linear regression or quadratic calibration curve. Forcing the calibration curve 
536 -19

through the origin is not recommended. The MS/MS instrument used during method 

development was calibrated using weighted (1/X) quadratic curves. Data may be fit with 

either a linear regression (response vs. concentration) or quadratic fit (response vs. 

concentration) and weighting may be used, if necessary. 

10.3.3 CALIBRATION ACCEPTANCE CRITERIA – The validation of the calibration is 

determined by calculating the concentration of the analyte from each of the analyses used 

to generate the calibration curve. Calibration points that are ≤ MRL should calculate to 

be within + 50% of their true value. All other calibration points should calculate to be 

within + 30% of their true value. If these criteria cannot be met, the analyst will have 

difficulty meeting ongoing QC criteria. In this case, corrective action should be taken to 

reanalyze the calibration standards, restrict the range of calibration, or select an alternate 

method of calibration. 

10.4 CONTINUING CALIBRATION CHECKS (CCCs) – The CCC verifies the calibration at the 

beginning, after every tenth field sample and at the end of each Analysis Batch. CCCs are 

not counted as samples. The beginning CCC for each Analysis Batch must be at or 

below the MRL. This CCC verifies instrument sensitivity prior to the analysis of samples. 

Subsequent CCCs should alternate between a medium and high concentration CAL standard. 

10.4.1 Inject an aliquot of the appropriate concentration CAL standard and analyze with the 

same conditions used during the initial calibration. 

10.4.2 Determine that the absolute areas of the quantitation ions of each of the internal standards 

have not changed by more than ± 50% from the average areas measured during the initial 

calibration. If IS areas have changed by more than + 50%, remedial action is necessary 

(Sect. 10.4.4). Recalibrate according to Section 10.3 and verify sensitivity by analyzing a 

CCC at or below the MRL. 

10.4.3 Calculate the concentration of each analyte in the CCC. The CCC fortified at ≤ MRL 

must calculate to be within + 50% of its true value. CCCs fortified at all other levels 

must calculate to be within + 30%. If these conditions do not exist, then all data for the 

analyte must be considered invalid, and remedial action (Sect. 10.4.4) must be taken. 

The remedial action may require recalibration. Any field samples that have been 

analyzed since the last acceptable CCC that are still within holding time should be 

reanalyzed after adequate calibration has been restored. 

10.4.4 REMEDIAL ACTION – Failure to meet CCC QC performance criteria requires remedial 

action. Maintenance such as cleaning an ion source probe, sample cone, ion lenses 

and/or replacing LC columns may be required. Following major maintenance, the analyst 

must return to the initial calibration step (Sect. 10.3). 

11. PROCEDURE

11.1 Important aspects of this analytical procedure include proper sample collection and storage 

(Section 8), ensuring that the instrument is properly calibrated (Section 10) and that all 

required QC are met (Section 9). This section describes the procedures for sample 

preparation and analysis. 
536 -20

11.2 SAMPLE PREPARATION 

11.2.1 Samples are dechlorinated, preserved, collected and stored as described in Section 8.1. 

All field and QC samples must contain the dechlorinating and preservation agents listed 

in Section 8.1.1, including the LRB. Allow field samples to reach room temperature 

prior to analysis. 

11.2.2 Transfer a 1.0 mL aliquot of each field or QC sample to an autosampler vial; as 

appropriate, samples that will be chromatographed using small particle diameter LC 

columns (< 2 μm) may be filtered prior to analysis (see note below). Add the IS PDS 

(Sect. 7.2.1.2), cap and mix well. Larger amounts of sample may be used, but the amount 

of internal standard must be increased to give a final concentration that is equal to the 

CAL IS concentration. 

NOTE: If filtering is incorporated as part of the sample preparation, the first lot of filters 

must be subjected to the procedures outlined in the IDC (Sect. 9.2) and meet the 

acceptance criteria defined in Table 9 to ensure that they do not introduce interferences or 

retain any of the target analytes. Verification of subsequent lots of filters can be 

accomplished by examining a LRB and duplicate samples of LFBs fortified at the MRL. 

The LFBs should calculate to be within + 50% of the true value. If the LRB or the LFBs 

fail this evaluation, the full IDC will need to be repeated with the new lot of filters. CAL 

standards and CCCs should not be filtered in order to identify potential losses associated 

with the sample filtration devices. 

11.3 SAMPLE ANALYSIS 

11.3.1 Establish MS operating conditions equivalent to those summarized in Tables 1–3 of 

Section 17 as per the guidance in Section 10.2. Column choice and instrument 

parameters should be optimized prior to initiation of the IDC. 

11.3.2 Establish an appropriate retention time window (Sect. 10.2.4) for each analyte to identify 

them in QC and field sample chromatograms. This should be based on measurements of 

actual retention time variation for each compound in standard solutions analyzed on the 

LC over the course of time. The suggested variation is plus or minus three times the 

standard deviation of the retention time for each compound for a series of injections. The 

injections from the initial calibration and from the IDC (Sect. 9.2) may be used to 

calculate a suggested window size. However, the experience of the analyst should weigh 

heavily on the determination of an appropriate retention window size. 

11.3.3 Establish a valid initial calibration following the procedures outlined in Section 10.3 or 

confirm that the calibration is still valid by running a CCC as described in Section 10.4. 

If establishing an initial calibration for the first time, complete the IDC as described in 

Section 9.2 prior to analyzing field samples. 

11.3.4 Analyze aliquots of field and QC samples at appropriate frequencies (Section 9) with the 

LC/MS/MS conditions used to acquire the initial calibration. 
536 -21

11.3.5 At the conclusion of data acquisition, use the same software settings that were used in the 

calibration procedure to identify peaks of interest in the predetermined retention time 

windows. Identify an analyte by comparison of its retention time with that of the 

corresponding analyte peak in a CAL standard or CCC. 

11.3.6 EXCEEDING CALIBRATION RANGE – The analyst must not extrapolate beyond the 

established calibration range. If an analyte result exceeds the range of the initial 

calibration curve, the sample may be diluted using reagent water containing 20 mM 

ammonium acetate and 64 μg/L sodium omadine and with the appropriate amount of 

internal standard added to match the original level. Re-inject the diluted sample. 

Incorporate the dilution factor into final concentration calculations. The resulting data 

should be documented as a dilution and the dilution will affect analyte MRLs. 

12. DATA ANALYSIS AND CALCULATIONS

12.1 While complete chromatographic resolution is not necessary for accurate and precise 

measurements of analyte concentrations using MS/MS in reagent water, it is important in real 

matrices. The analyst must avoid chromatographic modifications that result in suppression 

or enhancement of analyte responses due to co-eluting matrix components. This is especially 

a problem with samples that contain higher TOC levels (Sect. 13.4.2). In validating this 

method, concentrations were calculated using the product ions listed in Table 3 of Section 17. 

Other ions may be selected at the discretion of the analyst as discussed in Section 10.2.2. 

12.2 Calculate analyte concentrations using the multipoint calibration established in Section 10.3. 

Report only those values that fall between the MRL and the highest CAL standard. Samples 

with target analyte responses that exceed the highest CAL standard require dilution and 

reanalysis (Sect. 11.3.6). 

12.3 Calculations must use all available digits of precision, but final reported concentrations 

should be rounded to an appropriate number of significant figures (one digit of uncertainty), 

typically two, and not more than three significant figures. 

12.4 Prior to reporting the data, the chromatograms should be reviewed for any incorrect peak 

identifications or poor integrations. 

12.5 Prior to reporting data, the laboratory is responsible for assuring that QC requirements have 

been met or that any appropriate qualifier is documented. 

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY AND DETECTION LIMITS – Tables for these data are 

presented in Section 17. Instrumental conditions are presented in Tables 1 through 3. DLs 

and LCMRLs are presented in Table 4. LCMRLs were determined and calculated using a 

procedure described elsewhere.1 Single laboratory precision and accuracy data are presented 

for three water matrices: reagent water (Table 5); chlorinated (finished) surface water (Table 

6); and chlorinated (finished) groundwater (Table 7). Figure 1 shows an overlaid 

chromatogram of the normalized MS/MS responses for the analytes obtained under the 

conditions employed during method development. 
536 -22

13.2 SECOND LABORATORY EVALUATION – The performance of this method was 

demonstrated by a second laboratory, with results similar to those reported in Section 17. 

The authors wish to acknowledge the work of Dr. Jody Shoemaker of the U.S. EPA National 

Exposure Research Laboratory, Cincinnati, OH for conducting the second laboratory 

evaluation. 

13.3 ANALYTE STABILITY STUDIES – Chlorinated surface water samples, fortified with 

target analytes at 5 μg/L, were preserved as required in Section 8 and stored over a 28-day 

period. The accuracy and precision of three replicate analyses, conducted on day 0, 7, 14, 21 

and 28, are presented in Section 17, Table 8. These data were used to determine the 28-day 

holding time.

13.4 PROBLEM COMPOUNDS – During method development and the second laboratory 

evaluation, the following problems were observed for Atrazine-desethyl-desisopropyl 

(DACT):

13.4.1 DACT is subject to hydrolysis at both high (> 9) and low (< 5) pH but has acceptable 

stability in neutral aqueous solutions. DACT is also degraded in the presence of free 

available chlorine, but stable in the presence of chloramines. Ammonium acetate 

mitigates both of these modes of loss and has sufficient buffer capacity at 20 mM to 

buffer finished groundwaters with relatively high pH that also have buffer capacity. 

13.4.2 DACT and its internal standard (DACT-13C) exhibited ESI matrix suppression in the 

second laboratory evaluation after being fortified into a chlorinated surface water 

containing high total organic carbon (TOC) levels (9.5 mg/L). The DACT-13C response 

was suppressed 26% relative to the calibration; however, since DACT was affected by 

the suppression in an equivalent manner, the DACT recovery was 99%. The matrix 

suppression occurred due to co-eluting TOC in the early part of the chromatographic run 

and DACT is the analyte most susceptible to matrix effects. As discussed in Section 

4.4.1, good performance is achieved by optimizing chromatographic separation, and in 

particular, the separation of DACT from early eluting contaminants. Modifications that 

seek to shorten the chromatographic separation without accounting for the need to 

separate the analytes from early eluting interferences may result in failure to meet QC 

criteria.

14. POLLUTION PREVENTION

14.1 For information about pollution prevention that may be applicable to laboratory operations, 

consult “Less is Better: Laboratory Chemical Management for Waste Reduction” available 

from the American Chemical Society’s Department of Government Relations and Science 

Policy, 1155 16th Street N.W., Washington, D.C., 20036, or on-line at 

http://www.ups.edu/community/storeroom/Chemical_Wastes/wastearticles.htm. 

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts of 

waste since only small amounts of reagents and solvents are used. The matrices of concern 

are finished drinking water or source water. However, the Agency requires that laboratory 

waste management practices be conducted consistent with all applicable rules and 
536 -23

regulations, and that laboratories protect the air, water, and land by minimizing and 

controlling all releases from fume hoods and bench operations. Also, compliance is required 

with any sewage discharge permits and regulations, particularly the hazardous waste 

identification rules and land disposal restrictions. For further information on waste 

management, see the publications of the American Chemical Society’s Laboratory 

Environment, Health & Safety Task Force on the Internet at 

http://membership.acs.org/c/ccs/publications.htm. Additional waste management 

information can be found in “Laboratory Waste Minimization and Pollution Prevention,” 

Copyright © 1996 Battelle Seattle Research Center, which can be located at 

http://www.p2pays.org/ref/01/text/00779/index2.htm. 

16. REFERENCES

1. Winslow, S.D.; Pepich, B.V.; Martin, J.J.; Hallberg, G.R.; Munch D.J.; Frebis, C.P.; Hedrick, E.J.; 

Krop, R.A. Statistical Procedures for Determination and Verification of Minimum Reporting Levels 

for Drinking Water Methods. Environ. Sci. Technol. 2006, 40, 281-288. 

2. Glaser, J.A.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. Trace Analyses for 

Wastewaters. Environ. Sci. Technol. 1981, 15, 1426-1435. 

3. “Guidelines, Recommendations, and Regulations for Handling Antineoplastic Agents”, Center for 

Disease Control, National Institute for Occupational Safety and Health, 

http://www.cdc.gov/niosh/topics/antineoplastic/pubs.html#b. 

4. “OSHA Safety and Health Standards, General Industry,” (29CFR1910), Occupational Safety and 

Health Administration, OSHA 2206 (Revised, January 1976). 

5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, Committee 

on Chemical Safety, 3rd Edition, 1979. 

6. Steen, Ruud J.C.A.; Hogenboom, Ariadne C.; Leonards, Pim E.G.; Peerboom, Renee A.L.; Cofino, 

Wim P.; Brinkman, Udo A.Th. Ultra-trace Level Determination of Polar Pesticides and Their 

Transformation Products in Surface and Estuarine Water Samples Using Column Liquid 

Chromatography-Electrospray Tandem Mass Spectrometry. J. Chromatogr. A 1999, 857, 157-166. 

7. Huang, S.; Mayer, T.J.; Yokley, R.A.; Perez, R. Direct Aqueous Injection Liquid 

Chromatography/Electrospray Ionization-Mass Spectrometry/Mass Spectrometry Analysis of Water 

for Atrazine, Simazine, and their Chlorotriazine Metabolites. J. Agric. Food Chem. 2006, 54, 713-

719. 

8. Kloepfer, A.; Quintana, J.B.; Reemtsma, T. Operational Options to Reduce Matrix Effects in Liquid 

Chromatrography-Electrospray Ionization-Mass Spectrometry Analysis of Aqueous Environmental 

Samples. J. Chromatogr. A 2005, 1067, 153-160. 
536 -24

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. LC CONDITIONS a

Time (min.) %5 mM NH4OAc % Methanol 

Initial 90 10 

5.0 90 10 

25.0 70 30 

25.1 60 40 

35.0 60 40 

35.1 50 50 

45.0 50 50 

45.1 90 10 

65.0 90 10 

a

 Chromatographic conditions: Column: Waters Xterra® RP18, 2.1 x 150 mm, 3.5 μm particle 

diameter; Injection Volume: 100 μL; Flow Rate: 0.250 mL/min. 

TABLE 2. ESI-MS/MS METHOD CONDITIONS 

MS Parameter Setting 

Polarity Positive Ion Mode 

Capillary Voltage 0.5 kV 

Source Temperature 100 oC 

Desolvation Temperature 260 oC 

Desolvation Gas Flow 616 L/hr 

Cone Gas Flow 26 L/hr 

Collision Cell Pressure 2.1 e-3 mbar 

Collision Gas Argon 
536 -25

TABLE 3. LC/ESI-MS/MS RETENTION TIMES, PRECURSOR AND PRODUCT IONS, 

AND COLLISION ENERGIES 

MRM 

Segment 

# 

Analyte Ret. 

Time 

(min.) 

Precursor 

Ion 

(m/z) 

Product 

Ion 

(m/z) 

Collision 

Energy 

(eV) 

1 Atrazine-desethyl-desisopropyl-13C (IS) 3.78 149 105.5 22 

1 Atrazine-desethyl-desisopropyl 3.78 146 103.5 22 

2 Atrazine-desisopropyl-d5 (IS) 11.26 179 137 22 

2 Atrazine-desisopropyl 11.57 174 132 22 

3 Atrazine-desethyl-d7 (IS) 18.66 195 147 22 

3 Atrazine-desethyl 19.09 188 146 22 

4 Simazine-d10 (IS) 30.48 212 137 20 

4 Simazine 30.96 202 132 20 

4 Cyanazine-d5 (IS) 32.03 246 219 20 

4 Cyanazine 32.20 241 214 20 

5 Atrazine-d5 (IS) 37.03 221 179 23 

5 Atrazine 37.37 216 174 23 

6 Propazine-d14 (IS) 42.87 244 196 20 

6 Propazine 43.51 230 188 20 

TABLE 4. LC/ESI-MS/MS LOWEST CONCENTRATION MRLs AND DLs a

Analyte LCMRL Concentration 

Levels (μg/L) 

Calculated 

LCMRL (μg/L) 

Calculated 

DL (μg/L) 

Atrazine-desethyl-desisopropyl 0.104, 0.208, 0.260, 0.520 0.14 0.028 

Atrazine-desisopropyl 0.102, 0.204, 0.255, 0.510 0.11 0.043 

Atrazine-desethyl 0.021, 0.052, 0.104, 0.130 0.026 0.017 

Simazine 0.020, 0.050, 0.100, 0.125 0.020 0.010 

Cyanazine 0.019, 0.048, 0.096, 0.120 0.027 0.010 

Atrazine 0.021, 0.052, 0.104, 0.130 0.037 0.010 

Propazine 0.020, 0.050, 0.100, 0.125 0.028 0.010 a DLs calculated using data acquired on a single day at the lowest LCMRL concentration level (n=7 sample 

replicates).
536 -26

TABLE 5. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES 

FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN REAGENT WATER (n=7 SAMPLES)

Fortified Conc. = 0.5 μg/L 

(n=7) 

Fortified Conc. = 5.0 μg/L 

(n=7) 

Analyte 

Mean % 

Recovery 

Relative Standard 

Deviation (%RSD)

Mean % 

Recovery 

Relative Standard 

Deviation 

(%RSD) 

Atrazine-desethyl-desisopropyl 98.9 4.9 89.4 4.2 

Atrazine-desisopropyl 100 3.4 96.1 3.7 

Atrazine-desethyl 99.4 2.7 97.6 1.3 

Simazine 101 3.0 101 1.9 

Cyanazine 97.7 1.3 99.3 1.4 

Atrazine 99.4 2.5 99.7 1.6 

Propazine 103 2.9 99.0 2.0 

TABLE 6. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES 

FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN A CHLORINATED SURFACE 

WATER a

 (n=7 SAMPLES)

Fortified Conc. = 0.5 μg/L 

(n=7) 

Fortified Conc. = 5.0 μg/L 

(n=7) 

Analyte 

Mean % 

Recovery b

Relative Standard 

Deviation (%RSD)

Mean % 

Recoveryb

Relative Standard 

Deviation 

(%RSD) 

Atrazine-desethyl-desisopropyl 102 8.4 103 2.7 

Atrazine-desisopropyl 100 6.6 101 3.7 

Atrazine-desethyl 95.5 1.5 97.9 0.9 

Simazine 94.5 3.7 99.4 1.7 

Cyanazine 95.3 4.2 99.4 1.7 

Atrazine 97.4 6.1 101 0.9 

Propazine 94.9 6.0 105 1.5 a Surface water physical parameters: pH=7.1; Hardness = 154 mg/L; Free chlorine = 1.5 mg/L (Hach). 

b Recoveries corrected for native levels in the unfortified matrix: Atrazine-desethyl-desisopropyl (0.043 μg/L), 

Atrazine-desisopropyl (0.001 μg/L), Atrazine-desethyl (0.031 μg/L), Simazine (0.054 μg/L), Cyanazine (0.018 

μg/L), Atrazine (0.316 μg/L) and Propazine (0.038 μg/L). 
536 -27

TABLE 7. LC/ESI-MS/MS PRECISION AND ACCURACY OF METHOD ANALYTES 

FORTIFIED AT 0.5 μg/L AND 5.0 μg/L IN A CHLORINATED 

 GROUNDWATER a (n=7 SAMPLES)

Conc. = 0.5 μg/L 

(n=7) 

Conc. = 5.0 μg/L 

(n=7) 

Analyte 

Mean % 

Recovery b

Relative Standard 

Deviation 

(%RSD) 

Mean % 

Recoveryb

Relative Standard 

Deviation (%RSD)

Atrazine-desethyl-desisopropyl 106 3.5 95.7 2.5 

Atrazine-desisopropyl 99.6 5.2 103 3.0 

Atrazine-desethyl 95.7 1.7 98.6 1.5 

Simazine 94.6 3.1 98.0 1.5 

Cyanazine 93.5 1.8 101 1.3 

Atrazine 96.7 0.9 100 1.5 

Propazine 91.7 3.7 99.7 1.4 a Groundwater physical parameters: pH = 7.5; Hardness = 360 mg/L; Free chlorine = 0.49 mg/L. 

b Recoveries corrected for native levels in the unfortified matrix: Atrazine-desethyl-desisopropyl (0.032μg/L), 

Atrazine-desisopropyl (not detected), Atrazine-desethyl (0.036 μg/L), Simazine (0.030 μg/L), Cyanazine (0.018 

μg/L), Atrazine (0.057 μg/L), and Propazine (0.036 μg/L). 
536 -28

TABLE 8. LC/ESI-MS/MS SAMPLE HOLDING TIME DATA FOR METHOD ANALYTES FORTIFIED A

CHLORINATED SURFACE WATER a (n=3 SAMPLES)

Analyte Day 0 Day 7 Day 14 Day 21 

%Rec %RSD %Rec %RSD %Rec %RSD %Rec %RSD 

Atrazine 98.0 4.1 97.4 2.9 99.4 1.1 94.3 7.0 Atrazine-desethyl 107 2.5 104 4.1 111 1.7 103 4.7 Atrazine-desethyl-desisopropyl 95.9 7.7 93.9 6.2 102 1.2 90.8 6.6 Atrazine-desisopropyl 105 3.5 89.8 7.6 95.4 1.1 93.5 7.9 Cyanazine 99.3 4.7 94.0 1.2 94.8 1.6 87.8 4.7 Propazine 112 2.9 102 4.8 109 2.5 103 6.1 Simazine 94.2 6.0 99.6 4.8 93.6 6.8 89.1 11.8 

a Surface water physical parameters: pH = 7.0; Hardness=154 mg/L; Free chlorine = 1.2 mg/L (Hach). 
536 -29

TABLE 9. INITIAL DEMONSTRATION OF CAPABILITY (IDC) QUALITY CONTROL 

REQUIREMENTS 

Method 

Reference 

Requirement Specification and 

Frequency 

Acceptance Criteria 

Section 

9.2.1 

Demonstration of 

Low System 

Background 

Analyze a LRB prior to 

any other IDC steps. 

Demonstrate that all target 

analytes are < 1/3 of the MRL 

and that possible interferences 

from reagents and glassware 

do not prevent the 

identification and quantitation 

of target analytes. 

Section 

9.2.1 

Test For System 

Carryover 

Analyze a reagent water 

blank after the high CAL 

standard during the IDC 

calibration. 

Demonstrate that the target 

analytes are < 1/3 of the MRL 

and that carry-over from 

previous samples does not 

prevent the identification and 

quantitation of target analytes. 

Section 

9.2.2 

Demonstration of 

Precision 

Analyze 7 replicate CCCs 

fortified near the midrange 

concentration. 

%RSD must be ≤ 20% 

Section 

9.2.3 

Demonstration of 

Accuracy 

Calculate average recovery 

for replicates used in 

Section 9.2.2. 

Mean recovery within + 30% 

of the true value. 

Section 

9.2.4 

Minimum 

Reporting Limit 

(MRL) 

Confirmation 

Fortify and analyze 7 

replicate CCCs at the 

proposed MRL 

concentration. Calculate 

the mean and the Half 

Range (HR). Confirm that 

the Upper PIR and Lower 

PIR (Sect. 9.2.4.2) meet 

the recovery criteria. 

Upper PIR ≤ 150% 

Lower PIR ≥ 50% 

Section 

9.2.5 

Quality Control 

Sample (QCS) 

Analyze mid-level QCS 

sample. 

Results must be within + 30% 

of the true value. 
536 -30

TABLE 10. ONGOING QUALITY CONTROL REQUIREMENTS 

Method 

Reference 

Requirement Specification and 

Frequency 

Acceptance Criteria 

Section 

8.3 

Sample Holding 

Time 

28 days when processed and 

stored according to Sections 

8.1 and 8.2. 

Sample results are valid only if 

samples are analyzed within the 

sample holding time. 

Section 

10.3 

Initial Calibration Use the internal standard 

calibration technique to 

generate a linear or quadratic 

calibration curve. Use at 

least 5 standard 

concentrations. Validate the 

calibration curve as 

described in Section 10.3.3. 

When each calibration standard 

is calculated as an unknown 

using the calibration curve, the 

lowest level standard should be 

within + 50% of the true value. 

All other points should be 

within + 30% of the true value. 

Section 

9.3.1 

Laboratory Reagent 

Blank (LRB) 

Daily, or with each Analysis 

Batch of up to 20 field 

samples, whichever is more 

frequent. 

Demonstrate that all target 

analytes are below 1/3 the 

MRL, and that possible 

interference from reagents and 

glassware do not prevent 

identification and quantitation 

of target analytes. 

Section 

10.4 

Continuing 

Calibration Check 

(CCC) 

Verify initial calibration by 

analyzing a low-level CCC at 

the beginning of each 

Analysis Batch. Subsequent 

CCCs are required after 

every 10 field samples, and 

after the last field sample in a 

batch. 

The lowest level CCC must be 

within + 50% of the true value. 

All other points must be within 

+ 30% of the true value. 

Results for field samples that 

are not bracketed by acceptable 

CCCs are invalid. 
536 -31

TABLE 10. ONGOING QUALITY CONTROL REQUIREMENTS (CONTINUED) 

Method 

Reference 

Requirement Specification and 

Frequency 

Acceptance Criteria 

Section 

9.3.4 

Internal Standard 

(IS) 

Isotopically labeled internal 

standards are added to all 

standards and samples. 

Peak area counts for each IS 

must be within ± 50% of the 

average peak areas in the initial 

calibration. 

Section 

9.3.5 

Laboratory 

Fortified Sample 

Matrix (LFSM) 

Analyze one LFSM per 

Analysis Batch. Fortify the 

LFSM with target analytes at 

a concentration close to but 

greater than the native 

concentrations (if known). 

Calculate LFSM recoveries. 

For LFSMs fortified at 

concentrations ≤ MRL, the 

result should be within + 50% 

of the true value. All other 

LFSMs should be within + 30% 

of the true value. 

Section 

9.3.6 

Laboratory 

Fortified Sample 

Matrix Duplicate 

(LFSMD) or 

Laboratory 

Duplicate(LD) 

Analyze at least one LFSMD 

or LD with each Analysis 

Batch of up to 20 field 

samples. 

For LFSMDs or LDs, RPDs 

should be ≤ 30%. 

Sections 

9.3.7 

Quality Control 

Sample (QCS) 

Analyze mid-level QCS 

sample when new PDS 

solutions are prepared, or at 

least quarterly if PDS 

solutions are prepared less 

frequently. 

Results must be + 30% of the 

true value. 
FIGURE 1. NORMALIZED, OVERLAID CHROMATOGRAMS OF LC/ESI-MS/MS TRANSITIONS FOR M

ANALYTES FORTIFIED AT 0.5 μg/L IN REAGENT WATER a

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45

0 

100

%

3.82

DACT 

DIA 

DEA 

Simazine 

Cyanazine 

Atrazine 

Propazine 

a The chromatograms for the isotopically-enriched internal standards are not included because they co-elute with 

analytes. 

536 -32