EPA # 815-B-01-002


METHOD 531.2.	 MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND 

N-METHYLCARBAMATES IN WATER BY DIRECT AQUEOUS 

INJECTION HPLC WITH POSTCOLUMN DERIVATIZATION 

Revision 1.0 

September 2001 

M.V. Bassett, S.C. Wendelken, and B.V. Pepich (IT Corporation) 

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

L. Henry (American Water Works Service Company) 

R.L. Graves - Method 531.1, Revision 3.0 (1989)


T. Engels (Battelle Columbus Laboratories)


D.J. Munch (US EPA, Office of Water) - National Pesticide Survey Method 5, Revision 2.0


(1987)


D.L. Forest - Method 531, Revision 1.0 (1985)


TECHNICAL SUPPORT CENTER


OFFICE OF GROUND WATER AND DRINKING WATER


U. S. ENVIRONMENTAL PROTECTION AGENCY


CINCINNATI, OHIO 45268


531.2 - 1

MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND 

N-METHYLCARBAMATES IN WATER BY DIRECT AQUEOUS INJECTION HPLC 

WITH POSTCOLUMN DERIVATIZATION 

1. SCOPE AND APPLICATION 

1.1 	 This is a high performance liquid chromatographic (HPLC) method applicable to 

the determination of certain N-methylcarbamoyloximes and N-methylcarbamates 

in finished drinking waters. The following compounds can be determined using 

this method:


Analyte


Aldicarb


Aldicarb sulfone


Aldicarb sulfoxide


Carbaryl


Carbofuran


3-Hydroxycarbofuran


Methiocarb


Methomyl


1-Naphthol


Oxamyl


Propoxur


Chemical Abstracts Service (CAS) 

Registry Number 

116-06-3 

1646-88-4 

1646-87-3 

63-25-2 

1563-66-2 

16655-82-6 

2032-65-7 

16752-77-5 

90-15-3 

23135-22-0 

114-26-1 

1.2 	 Detection Limits are compound, instrument, and matrix dependent. The 

Detection Limit is defined as the statistically calculated minimum concentration 

that can be measured with 99% confidence that the reported value is greater than 

zero.(1) Experimentally determined Detection Limits for the above listed analytes 

are provided in Section 17, Tables 2 - 4. The Detection Limit differs from, and is 

lower than the Minimum Reporting Level (MRL) (Sect. 3.15). The concentration 

range for target analytes in this method was evaluated between 0.2 ug/L and 10 

ug/L. Precision and accuracy data and sample holding time data are presented in 

Section 17, Tables 5 - 8. 

531.2 - 2

1.3 	 This method is restricted to use by or under the supervision of analysts skilled in 

HPLC analysis and the interpretation of HPLC chromatograms. 

2. SUMMARY OF METHOD 

2.1	 A water sample is filtered. Method analytes are chromatographically separated by 

injecting an aliquot (up to 1000 uL) into a high performance liquid 

chromatographic (HPLC) system equipped with a reversed phase (C18) column. 

After elution from the column, the analytes are hydrolyzed in a postcolumn 

reaction with 0.075 N sodium hydroxide (NaOH) at 80 to 100 °C to form methyl 

amine. The methyl amine is reacted with o-phthalaldehyde (OPA) and 2-

mercaptoethanol (or N,N-dimethyl-2-mercaptoethylamine) to form a highly 

fluorescent isoindole which is detected by a fluorescence detector.(2) Analytes are 

quantitated using the external standard technique. 

3. DEFINITIONS 

3.1	 ANALYSIS BATCH – A set of samples prepared and analyzed on the same 

instrument during a 24-hour period. An analysis batch begins with a Continuing 

Calibration Check (CCC) at or below the MRL. Subsequent CCCs are analyzed 

every 10 samples, should alternate between medium and high concentrations, and 

must end the analysis batch. An analysis batch is limited to 20 field samples. 

Laboratory Reagent Blanks (LRBs), Laboratory Fortified Sample Matrices 

(LFSMs), Laboratory Fortified Sample Matrix Duplicates (LFSMDs), Field 

Duplicates (FDs), and CCCs are not counted as samples. Required batch QC 

samples include: LRB, CCC, LFSM, and either a FD or a LFSMD. 

If a sample(s) in an analytical batch needs to be reloaded for analysis within 48 

hours of the start of the original analysis batch, it may be considered part of the 

original analysis batch. The reanalyzed sample(s) have the same QC requirements 

as above. However, reanalysis of the LFSM, and FD or LFMD are not required if 

they were already analyzed and passed QC requirements. Any newly prepared 

samples would require a full set of QC samples. 

3.2	 SURROGATE ANALYTE (SUR) – A pure analyte, which chemically resembles 

target analytes and is extremely unlikely to be found in any sample. This analyte 

is added to a sample aliquot in known amount(s) before filtration or other 

processing and is measured with the same procedures used to measure other 

sample components. The purpose of the SUR is to monitor method performance 

with each sample. 

3.3	 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or 

other blank matrix that is treated exactly as a sample including exposure to all 

531.2 - 3

glassware, equipment, solvents and reagents, sample preservatives, and 

surrogates that are used in the analysis batch. The LRB is used to determine if 

method analytes or other interferences are present in the laboratory environment, 

the reagents, or the apparatus. 

3.4	 LABORATORY FORTIFIED BLANK (LFB) – An aliquot of reagent water or 

other blank matrix to which known quantities of the method analytes and all the 

preservation compounds are added in the laboratory. The LFB is analyzed exactly 

like a sample, and its purpose is to determine whether the methodology is in 

control, and whether the laboratory is capable of making accurate and precise 

measurements. For this direct injection method, a LFB is the same as a 

Continuing Calibration Check standard (CCC - Sect. 3.12). 

3.5	 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of a 

preserved field sample to which known quantities of the method analytes are 

added in the laboratory. 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. 

3.6	 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A 

second aliquot of the field sample used to prepare the LFSM, fortified, processed, 

and analyzed identically to the LFSM. The LFSMD is used instead of the Field 

Duplicate to access method precision when the occurrence of target analytes are 

low. 

3.7	 LABORATORY DUPLICATES (LD1 and LD2) – Two aliquots of the same 

sample taken in the laboratory and analyzed separately with identical procedures. 

Analyses of LD1 and LD2 indicate precision associated with laboratory 

procedures, but not with sample collection, preservation, or storage procedures. 

3.8	 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the 

same time and place under identical circumstances, and treated exactly the same 

throughout field and laboratory procedures. Analyses of FD1 and FD2 give a 

measure of the precision associated with sample collection, preservation, and 

storage, as well as laboratory procedures. 

3.9	 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing 

one or more method analytes prepared in the laboratory using assayed reference 

materials or purchased from a reputable commercial source. 

3.10	 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution 

containing the analytes prepared in the laboratory from stock standard solutions 

531.2 - 4

and diluted as needed to prepare calibration solutions and other needed analyte 

solutions. 

3.11	 CALIBRATION STANDARD (CAL) – A solution prepared from the primary 

dilution standard solution and/or stock standard solution, and the surrogate 

analytes. The CAL solutions are used to calibrate the instrument response with 

respect to analyte concentration. 

3.12	 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard 

containing the method analytes and surrogate(s), which is analyzed periodically to 

verify the accuracy of the existing calibration for those analytes. 

3.13	 QUALITY CONTROL SAMPLE (QCS) – A sample prepared using a PDS of 

methods analytes that is obtained from a source external to the laboratory and 

different from the source of calibration standards. The second source PDS and the 

surrogate PDS are used to fortify the QCS at a known concentration. The QCS is 

used to check calibration standard integrity. 

3.14	 DETECTION LIMIT – 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 of precision 

(Sect. 9.2.4), and accurate quantitation is not expected at this level. (1) 

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

be reported as a quantitated value for a target analyte in a sample following 

analysis. This defined concentration can be no lower than the concentration of the 

lowest continuing calibration standard for that analyte and can only be used if 

acceptable quality control criteria for this standard are met. 

3.16	 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided 

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

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

4. INTERFERENCES 

4.1	 All glassware must be meticulously cleaned. Wash glassware with detergent and 

tap water, rinse with tap water, followed by reagent water. A final rinse with 

solvents may be needed. In place of a solvent rinse, non-volumetric glassware can 

be heated in a muffle furnace at 400 o

C for 2 hours. Volumetric glassware should 

not be heated above 120 o

C. 

4.2	 Method interferences may be caused by contaminants, especially amines and 

ammonia, in solvents, reagents (including reagent water), sample bottles and caps, 

and other sample processing hardware that lead to discrete artifacts and/or 

531.2 - 5

elevated baselines in the chromatograms. The samples or analytical system may 

be contaminated from being handled with bare fingers. All items such as these 

must be routinely demonstrated to be free from interferences (less than 1/3 the 

MRL for each target) under the conditions of the analysis by analyzing laboratory 

reagent blanks as described in Section 9. Subtracting blank values from sample 

results is not permitted. 

5. SAFETY 

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

precisely defined; each chemical compound should be treated as a potential health 

hazard, and exposure to these chemicals should be minimized. The laboratory is 

responsible for maintaining a current awareness file of OSHA regulations 

regarding the safe handling of the chemicals specified in this method. A reference 

file of MSDSs should also be made available to all personnel involved in the 

chemical analysis. Additional references to laboratory safety are available.(3-5) 

5.2	 Pure standard materials and stock standards of these compounds should be 

handled with suitable protection to skin and eyes. Care should be taken not to 

breathe the vapors or ingest the materials. 

6. 	 EQUIPMENT AND SUPPLIES (All specifications are suggested. Brand names and/or 

catalog numbers are included for illustration only.) 

6.1	 SAMPLE CONTAINERS – Amber glass bottles fitted with PTFE 

(polytetrafluoroethylene) lined screw caps. 

6.2	 VIALS – Screw cap or crimp top glass autosampler vials with PTFE faced septa, 

amber or clear. 

6.3	 VOLUMETRIC FLASKS – Class A, various sizes used for preparation of 

standards. 

6.4 GRADUATED CYLINDERS – Various sizes. 

6.5 MICRO SYRINGES – Various sizes. 

6.6 BALANCE – Analytical, capable of accurately weighing to 0.0001 g. 

6.7	 DISPOSABLE SYRINGES – 5 to 30 mL (B-D Cat.#: 309603, 309650 or 

equivalent) size, used to filter sample extracts before analysis. 

531.2 - 6

6.8	 FILTERS – Disposable filters used to filter samples before analysis (Millipore 

0.22 um PVDF membrane, Cat. #: SLGV 013 NL or equivalent). 

6.9	 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

INSTRUMENTATION 

6.9.1	 HPLC SYSTEM – Capable of reproducibly injecting up to 1000-uL 

aliquots, and performing binary or ternary linear gradients at a constant 

flow rate near the flow rate used for development of this method, e.g., 1.5 

mL/min. A ternary gradient was used to achieve the separation pictured in 

Figure 1. However, a binary gradient may be used as long as the minimum 

resolution is achieved as specified in Sect. 9.10. The use of a column 

heater is strongly recommended. During method development, the column 

was heated to 30 °C to help insure stable retention times. Column 

temperature may be adjusted as long as column performance is not 

compromised. 

6.9.2	 POSTCOLUMN REACTION SYSTEM – Capable of mixing reagents 

into the mobile phase. Reactor should be constructed using 

polyetheretherketone (PEEK) or PTFE tubing and equipped with two 

pumps capable of delivering 0.5 mL/min of each reagent; mixing tees; and 

two reaction coils. Postcolumn system manufacturers recommend 

different reaction coil temperatures for the carbamate hydrolysis reaction. 

Therefore, the first reaction coil temperature may range from 80 to 100 °C. 

Analyte signal can increase with temperature over this temperature range; 

however, analysts should be aware that with some systems, baseline noise 

can also increase with increasing temperatures. If temperatures greater 

than 95 °C are used, a slight backpressure must be maintained on the 

system to prevent boiling of the post column eluent. This may be 

accomplished by use of a restrictor placed in line after the detector. The 

second reaction takes place at ambient temperatures. Method performance 

data were collected with two postcolumn reaction modules: the Waters 

Postcolumn Carbamate System and the Pickering Model 5200 Carbamate 

System, both with a reactor temperature of 80 o

C. 

6.9.3	 HPLC DETECTOR – A fluorescence detector capable of excitation at 

approximately 340 nm and detection of emission energy at approximately 

465 nm. Optimum excitation and emission wavelengths may vary slightly 

for each system. For the development of this method, Waters Model 474 

and Model 2475 scanning fluorescence detectors were used. 

6.9.4	 ANALYTICAL COLUMN – For the development of this method, an 

HPLC “carbamate” column (3.9 × 150 mm) packed with 4 um d

phase particles (Waters Cat. # : WAT035577) was used. Any column that 

p C18 solid 

531.2 - 7

provides adequate resolution, peak shape, capacity, accuracy, and 

precision (Sect. 9) may be used. 

6.9.5	 HPLC DATA SYSTEM - A computerized data system is recommended 

for data acquisition and processing. A Waters Millennium software 

system was used to generate all data contained in the Section 17 tables. 

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 shall conform to 

the specifications of the Committee on Analytical Reagents of the American 

Chemical Society, where such specifications are available. Other grades may be 

used, provided it is first determined that the reagent is of sufficiently high purity 

to permit its use without lessening the quality of the determination. Reagents of 

lesser purity may also affect instrument performance, such as clogging of the post 

column reactor. 

7.1.1	 REAGENT WATER – Purified water which does not contain any 

measurable quantities of any target analytes or interfering compounds 

greater than 1/3 the MRL for each compound of interest. 

7.1.2	 PRESERVED REAGENT WATER – Reagent water which has sample 

preservation reagents added in the same concentrations as the samples. To 

prepare 1 liter of preserved reagent water, add a sufficient amount of 

potassium dihydrogen citrate (Sect. 7.1.11.1) to yield a concentration of 

9.2 to 9.5 g/L and sodium thiosulfate (Sect. 7.1.11.2) to yield a 

concentration in the range of 80 to 320 mg/L to a graduated bottle or 

volumetric flask. Fill to the 1 L mark with reagent water. 

7.1.3	 ACETONITRILE (CH3CN, CAS#: 75-05-8) – High purity, demonstrated 

to be free of analytes and interferences (HPLC grade or better). 

7.1.4	 METHANOL (CH3OH, CAS#: 67-56-1) – High purity, demonstrated to 

be free of analytes and interferences (HPLC grade or better). 

7.1.5	 SODIUM HYDROXIDE (NaOH, CAS#: 1310-73-2), 50% (w/w) solution 

(Fisher Cat. #: SS254-500 or equivalent). Due to the hygroscopic nature 

of NaOH pellets, a 50% (w/w) solution is used in this method for the 

preparation of reagents. 

7.1.6	 POSTCOLUMN REAGENT #1 (hydrolysis solution) – Sodium 

hydroxide, 0.075 N. Dilute 4 mL of 50% w/w sodium hydroxide (NaOH) 

531.2 - 8

solution to 1 L with reagent water. The concentration of the hydrolysis 

solution can dramatically effect the analyte response. Filter and degas 

with helium just before use. 

7.1.7	 PREPARATION OF THE OPA SOLUTION (to be used in preparation of 

Postcolumn Reagent #2, Sect. 7.1.10) – Dissolve 100 + 10 mg of ophthaldehyde (OPA, CAS#: 643-79-8) in 5 - 10 mL of methanol. 

7.1.8	 PREPARATION OF THE OPA DILUTENT (to be used in preparation of 

Postcolumn Reagent #2, Sect. 7.1.10) – Use one of the following 

formulations in the preparation of the preliminary OPA dilutent. 

Performance data presented in Section 17 were collected using the sodium 

tetraborate formulation. Method performance was checked with the boric 

acid formulation and found to be equivalent. OPA dilutent is 

commercially available. 

7.1.8.1 Prepared using sodium tetraborate : Dissolve 19.1 g of sodium 

tetraborate decahydrate (Na2B4O7�10H2O, CAS#: 1303-96-4) in a 1 

L volumetric flask. Bring the volume up to 1.0 L with reagent 

water to make a 0.05 M solution. The sodium borate will dissolve 

in less than 2 hours if a stir bar is used. Filter and degas prior to 

preparation of Postcolumn Reagent #2 (Sect. 7.1.10). 

OR 

7.1.8.2 Prepared using boric acid: Dissolve 3.0 g of boric acid (H3BO3, 

CAS#: 10043-35-3) in a 1 L volumetric flask in approximately 800 

mL of reagent water. Add 1.2 mL of a 50% (w/w) NaOH solution. 

Bring the volume up to 1.0 L with reagent water. Filter and degas 

prior to preparation of Postcolumn Reagent #2 (Sect. 7.1.10). 

7.1.9	 PREPARATION OF THE NUCLEOPHILIC SOLUTION (to be used in 

preparation of Postcolumn Reagent #2, Sect. 7.1.10) – Use one of the 

following formulations in the preparation of the nucleophilic solution. 

Either one of these compounds reacts with OPA and the target 

methylamine to form the isoindole detected by the fluorescence detector. 

Both reagents have characteristic strong odors and should be handled in a 

fume hood. 

7.1.9.1	 Prepared using 2-mercaptoethanol (CAS#: 60-24-2): This 

compound is in liquid form and may be used directly in the 

preparation of the derivatization solution, Postcolumn Reagent 

#2 (Sect 7.1.10). Use 1 mL of 2-mercaptoethanol per liter of 

Postcolumn Reagent #2. 

OR 

7.9.1.2 Prepared using N,N-dimethyl-2-mercaptoethylamine531.2 - 9

hydrochloride (CAS#: 13242-44-9): Use approximately 10 mL of 

OPA dilutent (Sect. 7.1.8) to dissolve 2.0 + 0.2 g of N,Ndimethyl-2-mercaptoethylamine-hydrochloride. 

7.1.10	 FINAL PREPARATION OF POSTCOLUMN REAGENT #2 (OPA 

derivatization solution), – Studies have shown that this reagent made with 

either of the two nucleophiles listed above (Sect. 7.1.9) is stable for a 

period of at least 36 hours. However, individual laboratory conditions vary 

and daily preparation of this solution may be necessary. 

7.1.10.1	 Add the dissolved OPA (Sect. 7.1.7) to 1 L of either formulation 

of the OPA dilutent (Sect. 7.1.8.1 or 7.1.8.2), which has been 

filtered and degassed. 

7.1.10.2	 To the 1 liter flask, add either one of the 2 nucleophiles specified 

in Section 7.1.9 (7.1.9.1 or 7.1.9.2). 

7.1.11 SAMPLE PRESERVATION REAGENTS ( See also Sect. 8.1.1) 

7.1.11.1	 Potassium dihydrogen citrate (C6H7KO7, CAS #: 866-83-1) – 

Added to adjust sample pH to ~ 3.8, which acts as a biocide to 

guard against potential degradation of method analytes by 

microorganisms and to prevent chemical degradation. Oxamyl, 

3-hydroxycarbofuran, carbaryl, and methiocarb all hydrolyze in 

neutral or basic waters even when held at refrigerated 

temperatures. 

7.1.11.2	 Sodium thiosulfate (Na2S2O3, CAS#: 7772-98-7) – Acts as a 

dechlorinating agent. Aldicarb and methiocarb are rapidly 

degraded in waters that are not dechlorinated. 

7.2	 STANDARD SOLUTIONS – Standard Solutions may be prepared from certified, 

commercially available solutions or from neat compounds. Compounds used to 

prepare solutions must be 96% pure or greater and the weight may be used 

without correction for purity to calculate the concentration of the stock standard. 

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

and are included as an example. Standards for sample fortification generally 

should be prepared in the smallest volume that can be accurately measured to 

minimize the addition of organic solvent to aqueous samples. Even though 

stability times for standard solutions are suggested in the following sections, 

laboratories should use standard QC practices to determine when Standard 

Solutions described in this section need to be replaced. 

531.2 - 10

7.2.1	 SURROGATE ANALYTE (SUR) STANDARD SOLUTION, 4-

BROMO-3,5-DIMETHYLPHENYL N-METHYLCARBAMATE 

(BDMC, CAS #: 672-99-1) 

7.2.1.1 SUR STOCK SOLUTION – May be purchased as certified 

standard or prepared from neat material. If preparing from neat 

material: accurately weigh approximately 25 to 35 mg of the neat 

SUR to the nearest 0.1 mg into a tared, 5-mL volumetric flask. 

Dilute to the mark with methanol. Stock solutions have been 

shown to be stable for 12 months when stored at -10°C or less. 

7.2.1.2 SUR PRIMARY DILUTION STANDARD – Prepare the SUR 

Primary Dilution Standard (PDS) by adding enough of the SUR 

stock standard to a volumetric flask partially filled with methanol 

to make a final concentration near 10 ug/mL when filled to the 

mark with methanol. The PDS has been shown to be stable for 6 

months when stored at -10 °C or less. 

7.2.2	 ANALYTE STANDARD SOLUTIONS – Obtain the analytes listed in the 

table in Section 1.1 as neat or solid standards or as commercially prepared 

ampulized solutions from a reputable standard manufacturer. Prepare the 

Analyte Stock and Primary Dilutions Standards as described below. 

7.2.2.1 ANALYTE STOCK STANDARD SOLUTION – If preparing 

from neat material, accurately weigh approximately 25 to 35 mg of 

pure material to the nearest 0.1 mg into a tared, 5-mL volumetric 

flask. Dilute to the mark with methanol. Repeat for each target 

analyte. 

7.2.2.2 ANALYTE PRIMARY DILUTION STANDARD (PDS) 

SOLUTION – Prepare the analyte PDS by dilution of the target 

analyte stock standards. Add enough of each target stock standard 

to a volumetric flask partially filled with methanol to make a final 

concentration near 10 ug/mL when filled to the mark with 

methanol. A serial dilution of this PDS to make a 1.0 ug/mL 

solution is useful for low level spiking. The PDSs have been 

shown to be stable for 6 months when stored at -10 °C or less. 

7.2.3	 CALIBRATION STANDARDS (CAL) – At least 5 calibration 

concentrations will be required to prepare the initial calibration curve 

(Sect. 10.2). Prepare the calibration standards over the concentration 

range of interest from dilutions of the Analyte PDSs in preserved reagent 

531.2 - 11

1

2

3

4

5

6

water (7.1.2), filtering the CAL standards in the same manner as the 

samples (Sect. 11.1.4). The lowest concentration of calibration standard 

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

The calibration standards for the development of this method were 

prepared as specified below. Calibration standards must be prepared using 

preserved reagent water (Sect. 7.1.2). An example of the dilutions used to 

prepare the CALs used to collect the data in Section 17 are shown in the 

table below. These standards may be also be used as CCCs. If stored, the 

aqueous standards must be stored in amber glass and refrigerated in the 

same manner as the samples. 

PREPARATION OF CALIBRATION (CAL) CURVE STANDARDS 

CAL 

Level 

Analyte 

PDS Conc. 

(ug/mL) 

Vol. of 

Analyte PDS 

(uL) 

Vol. of 

10 ug/mL 

SUR PDS 

(uL) 

Final Vol. 

of 

CAL Std. 

(mL) 

Final Conc. 

of CAL 

Std. 

(ug/L) 

Final Conc. 

of SUR 

Std.

(ug/L) 

1.0 5.0 5.0 25 0.20 2.00 

1.0 12.5 5.0 25 0.50 2.00 

1.0 25.0 5.0 25 1.00 2.00 

10.0 5.0 5.0 25 2.00 2.00 

10.0 12.5 5.0 25 5.00 2.00 

10.0 25.0 5.0 25 10.0 2.00 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

8.1 SAMPLE BOTTLE PREPARATION 

8.1.1	 Prior to shipment to the field, preservatives listed in Section 7.1.11 must 

be added as dry solids to each amber bottle fitted with a PTFE lined screw 

cap. A 40 or 60-mL sample bottle is recommended. Add a sufficient 

amount of potassium dihydrogen citrate to yield a concentration in the 

sample of 9.2 to 9.5 g/L. Potassium dihydrogen citrate buffers sample pH 

to ~3.8 to prevent hydrolysis of oxamyl, 3-hydroxycarbofuran, carbaryl, 

and methiocarb. Add sodium thiosulfate to yield a sample concentration 

in the range of 80 to 320 mg/L. The concentration of 80 mg/L of sodium 

thiosulfate is adequate for dechlorination, but difficult to measure if a 

small (e.g., 40-mL size) sample bottle is chosen. Method performance 

was tested using up to 320 mg/L without any adverse effect. Sodium 

thiosulfate eliminates the residual free chlorine in the samples, which 

531.2 - 12

rapidly degrades aldicarb and methiocarb. 

8.1.2	 Grab samples must be collected in accordance with conventional sampling 

practices (6)

. Sample bottles must not be prerinsed with sample before 

collection. Doing so will wash out the preservatives added to the bottles 

prior to shipment. 

8.2 SAMPLE COLLECTION 

8.2.1	 When sampling from a cold water tap, remove the aerator so that no air 

bubbles will be trapped in the sample. Open the tap, and allow the system 

to flush until the water temperature has stabilized (usually about 3-5 

minutes). Collect samples from the flowing system. 

8.2.2	 When sampling from an open body of water, fill a wide-mouth bottle or 

beaker with sample from a representative area, and carefully fill sample 

bottles from the container. Sampling equipment, including automatic 

samplers, must be free of plastic tubing, gaskets, and other parts that may 

leach interfering analytes into the water sample. 

8.2.3	 If sampling waters high in colloidal iron, filtration of the sample may be 

necessary prior to preservation in the field to help prevent the precipitation 

of the iron in the reactor. 

8.2.4	 Fill sample bottles, taking care not to flush out the sample preservation 

reagents. Samples do not need to be collected headspace free. 

8.2.5	 After collecting the sample, cap carefully to avoid spillage, and agitate by 

hand for 1 minute. Keep samples sealed from collection time until 

analysis. 

8.3	 SAMPLE SHIPMENT AND STORAGE – All samples should be iced during 

shipment and must not exceed 10 o

C during the first 48 hours after collection. 

Samples should be confirmed to be at or below 10 o

C when they are received at 

the laboratory. Samples stored in the lab must be held at or below 6 o

C and 

protected from light until analysis. Samples should not be frozen. 

8.4	 SAMPLE AND EXTRACT HOLDING TIMES – Results of the sample storage 

stability study of all method analytes indicated that all compounds are stable for 

28 days in water samples that are collected, dechlorinated, preserved, shipped and 

stored as described in Sections 8.1 - 8.3. Samples must be analyzed within 28 

days. 

9. QUALITY CONTROL 

531.2 - 13

9.1	 Quality control (QC) requirements include the Initial Demonstration of 

Capability, the determination of the Detection Limit, and subsequent analysis in 

each analysis batch of a Laboratory Reagent Blank (LRB), Continuing Calibration 

Check Standards (CCC), a Laboratory Fortified Blank (LFB), a Laboratory 

Fortified Sample Matrix (LFSM), and either a Laboratory Fortified Sample Matrix 

Duplicate (LFSMD) or a Field Duplicate Sample. This section details the specific 

requirements for each QC parameter. The QC criteria discussed in the following 

sections are summarized in Section 17, Tables 10 and 11. These criteria are 

considered the minimum acceptable QC criteria, and laboratories are encouraged 

to institute additional QC practices to meet their specific needs. 

9.2	 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – Requirements for the 

Initial Demonstration of Capability are described in the following sections and 

summarized in Section 17, Table 10. 

9.2.1	 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND – 

Before any field samples are analyzed, and any time a new set of reagents 

is used, it must be demonstrated that a laboratory reagent blank is 

reasonably free of contamination and that the criteria in Section 9.4 are 

met. 

9.2.2	 INITIAL DEMONSTRATION OF ACCURACY – Prior to the analysis of 

the IDC samples, verify calibration accuracy with the preparation and 

analysis of a mid-level QCS as defined in Section 9.11. If the analyte 

recovery is not + 30% of the true value, the accuracy of the method is 

unacceptable. The source of the problem must be identified and corrected. 

After the accuracy of the calibration has been verified, prepare and analyze 

4-7 replicate LFBs (or CCCs in this method) fortified at 2 ug/L, or near the 

mid-range of the initial calibration curve, according to the procedure 

described in Section 11. Sample preservatives as described in Section 

8.1.1 must also be added to these samples. The average recovery of the 

replicate values must be within ± 20% of the true value. 

9.2.3 INITIAL DEMONSTRATION OF PRECISION – Using the same set of 

replicate data generated for Section 9.2.2, calculate the standard deviation 

and percent relative standard deviation of the replicate recoveries. The 

relative standard deviation (%RSD) of the results of the replicate analyses 

must be less than 20%. 

9.2.4	 DETECTION LIMIT DETERMINATION – Prepare and analyze at least 7 

replicate LFBs at a concentration estimated to be near the Detection Limit 

over at least 3 days using the procedure described in Section 11. This 

fortification level may be estimated by selecting a concentration with a 

531.2 - 14

signal of 2 to 5 times the noise level. The appropriate concentration will 

be dependent upon the sensitivity of the HPLC system being used. Sample 

preservatives as described in Section 8.1.1 must be added to these 

samples. Calculate the Detection Limit using the equation 

Detection Limit = St( n - 1, 1 - alpha = 0.99) 

where 

t(n-1,1-alpha = 0.99) = Student's t value for the 99% confidence level 

with n-1 degrees of freedom 

n = number of replicates, and 

S = standard deviation of replicate analyses. 

NOTE: Calculated Detection Limits need only be less than 1/3 of the 

laboratory’s MRL to be considered acceptable. Do not subtract 

blank values when performing Detection Limit calculations. The 

Detection Limit is a statistical determination of precision only.(1) 

If the Detection Limit replicates are fortified at a low enough 

concentration, it is likely that they will not meet the precision and 

accuracy criteria for CCCs, and may result in a calculated 

Detection Limit that is higher than the fortified concentration. 

Therefore, no precision and accuracy criteria are specified. 

9.2.5	 METHOD MODIFICATIONS – The analyst is permitted to modify HPLC 

columns and conditions prior to the postcolumn reaction. The analyst is 

also allowed to modify the surrogate standard. Each time such method 

modifications are made, the analyst must repeat the procedures of the IDC 

(Sect. 9.2). 

9.3	 Minimum Reporting Level (MRL) – The MRL is the threshold concentration of 

an analyte that a laboratory can expect to accurately quantitate in an unknown 

sample. The MRL should not be established at an analyte concentration that is 

less than either three times the Detection Limit or a concentration which would 

yield a response less than a signal-to-noise (S/N) ratio of five. Depending upon 

the study’s data quality objectives it may be set at a higher concentration. 

Although the lowest calibration standard must be at or below the MRL, the 

MRL must never be established at a concentration lower than the lowest 

calibration standard. 

9.4	 LABORATORY REAGENT BLANK (LRB) – This is a direct injection method 

without a conventional extraction. An LRB is required with each analysis batch 

(Sect. 3.1) of samples to determine any background system contamination. If 

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

531.2 - 15

would prevent the determination of that analyte, determine the source of 

contamination and eliminate the interference before processing samples. 

Background contamination must be reduced to an acceptable level before 

proceeding. Background from method analytes or contaminants that interfere 

with the measurement of method analytes must be below 1/3 the MRL. If the 

target analytes are detected in the LRB at concentrations equal to or greater than 

this level, then all data for the problem analyte(s) must be considered invalid for 

all samples in the analysis batch. 

9.5	 CONTINUING CALIBRATION CHECK (CCC) – A CCC is prepared in the 

same manner as the initial calibration solutions (Sect. 7.2.3), using preserved 

reagent water (Sect. 7.1.2) and filtering in the same manner as the samples 

(11.1.4). It is analyzed during an analysis batch at a required frequency to confirm 

that the instrument meets initial calibration criteria. See Section 10.3 for 

concentration requirements, frequency requirements, and acceptance criteria. 

9.6	 LABORATORY FORTIFIED BLANKS – For this direct injection method, a 

CCC is the same as an LFB. Consequently, the analysis of an LFB is not required. 

9.7	 SURROGATE RECOVERY – The surrogate standard is fortified into all samples, 

blanks, LRBs, and LFSMs and LFSMDs prior to sample filtration. They are also 

added to the calibration curve and calibration check standards. The surrogate is a 

means of assessing method performance from preparation and filtration to final 

chromatographic measurement. 

9.7.1	 When surrogate recovery from a sample, blank, or CCC is less than 70% 

or greater than 130%, check (1) calculations to locate possible errors, (2) 

standard solutions for degradation, (3) contamination, and (4) instrument 

performance. If those steps do not reveal the cause of the problem, 

reanalyze the sample. 

9.7.2	 If the reanalysis meets the surrogate recovery criterion, report only data for 

the reanalyzed sample. 

9.7.3	 If the sample reanalysis fails the 70-130% recovery criterion, the analyst 

should check the calibration by reinjecting the most recently acceptable 

calibration standard. If the calibration standard fails the criteria of Section 

10.3, recalibration is in order per Section 10.2. If the calibration standard 

is acceptable, preparation (which includes spiking with surrogate and 

filtration) and analysis of the sample should be repeated provided the 

sample is still within the holding time. If this sample reanalysis also fails 

the recovery criterion, report all data for that sample as suspect due to 

surrogate recovery. 

531.2 - 16

9.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of an 

LFSM is required in each analysis batch and is used to determine that the sample 

matrix does not adversely affect method accuracy. 

precision is accomplished by analysis of a Field Duplicate (Sect. 9.9), however, 

infrequent occurrence of target analytes would hinder this assessment. 

occurrence of target analytes in the samples is infrequent, or if historical trends are 

unavailable, a second LFSM, or LFMSD, must be prepared and analyzed from a 

duplicate of the field sample. sis batches that contain LFSMDs will not 

require the analysis of a Field Duplicate. a variety of different sample matrices 

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

water sources, method performance should be established for each. 

LFSM data should be documented for all routine sample sources for the 

laboratory. 

9.8.1 Within each analysis batch, a minimum of one field sample is fortified as 

an LFSM for every 20 samples processed. d by 

spiking a sample with an appropriate amount of the appropriate Analyte 

PDS (Sect. 7.2.2.2). 

matrix background concentration, if known. Use historical data or rotate 

through a range of concentrations when selecting a fortifying 

concentration. mple that has already 

been analyzed aids in the selection of appropriate spiking levels. 

9.8.2 Calculate the (R) for each analyte using the equation 

where 

A = measured concentration in the fortified sample 

B = measured concentration in the unfortified sample, and 

C = fortification concentration. 

9.8.3 Analyte recoveries may exhibit a matrix bias. 

above their native concentration, recoveries should range between 70 -

130%. tion at the MRL, 50 to 150% recoveries are 

acceptable. If the accuracy of 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. 

9.9 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX 

Assessment of method 

If the 

Analy

If

Over time, 

The LFSM is prepare

Select a spiking concentration at least twice the 

Selecting a duplicate bottle of a sa

percent recovery

For samples fortified at or 

For LFSM fortifica

531.2 - 17

DUPLICATE (FD or LFSMD) – Within each analysis batch, a minimum of one 

Field Duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD) 

must be analyzed. Duplicates check the precision associated with sample 

collection, preservation, storage, and laboratory procedures. If target analytes are 

not routinely observed in field samples, an LFSMD must be analyzed rather than a 

FD. 

9.9.1	 Calculate the relative percent difference (RPD) for duplicate 

measurements (FD1 and FD2) using the equation 

9.9.2	 If an LFSMD is analyzed instead of a Field Duplicate, calculate the 

relative percent difference (RPD) for duplicate LFSMs (LFSM and 

LFSMD) using the equation 

9.9.3	 RPDs for FDs and duplicate LFSMs should fall in the range of ± 30% for 

samples fortified at or above their native concentration. Greater variability 

may be observed when LFSMs are spiked near the MRL. At the MRL, 

RPDs should fall in the range of 0 to 50% for samples fortified at or above 

their native concentration. If the accuracy of any analyte falls outside the 

designated range, and the laboratory performance for that analyte is shown 

to be in control in the LFB, 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 

9.10	 RESOLUTION CHECK – The resolution of peaks in a calibration standard or 

CCC near the mid level of calibration must be monitored in each analytical batch. 

During the development of this method, the 2 ug/L level was monitored. Closely 

eluting peaks that are not baseline resolved must have a resolution (Rs) of 1.0 or 

greater using the equation (7) 

531.2 - 18

where 

t1 and t2 = retention times of the first and second adjacent peaks 

W0.5,1 and W0.5,2 = widths of the adjacent peaks at half height. 

Resolution must be monitored once for every 24-hour analytical batch and may be 

monitored at any time during the 24-hour period. It is recommended that 

resolution be checked prior to sample analysis, especially if the system in use has 

a history of resolution problems. If a resolution check fails, all samples must be 

reanalyzed after the problem is corrected, including the QC samples. 

9.11	 QUALITY CONTROL SAMPLE (QCS) – During the analysis of the IDC (Sect. 

9.2), each time that new analyte standard solutions (Sects. 7.2.2.1 and 7.2.2.2) are 

prepared, or at least quarterly, analyze a QCS from a source different from the 

source of the calibration standards. The QCS is fortified into preserved reagent 

water (Sect. 7.1.2) and analyzed as a CCC. The acceptance criteria is the same as 

the CCC criteria at mid-level; the calculated amount for each analyte must be + 

30% of the true value. If measured analyte concentrations are not of acceptable 

accuracy, check the entire analytical procedure to locate and correct the problem 

source. 

10. CALIBRATION AND STANDARDIZATION 

10.1	 After initial calibration is successful, a Continuing Calibration Check (CCC) is 

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

sample (Sect. 10.3). Initial calibration should be repeated each time a major 

instrument modification or maintenance is performed. 

10.2 INITIAL CALIBRATION 

10.2.1	 Establish HPLC operating parameters equivalent to the suggested 

conditions in Section 17, Table 1, including the postcolumn reactor. The 

system is calibrated using the external standard technique. A fluorescence 

detector was used with the excitation and emission wavelengths optimized 

at 340 and 465 nm, respectively. Other HPLC conditions may be used as 

long as all QC requirements in Section 9 are met. 

10.2.2	 Prepare a set of at least 5 calibration standards as described in Section 

7.2.3. The lowest concentration calibration standard must be at or below 

the MRL, which may depend on system sensitivity. It is recommended 

that at least four of the Calibration Standards are at a concentration greater 

than or equal to the MRL. 

10.2.3 Generate a calibration curve for each analyte by plotting the peak response 

531.2 - 19

(area is recommended) against analyte concentration. The instrument used 

during method development yielded linear curves for the target analytes 

over the concentration range of interest. However, data may be fit with 

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

(response vs. concentration). Alternately, if the ratio of the analyte peak 

area to concentration (or response factor) is relatively constant (%RSD < 

30%) an average response factor may be used to calculate analyte 

concentration. 

10.2.4	 When quantitated using the initial calibration curve, each calibration point, 

except the lowest point, for each analyte should calculate to be 70-130% of 

its true value. The lowest calibration point should calculate to be 50-150% 

of its true value. Failure to meet this criteria may indicate future difficulty 

in meeting CCC QC requirements during the analysis batch. 

10.3	 CONTINUING CALIBRATION CHECK (CCC) – Minimum daily calibration 

verification is as follows. Verify the initial calibration at the beginning and end of 

each group of analyses, and after every tenth sample during analyses. (In this 

context, a “sample” is considered to be a field sample. LRBs, CCCs, LFSMs, and 

LFSMDs are not counted as samples. The beginning CCC each day must be at or 

below the MRL in order to verify instrument sensitivity prior to any analyses. 

Subsequent CCCs should alternate between a medium and high concentration 

standard. 

10.3.1	 Inject an aliquot of the appropriate concentration calibration solution and 

analyze with the same conditions used during the initial calibration. 

10.3.2	 Calculate the concentration of each analyte and surrogate in the check 

standard. The calculated amount for each analyte for medium and high 

level CCCs must be ± 30% of the true value. The calculated amount for 

the lowest calibration point for each analyte must be within ± 50% of the 

true value. If these conditions do not exist, then all data for the problem 

analyte must be considered invalid, and remedial action should be taken 

which may require recalibration. Any field or QC samples that have been 

analyzed since the last acceptable calibration verification should be 

reanalyzed after adequate calibration has been restored. 

11. PROCEDURE 

11.1 SAMPLE PREPARATION 

11.1.1	 Samples are preserved, collected and stored as presented in Section 8. All 

field and QC samples must contain the preservatives listed in Section 

8.1.1. Measure a 25-mL aliquot of sample into a volumetric flask. 

531.2 - 20

Alternate volumes are allowed as long as they are determined using a glass 

volumetric flask or pipette. When the volumes of SUR and analyte PDS 

added to the field and QC samples are kept to a minimum, as described 

below, no volume adjustment is necessary. 

11.1.2	 Add an aliquot of the SUR PDS (Sect. 7.2.1.2) to all samples and mix by 

capping and inverting the sample. If the SUR PDS volume is less than 1% 

of the total volume, no volume adjustment is necessary. For the 

development of this method, the addition of 5.0 uL of a 10 ug/mL SUR 

PDS to a 25-mL sample resulted in a SUR concentration of 2.0 ug/L. 

11.1.3	 If the sample is an LFSM, LFSMD or LFB/CCC, add the necessary 

amount of analyte PDS. If the PDS volume is less than 1% of the total 

volume, no volume adjustment is necessary. Cap and invert each sample 

to ensure all components are properly mixed. 

11.1.4	 Filter samples prior to filling appropriate autosampler vials (Filters as 

specified in Section 6.8 are recommended.). 

11.2 ANALYSIS OF SAMPLES 

11.2.1	 Establish operating conditions as summarized in Table 1 of Section 17 for 

the HPLC system, including the postcolumn reactor. HPLC conditions 

and columns should be optimized prior to the initiation of the IDC. 

Confirm that resolution of target peaks meets the requirements of Section 

9.10. Retention times are given for illustration in Table 2. Specific 

retention times will depend on the column and chromatographic conditions 

chosen and may be modified by the user as long as adequate sensitivity 

and peak resolution are achieved. 

11.2.2	 Load filled autosampler vials into the HPLC autosampler. Start the 

injection sequence. For the development of this method, the injection 

volume ranged between 200 and 1000 uL and depended on system 

sensitivity. 

11.2.3	 Determine optimal excitation and emission wavelengths (if necessary). 

Complete the IDC requirements described in Section 9.2. 

11.2.4	 Establish an appropriate retention time window for each target and 

surrogate to identify them in the QC and field samples. This should be 

based on measurements of actual retention time variation for each 

compound in standard solutions analyzed on the HPLC over the course of 

time. Plus or minus three times the standard deviation of the retention 

time for each compound while establishing the initial calibration and 

531.2 - 21

completing the IDC can be used to calculate a suggested window size; 

however, the experience of the analyst should weigh heavily on the 

determination of the appropriate retention window size. 

11.2.5 Calibrate the system by either the analysis of a calibration curve (Sect. 10. 

2) or by confirming the initial calibration is still valid by analyzing a 

continuing calibration check as described in Section 10.3. Begin 

analyzing field and QC samples at their appropriate frequency by injecting 

the same size aliquots under the same conditions used to analyze the initial 

calibration. Confirm that the system meets the resolution check criteria as 

described in Section 9.10 once for every 24 hour analytical batch. 

11.2.6	 The analyst must not extrapolate beyond the established calibration range. 

If an analyte peak area exceeds the range of the initial calibration curve, 

the extract may be diluted with preserved reagent water (Sect. 7.1.2). 

Acceptable surrogate performance (Sect. 9.7) should be determined from 

the undiluted sample extract. Any dilutions will also affect analyte MRL. 

12. DATA ANALYSIS AND CALCULATION 

12.1	 Identify the method analytes in the sample chromatogram by comparing the 

retention time of the suspect peak to the retention time of an analyte peak in a 

calibration standard. Surrogate retention times should be confirmed to be within 

acceptance limits (Sect. 11.2.3) even if no target compounds are detected. 

Surrogate concentrations need to be calculated and determined to be within QC 

limits (Sect. 9.7). 

12.2	 Calculate the analyte concentrations using the initial calibration curve generated 

as described in Section 10.2. Quantitate only those values that fall between the 

MRL and the highest calibration standard. Samples with target analyte responses 

that exceed the highest standard require dilution and reanalysis (Sect. 11.2.5). 

12.3	 Adjust the calculated concentrations of the detected analytes to reflect the initial 

sample volume and any dilutions performed. 

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

peak identification or poor integration. 

12.5 Analyte concentrations are reported in ug/L (usually to two significant figures). 

13. METHOD PERFORMANCE 

13.1	 PRECISION, ACCURACY, AND DETECTION LIMITS – Detection Limits are 

presented in Section 17, Tables 3-5 and were calculated using the formula present 

531.2 - 22

in Section 9.2.4. Detection Limits were evaluated using two commercially 

available postcolumn systems and two different fluorescence detectors. Single 

laboratory precision and accuracy are presented for three water matrices: reagent 

water (Table 6); chlorinated (finished) surface water (Table 7); and chlorinated 

(finished) ground water (Table 8). 

13.2 	 SAMPLE STORAGE STABILITY STUDIES – Chlorinated (finished) ground 

water samples, fortified with method analytes at 2.0 ug/L, were preserved and 

stored as required in Section 8. The average of triplicate analyses, conducted on 

days 0, 2, 7, 14, and 28 days are presented in Section 17, Table 9. These data 

document the 28-day sample holding time. 

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 NW, Washington, 

D.C., 20036. 

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. However, the Agency 

requires that laboratory waste management practices be conducted consistent with 

all applicable rules and 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, consult “The Waste 

Management Manual for Laboratory Personnel” also available from the American 

Chemical Society at the address in Section 14.1. 

531.2 - 23

16. REFERENCES 

1.	 Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for 

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

2.	 Moye, H.A., Sherrer, S.J., and St. John, P.A., “Dynamic Labeling of Pesticides for High 

Performance Liquid Chromatography: Detection of N-Methylcarbamates and oPhthalaldehyde,” Anal. Lett. 1977, 10, 1049. 

3.	 "OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational 

Safety and Health Administration, OSHA 2206, (Revised, Jan.1976). 

4.	 “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of Health, 

Education, and Welfare, Public Health Service, Center for Disease Control, National 

Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977. 

5.	 “Safety In Academic Chemistry Laboratories,” 3rd Edition, American Chemical Society 

Publication, Committee on Chemical Safety, Washington, D.C., 1979. 

6.	 ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for 

Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986. 

7.	 Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, 2nd 

ed.;Wiley: New York, 1997; pp 22-25. 

531.2 - 24

17.	 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA* 

TABLE 1. INSTRUMENT METHOD CONDITIONS 

Time 

(min) 

%Water %Methanol %Acetonitrile 

initial 88.0 12.0 0.0 

5.30 88.0 12.0 0.0 

5.40 68.0 16.0 16.0 

14.00 68.0 16.0 16.0 

16.10 50.0 25.0 25.0 

20.00 50.0 25.0 25.0 

22.00 88.0 12.0 0.0 

30.00 88.0 12.0 0.0 

Instrument Method Conditions 

Column:	 Waters Carbamate 3.9 × 150 mm packed with 4.0 um C18 

stationary phase. 

Postcolumn Reactor:	 Reaction coil set at 80 oC, flow rate for Postcolumn Reagent #1 

and #2 = 0.5 mL/min (each) for Waters unit, 0.3 mL/min for the 

Pickering unit. 

Fluorescence Detector:	 340 nm excitation, 465 nm emission with a 18 nm band width; 

Gain = 100; Attn. = 16; Response = Standard; 16 uL flow cell. 

HPLC:	 A ternary gradient comprised of water, methanol, and acetonitrile 

with a flow of 1.5 mL/min as shown in the table above. 

*Instrumentation, when specified, does not constitute endorsement. Brand names are included for illustration only. 

531.2 - 25

TABLE 2. RETENTION TIME DATA*


Analyte Retention 

Time (min.) 

STD 

DEV %RSD 

Aldicarb sulfoxide 4.36 0.0092 0.21 

Aldicarb sulfone 5.07 0.0089 0.17 

Oxamyl 5.74 0.0095 0.17 

Methomyl 6.53 0.0077 0.12 

3-Hydroxycarbofuran 9.82 0.013 0.13 

Aldicarb 11.5 0.013 0.11 

Propoxur 14.3 0.020 0.14 

Carbofuran 14.8 0.024 0.16 

Carbaryl 17.0 0.026 0.16 

1-Naphthol 18.6 0.019 0.10 

Methiocarb 21.8 0.015 0.07 

BDMC (SUR) 22.3 0.015 0.07 

*Retention time data is calculated from precision and accuracy data results presented in 

Table 6 and the calibration curve used to quantitate the data. Retention times may differ 

depending on the chromatographic conditions and columns used. 

531.2 - 26

TABLE 3.	 DETECTION LIMITS IN REAGENT WATER USING THE WATERS 

POSTCOLUMN CARBAMATE SYSTEM AND THE WATERS MODEL 

474 DETECTOR


Analyte Fortification 

Level (ug/L) 

Detection 

Limita

 (ug/L) 

Signal to Noise 

Ratio 

Aldicarb sulfoxide 0.20 0.059 8:1 

Aldicarb sulfone 0.10 0.057 3:1 

Oxamyl 0.20 0.065 10:1 

Methomyl 0.20 0.050 10:1 

3-Hydroxycarbofuran 0.20 0.029 18:1 

Aldicarb 0.20 0.026 9:1 

Propoxur 0.20 0.037 6:1 

Carbofuran 0.20 0.043 9:1 

Carbaryl 0.20 0.045 13:1 

1-Naphthol 0.20 0.063 10:1 

Methiocarb 0.20 0.061 11:1 

a

Detection Limits were determined by analyzing 7 replicates over 3 days using the 

conditions outlined in Table 1 with a 1000-uL injection. 

531.2 - 27

TABLE 4.	 DETECTION LIMITS IN REAGENT WATER USING THE 

PICKERING MODEL PCX5200 POSTCOLUMN SYSTEM AND THE 

WATERS MODEL 474 DETECTOR


Analyte Fortification 

Level (ug/L) 

Detection Limit

a 

(ug/L) 

Signal to 

Noise Ratio 

Aldicarb sulfoxide 0.20 0.056 13:1 

Aldicarb sulfone 0.20 0.026 15:1 

Oxamyl 0.20 0.045 9:1 

Methomyl 0.20 0.045 11:1 

3-Hydroxycarbofuran 0.20 0.041 11:1 

Aldicarb 0.20 0.042 7:1 

Propoxur 0.20 0.040 11:1 

Carbofuran 0.20 0.058 7:1 

Carbaryl 0.20 0.065 22:1 

1-Naphthol 0.20 0.034 9:1 

Methiocarb 0.20 0.036 5:1 

a

Detection Limits were determined by analyzing 7 replicates over 3 days using the 

conditions outlined in Table 1 with a 250-uL injection. 

531.2 - 28

TABLE 5.	 DETECTION LIMITS IN REAGENT WATER USING THE WATERS 

POSTCOLUMN CARBAMATE ANALYSIS SYSTEM AND THE 

WATERS MODEL 2475 DETECTOR


Analyte Fortification 

Level (ug/L) 

Detection 

Limita,b (ug/L) 

Signal to 

Noise Ratio 

Aldicarb sulfoxide 0.20 0.038 14:1 

Aldicarb sulfone 0.20 0.033 9:1 

Oxamyl 0.10 0.044 4:1 

Methomyl 0.20 0.054 24:1 

3-Hydroxycarbofuran 0.20 0.038 7:1 

Aldicarb 0.20 0.049 12:1 

Propoxur 0.20 0.061 10:1 

Carbofuran 0.20 0.050 12:1 

Carbaryl 0.10 0.043 9:1 

1-Naphthol 0.20 0.115 3:1 

Methiocarb 0.20 0.055 5:1 

a

Detection Limits were determined by analyzing 7 replicates over 3 days using the


conditions outlined in Table 1 with a 200-uL injection.


b

These data were collected at American Water Works Service Company.


531.2 - 29

TABLE 6.	 PRECISION AND ACCURACY OF LOW AND HIGH LEVEL 

FORTIFIED REAGENT WATERa 

Analyte 

Concentration = 0.20 ug/L 

(n=7) 

Concentration = 10 ug/L 

(n=7) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Aldicarb sulfoxide 112 6.2 106 1.8 

Aldicarb sulfone 92 9.5 106 2.6 

Oxamyl 101 8.6 106 2.2 

Methomyl 101 6.5 106 2.9 

3-Hydroxycarbofuran 105 6.8 108 1.2 

Aldicarb 95 7.4 106 1.3 

Propoxur 109 5.9 109 2.0 

Carbofuran 112 6.7 110 2.2 

Carbaryl 112 7.0 107 2.1 

1-Naphthol 113 12.6 108 3.1 

Methiocarb 105 5.9 107 1.5 

BDMC (SUR)

b 108 4.3 101 2.3 

a

Data obtained using conditions in Table 1 using a 1000-uL injection. 

b

Surrogate concentration in all samples was 2.0 ug/L. 

531.2 - 30

TABLE 7.	 PRECISION AND ACCURACY OF LOW AND HIGH LEVEL 

FORTIFIED CHLORINATED SURFACE WATERa 

Analyte 

Concentration = 0.20 ug/L 

(n=7) 

Concentration = 10 ug/L 

(n=7) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Aldicarb sulfoxide 113 7.0 104 2.8 

Aldicarb sulfone 104 5.5 106 1.4 

Oxamyl 107 6.4 104 2.2 

Methomyl 110 9.8 104 1.6 

3-Hydroxycarbofuran 128 3.9 107 1.1 

Aldicarb 123 2.7 105 1.5 

Propoxur 128 6.0 106 2.1 

Carbofuran 140 5.6 105 2.5 

Carbaryl 112 9.7 106 0.9 

1-Naphthol 113 12.1 101 1.3 

Methiocarb 104 13.3 107 1.1 

BDMC (SUR)

b 108 2.1 96 3.9 

a

Data obtained using conditions in Table 1 using a 1000-uL injection. b

Surrogate concentration in all samples was 2.0 ug/L. 

531.2 - 31

TABLE 8. PRECISION AND ACCURACY OF LOW AND HIGH LEVEL


FORTIFIED CHLORINATED GROUND WATER


Analyte 

Concentration = 0.20 

(n=7) 

Concentration = 10 ug/L 

(n=7) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Mean % 

Recovery 

Relative 

Standard 

Deviation 

(%) 

Aldicarb sulfoxide 111 7.3 106 1.1 

Aldicarb sulfone 98 9.2 106 0.9 

Oxamyl 99 8.4 105 1.2 

Methomyl 99 10.2 105 1.4 

3-Hydroxycarbofuran 107 3.0 108 0.4 

Aldicarb 100 6.3 105 0.6 

Propoxur 112 6.1 107 0.8 

Carbofuran 112 4.1 107 1.6 

Carbaryl 119 5.1 108 1.3 

1-Naphthol 109 8.2 109 1.2 

Methiocarb 105 3.9 107 1.0 

BDMC (SUR)

b 109 2.0 97 4.3 

ug/L 

a

Data obtained using conditions in Table 1 using a 1000-uL injection. b

Surrogate concentration in all samples was 2.0 ug/L. 

531.2 - 32

TABLE 9.	 SAMPLE HOLDING TIME DATA FOR CHLORINATED GROUND 

WATER SAMPLES FORTIFIED WITH METHOD ANALYTES AT 2.0 

ug/L 

Analyte 

% Recovery for Samples Fortified at 2.0 ug/L 

Day 0 Day 2 Day 8 Day 15 Day 28 

Aldicarb sulfoxide 94 97 93 93 96 

Aldicarb sulfone 93 99 97 98 98 

Oxamyl 97 97 101 103 101 

Methomyl 96 95 99 98 97 

3-Hydroxycarbofuran 96 99 96 95 98 

Aldicarb 95 100 96 92 93 

Propoxur 95 98 97 98 99 

Carbofuran 96 99 97 97 100 

Carbaryl 96 97 98 94 100 

1-Naphthol 96 97 99 95 98 

Methiocarb 95 98 96 94 97 

BDMC (SUR)

b 99 105 100 99 99 

a

Data obtained using conditions in Table 1 using a 500-uL injection. b

Surrogate concentration in all samples was 2.0 ug/L. 

531.2 - 33

TABLE 10. INITIAL DEMONSTRATION OF CAPABILITY (IDC) 

REQUIREMENTS 

Method 

Reference 

Requirement Specification and 

Frequency 

Acceptance Criteria 

Sect. 9.2.1 Initial 

Demonstration of 

Low System 

Background 

Analyze LRB other 

IDC steps. 

Demonstrate that all target 

analytes are below 1/3 the 

intended MRL or lowest 

CAL standard, and that 

possible interference from 

reagents and glassware do 

not prevent the identification 

and quantitation of method 

analytes. 

prior to any

Sect. 9.2.2 Quality Control 

Sample (QCS) 

Second source standard, used to 

fortify preserved reagent water 

(Sect. 7.1.2). Analyze as a CCC 

after initial calibration but prior 

to the analysis of the IDA 

samples. 

Verifies initial calibration 

accuracy, recovery must be 

within + 30% of true value. 

Sect. 9.2.2 Initial 

Demonstration of 

Accuracy (IDA) 

Analyze 4-7 replicate 

LFBs/CCCs at 

midrange concentration. 

Mean recovery + 20% of true 

value. 

Sect. 9.2.3 Initial 

Demonstration of 

Precision (IDP) 

Calculate average recovery for 

replicates used in IDA. 

%RSD must be < 20%. 

Sect. 9.2.4 Detection Limit 

Determination 

Over a period of three days, 

prepare a minimum of 7 

replicate LFBs fortified at a 

concentration estimated to be 

near the Detection Limit. 

Analyze the replicates through 

all steps of the analysis. 

Calculate the Detection Limit 

using the equation in Sect. 9.2.4. 

Data from Detection Limit 

replicates are not required to 

meet method precision and 

accuracy criteria. f the 

Detection Limit replicates are 

fortified at a low enough 

concentration, it is likely that 

they will not meet precision 

and accuracy criteria. 

fortified 

I

531.2 - 34

TABLE 11. QUALITY CONTROL REQUIREMENTS (SUMMARY)


Method 

Reference 

Requirement Specification and Frequency Acceptance Criteria 

Sect. 8.3 

and Sect. 8.4 

Sample and 

Extract Holding 

Times 

Properly preserved samples must 

be shipped at or below 10 0C and 

may be held in the lab at or 

below 6 0

C for 28 days. Samples 

should not be frozen. 

Do not report data for samples 

that have not been properly 

preserved or stored, or that 

have exceeded their holding 

time. 

Sect. 9.4 Laboratory 

Reagent Blank 

(LRB) 

Include a LRB with each analysis 

batch (up to 20 samples). 

Analyze prior to analyzing 

samples and determine to be free 

from interferences. 

Demonstrate that all target 

analytes are below 1/3 the 

intended MRL, and that 

possible interference from 

reagents and glassware do not 

prevent the identification and 

quantitation of method 

analytes. 

If targets exceed 1/3 the MRL, 

results for all subject analytes 

in the analytical batch are 

invalid. 

Sect. 9.5 and 

Sect. 10.3 

Continuing 

Calibration 

Check (CCC) 

Verify initial calibration by 

analyzing a low level (at the 

MRL or below) CCC prior to 

analyzing samples. CCs are 

then injected after every 10 

samples and after the last sample, 

rotating concentrations to cover 

the calibrated range of the 

instrument. 

Recovery for each analyte 

must be 70-130% of the true 

value for all but the lowest 

level of calibration. The 

lowest calibration level CCC 

must be 50-150% of the true 

value. 

Results that are not bracketed 

by acceptable CCCs are 

invalid. 

C

Sect. 9.7 Surrogate 

Standards 

The surrogate, 4-bromo-3,5-

dimithylphenyl 

–methylcarbamate is added to all 

field and QC samples. 

Surrogate recovery must be 

70-130% of the true value. 

Samples that fail criteria, must 

be reported as suspect due to 

surrogate recovery. 

531.2 - 35

Method 

Reference 

Requirement Specification and Frequency Acceptance Criteria 

Sect. 9.8 Laboratory 

Fortified 

Sample Matrix 

(LFSM) and 

Laboratory 

Fortified 

Sample Matrix 

Duplicate 

(LFSMD) 

With each analysis batch (Sect. 

3.1), a minimum of one LFSM is 

extracted and analyzed. 

duplicate LFSM, or LFSMD, 

should be extracted when 

occurrence of target analytes is 

low. ield Duplicate analysis is 

not required for analysis batches 

containing an LFSMD. 

Recoveries not within 70-

130% (50-150% at the MRL) 

of the fortified amount may 

indicate a matrix effect. 

If an LFSMD is analyzed 

instead of a Laboratory 

Duplicate , target RPDs 

should be within + 30%. 

If all CCCs meet acceptance 

criteria, and 

LFSMD do not, sample is 

designated suspect/matrix. 

A 

F

LFSM or 

Sect. 9.9 Field Duplicates Analyze at least one duplicate 

with each analysis batch (20 

samples or less). Laboratory 

Fortified Sample Matrix 

Duplicate may be substituted for 

a Field Duplicate when the 

occurrence of target analytes is 

low. 

RPDs should be within + 

30%. 

If all CCCs meet acceptance 

criteria, and Field Duplicates 

do not, sample is designated 

suspect/matrix. 

A

Sect 9.10 Resolution 

Check 

Monitor once for every 24 hour 

analysis period. 

Closely eluting peaks that are 

not baseline resolved must 

have a resolution of > 1.0 or 

greater using the equation in 

Sect. 9.10 

Sect. 9.11 Quality Control 

Sample 

Analyze at least quarterly or 

when preparing new standards, as 

well as during the IDC. 

Same acceptance criteria as a 

mid-level CCC. 

Sect. 10.2 Initial 

Calibration 

Use external standard calibration 

technique to generate a 

calibration curve with at least 

five standards. 

When each calibration 

standard is calculated using 

the calibration curve, the 

results should be 70-130% of 

the true value for all but the 

lowest standard. est 

standard should be 50-150% 

of the true value. The lowest 

CAL standard concentration 

must be as low or lower than 

the intended MRL. 

The low

531.2 - 36

FIGURE 1. SAMPLE CHROMATOGRAM OF METHOD 531.2 ANALYTES


Reagent water, sample preservatives added, fortified at 2.0 ug/L. 

531.2 - 37

531.2 - 38