ASTM D7645-23

Standard Test Method for Determination of Aldicarb, Aldicarb Sulfone, Aldicarb Sulfoxide, Carbofuran, Methomyl, Oxamyl, and Thiofanox in Water by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)

SIGNIFICANCE AND USE
5.1 This test method has been developed by U.S. EPA Region 5 Chicago Regional Laboratory (CRL).  
5.2 The N-methyl carbamate (NMC) pesticides: aldicarb, carbofuran, methomyl, oxamyl, and thiofanox have been identified by EPA as working through a common mechanism. These affect the nervous system by reducing the ability of enzymes. Enzyme inhibition was the primary toxicological effect of regulatory concern to EPA in assessing the NMC’s food, drinking water, and residential risks. In most of the country, NMC residues in drinking water sources are at levels that are not likely to contribute substantially to the multi-pathway cumulative exposure. Shallow private wells extending through highly permeable soils into shallow, acidic ground water represent what the EPA believes to be the most vulnerable drinking water. Aldicarb sulfone and aldicarb sulfoxide are breakdown products of aldicarb and should also be monitored due to their toxicological effects.4  
5.3 This test method has been investigated for use with reagent, surface, and drinking water for the selected carbamates: aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox.
SCOPE
1.1 This test method covers the determination of aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox (referred to collectively as carbamates in this test method) in water by direct injection using liquid chromatography (LC) and detected with tandem mass spectrometry (MS/MS). These analytes are qualitatively and quantitatively determined by this test method. This test method adheres to multiple reaction monitoring (MRM) mass spectrometry.  
1.2 The Detection Verification Level (DVL) and Reporting Range for the carbamates are listed in Table 1.    
1.2.1 The DVL is required to be at a concentration at least 3 times below the Reporting Limit (RL) and have a signal/noise ratio greater than 3:1. Fig. 1 displays the signal/noise ratios of the primary single reaction monitoring (SRM) transitions, and Fig. 2 displays the confirmatory SRM transitions at the DVLs for the carbamates.  
1.3 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 14-Apr-2023
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: D19 - Water
• Drafting Committee: D19.06 - Methods for Analysis for Organic Substances in Water

Relations

• Refers: ASTM D3694-96(2024) - Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• Effective Date: 01-Apr-2024
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM E2554-18e1 - Standard Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method Using Control Chart Techniques
• Effective Date: 01-Apr-2018
• Refers: ASTM E2554-18 - Standard Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method Using Control Chart Techniques
• Effective Date: 01-Apr-2018
• Refers: ASTM E2554-13 - Standard Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method Using Control Chart Techniques
• Effective Date: 01-Apr-2013
• Refers: ASTM D4841-88(2013)e1 - Standard Practice for Estimation of Holding Time for Water Samples Containing Organic and Inorganic Constituents
• Effective Date: 01-Jan-2013
• Refers: ASTM D4841-88(2013) - Standard Practice for Estimation of Holding Time for Water Samples Containing Organic and Inorganic Constituents
• Effective Date: 01-Jan-2013
• Refers: ASTM D2777-12 - Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• Effective Date: 15-Jun-2012
• Refers: ASTM D3856-11 - Standard Guide for Management Systems in Laboratories Engaged in Analysis of Water
• Effective Date: 15-Nov-2011
• Refers: ASTM D3694-96(2011) - Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• Effective Date: 01-May-2011
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D4841-88(2008) - Standard Practice for Estimation of Holding Time for Water Samples Containing Organic and Inorganic Constituents
• Effective Date: 15-Jul-2008
• Refers: ASTM D2777-08 - Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• Effective Date: 15-Jan-2008
• Refers: ASTM E2554-07 - Standard Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method in a Single Laboratory Using a Control Sample Program
• Effective Date: 01-May-2007
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006

Overview

ASTM D7645-23 is a standard test method published by ASTM International for the determination of specific N-methyl carbamate pesticides and their breakdown products in water. The targeted analytes include aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox. This method employs liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) using the multiple reaction monitoring (MRM) mode for both qualitative and quantitative analysis.

This standard is essential for laboratories, regulatory agencies, and water utilities concerned with monitoring pesticide residues to ensure water safety and compliance with environmental regulations.

Key Topics

• Target Analytes: The method focuses on detecting seven carbamates-aldicarb, its sulfone and sulfoxide metabolites, carbofuran, methomyl, oxamyl, and thiofanox-collectively significant for water quality monitoring.
• Analytical Technique: Utilizes direct injection LC/MS/MS, offering sensitive, accurate, and selective quantification. The standard recommends external calibration with isotopically labeled surrogates for quality assurance.
• Significance: Developed in collaboration with the U.S. EPA, this method addresses regulatory concerns over N-methyl carbamates due to their neurotoxic effects resulting from enzyme inhibition. Monitoring both parent compounds and metabolites is critical due to their toxicological relevance.
• Sample Types: Applicable to reagent water, surface water, and drinking water. Special concern is highlighted for vulnerable sources, such as shallow private wells in permeable, acidic soils.
• Quality Control: Defines requirements for calibration, use of surrogates, blanks, laboratory control samples (LCS), matrix spikes (MS), and duplications for precision and recovery checks.

Applications

ASTM D7645-23 is widely applicable for:

• Drinking Water Monitoring: Ensures compliance with regulatory requirements by detecting carbamate pesticides and their metabolites in public and private water supplies.
• Environmental Surveillance: Used by environmental agencies and laboratories to assess surface and groundwater contamination, especially in agricultural regions.
• Quality Assurance: Provides guidance for laboratories aiming to achieve high standards of precision, bias, and accuracy in water analysis.
• Regulatory Compliance: Supports water utilities and environmental authorities in demonstrating regulatory conformity concerning pesticide residues.
• Water Safety Risk Assessment: Helps identify risks posed by N-methyl carbamates and informs decisions on water treatment and source protection.

Sample Preservation and Handling

• Samples must be collected in acidified, pre-cleaned amber glass bottles and stored between 0°C and 6°C.
• Colin sample with acetic acid to pH ≤3.8. For chlorinated water, dechlorinate with ascorbic acid to prevent analytic interference, especially for aldicarb.

Analytical Performance

• Detection Verification Levels (DVLs) are established at 250 ng/L, with a reporting range of 1-100 µg/L for all target compounds.
• Method validation requires meeting specific recovery and precision criteria, ensuring consistency across matrices such as reagent, surface, and drinking water.

Related Standards

• ASTM D1129: Terminology Relating to Water
• ASTM D1193: Specification for Reagent Water
• ASTM D2777: Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• ASTM D3694: Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• EPA Method 531, 531.2, 538: U.S. EPA procedures for carbamate pesticides in water, employing alternative or complementary analytical approaches

Practical Value

By following ASTM D7645-23, laboratories and regulatory bodies can confidently assess the presence of hazardous carbamate pesticides in water. The standard's rigorous quality assurance guidelines ensure data accuracy and support both routine monitoring and regulatory actions to protect public health and the environment. Employing LC/MS/MS provides the necessary selectivity and sensitivity for detecting trace contaminants, meeting modern analytical and regulatory expectations for water safety.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D7645 − 23
Standard Test Method for
Determination of Aldicarb, Aldicarb Sulfone, Aldicarb
Sulfoxide, Carbofuran, Methomyl, Oxamyl, and Thiofanox in
Water by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
This standard is issued under the fixed designation D7645; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This test method covers the determination of aldicarb,
aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl,
2. Referenced Documents
oxamyl, and thiofanox (referred to collectively as carbamates
in this test method) in water by direct injection using liquid
2.1 ASTM Standards:
chromatography (LC) and detected with tandem mass spec-
D1129 Terminology Relating to Water
trometry (MS/MS). These analytes are qualitatively and quan-
D1193 Specification for Reagent Water
titatively determined by this test method. This test method
D2777 Practice for Determination of Precision and Bias of
adheres to multiple reaction monitoring (MRM) mass spec-
Applicable Test Methods of Committee D19 on Water
trometry.
D3694 Practices for Preparation of Sample Containers and
for Preservation of Organic Constituents
1.2 The Detection Verification Level (DVL) and Reporting
Range for the carbamates are listed in Table 1. D3856 Guide for Management Systems in Laboratories
Engaged in Analysis of Water
1.2.1 The DVL is required to be at a concentration at least
3 times below the Reporting Limit (RL) and have a signal/ D4841 Practice for Estimation of Holding Time for Water
Samples Containing Organic and Inorganic Constituents
noise ratio greater than 3:1. Fig. 1 displays the signal/noise
ratios of the primary single reaction monitoring (SRM) D5847 Practice for Writing Quality Control Specifications
for Standard Test Methods for Water Analysis
transitions, and Fig. 2 displays the confirmatory SRM transi-
tions at the DVLs for the carbamates. E2554 Practice for Estimating and Monitoring the Uncer-
1.2.2 The reporting limit is the concentration of the Level 1
tainty of Test Results of a Test Method Using Control
calibration standard as shown in Table 2 for the carbamates. Chart Techniques
1.3 Units—The values stated in SI units are to be regarded
2.2 Other Documents:
as standard. No other units of measurement are included in this EPA Publication SW-846 Test Methods for Evaluating Solid
standard.
Waste, Physical/Chemical Methods
EPA Method 531 Measurement of N-Methyl Carbamoylox-
1.4 This standard does not purport to address all of the
imes and N-Methyl Carbamates in Drinking Water by
safety concerns, if any, associated with its use. It is the
Direct Aqueous Injection HPLC with Post Column De-
responsibility of the user of this standard to establish appro-
rivatization
priate safety, health, and environmental practices and deter-
EPA Method 531.2 Measurement of N-Methyl Carbamoy-
mine the applicability of regulatory limitations prior to use.
loximes and N-Methyl Carbamates in Water by Direct
1.5 This international standard was developed in accor-
Aqueous Injection HPLC with Post Column Derivatiza-
dance with internationally recognized principles on standard-
tion
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This test method is under the jurisdiction of ASTM Committee D19 on Water contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for Standards volume information, refer to the standard’s Document Summary page on
Organic Substances in Water. the ASTM website.
Current edition approved April 15, 2023. Published May 2023. Originally Available from National Technical Information Service (NTIS), U.S. Depart-
approved in 2010. Last previous edition approved in 2016 as D7645 – 16. DOI: ment of Commerce, 5285 Port Royal Road, Springfield, VA, 22161 or at http://
10.1520/D7645-23. www.epa.gov/epawaste/hazard/testmethods/index.htm.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7645 − 23
TABLE 1 Detection Verification Level and Reporting Range
3.3.20 SRM, n—Single Reaction Monitoring
DVL Reporting Range
3.3.21 SS, n—Surrogate Standard
Analyte
(ng/L) (μg/L)
3.3.22 TC, n—Target Compound
Aldicarb 250 1-100
Aldicarb Sulfone 250 1-100 -6
3.3.23 μM, n—micromolar, 1 × 10 moles/L
Aldicarb Sulfoxide 250 1-100
Carbofuran 250 1-100 3.3.24 VOA, n—Volatile Organic Analysis
Methomyl 250 1-100
Oxamyl 250 1-100
4. Summary of Test Method
Thiofanox 250 1-100
4.1 This is a performance-based method, and modifications
are allowed to improve performance.
4.2 For carbamate analysis, samples are shipped to the lab
EPA Method 538 Determination of Selected Organic Con-
acidified between above freezing and 6 °C and analyzed within
taminants in Drinking Water by Direct Aqueous Injection-
14 days of collection. In the lab, the samples are spiked with
Liquid Chromatography/Tandem Mass Spectrometry
surrogates, filtered using a syringe driven filter unit, and
(DAI-LC/MS/MS)
analyzed directly by LC/MS/MS.
13 15
3. Terminology
4.3 The carbamates, methomyl- C , N (surrogate) and
carbofuran- C (surrogate) are identified by retention time and
3.1 Definitions:
two SRM transitions. The target analytes and surrogate are
3.1.1 For definitions of terms used in this standard, refer to
quantitated using the primary SRM transitions utilizing an
Terminology D1129.
external calibration. The final report issued for each sample
3.2 Definitions of Terms Specific to This Standard:
lists the concentration of carbamates and the surrogate recov-
3.2.1 carbamates, n—in this test method, aldicarb, aldicarb
eries.
sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl,
and thiofanox collectively.
5. Significance and Use
3.2.2 detection verification level, DVL, n—a concentration
5.1 This test method has been developed by U.S. EPA
that has a signal/noise ratio greater than 3:1 and is at least 3
Region 5 Chicago Regional Laboratory (CRL).
times below the Reporting Limit (RL).
5.2 The N-methyl carbamate (NMC) pesticides: aldicarb,
3.2.3 independent reference material, IRM, n—a material of
carbofuran, methomyl, oxamyl, and thiofanox have been iden-
known purity and concentration obtained either from the
tified by EPA as working through a common mechanism. These
National Institute of Standards and Technology (NIST) or other
affect the nervous system by reducing the ability of enzymes.
reputable supplier; the IRM must be obtained from a different
Enzyme inhibition was the primary toxicological effect of
lot of material than is used for calibration.
regulatory concern to EPA in assessing the NMC’s food,
3.3 Acronyms:
drinking water, and residential risks. In most of the country,
3.3.1 CCC, n—Continuing Calibration Check
NMC residues in drinking water sources are at levels that are
3.3.2 IC, n—Initial Calibration
not likely to contribute substantially to the multi-pathway
cumulative exposure. Shallow private wells extending through
3.3.3 LC, n—Liquid Chromatography
highly permeable soils into shallow, acidic ground water
3.3.4 LCS/LCSD, n—Laboratory Control Sample/
represent what the EPA believes to be the most vulnerable
Laboratory Control Sample Duplicate
drinking water. Aldicarb sulfone and aldicarb sulfoxide are
3.3.5 MeOH, n—Methanol
breakdown products of aldicarb and should also be monitored
-3
3.3.6 mM, n—millimolar, 1 × 10 moles/L
due to their toxicological effects.
3.3.7 MRM, n—Multiple Reaction Monitoring
5.3 This test method has been investigated for use with
reagent, surface, and drinking water for the selected carbam-
3.3.8 MS/MSD, n—Matrix Spike/Matrix Spike Duplicate
ates: aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran,
3.3.9 NA, adj—Not Available
methomyl, oxamyl, and thiofanox.
3.3.10 ND, n—non-detect
3.3.11 P&A, n—Precision and Accuracy
6. Interferences
3.3.12 PPB, n—parts per billion
6.1 Method interferences may be caused by contaminants in
solvents, reagents, glassware, and other apparatus producing
3.3.13 PPT, n—parts per trillion
discrete artifacts or elevated baselines. All of these materials
3.3.14 QA, adj—Quality Assurance
are demonstrated to be free from interferences by analyzing
3.3.15 QC, adj—Quality Control
laboratory reagent blanks under the same conditions as
3.3.16 RL, n—Reporting Limit
samples.
3.3.17 RSD, n—Relative Standard Deviation
3.3.18 RT, n—Retention Time
Additional information about Carbamate pesticides area available from United
3.3.19 SDS, n—Safety Data Sheets States Environmental Protection Agency (EPA), http://www.epa.gov.
D7645 − 23
FIG. 1 Example Primary SRM Chromatograms Signal/Noise Ratios
FIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise Ratios
D7645 − 23
TABLE 2 Concentrations of Calibration Standards (PPB)
Analyte/Surrogate LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7 LV 8
Aldicarb 1 5 10 25 35 50 75 100
Aldicarb Sulfone 1 5 10 25 35 50 75 100
Aldicarb Sulfoxide 1 5 10 25 35 50 75 100
Carbofuran 1 5 10 25 35 50 75 100
Methomyl 1 5 10 25 35 50 75 100
Oxamyl 1 5 10 25 35 50 75 100
Thiofanox 1 5 10 25 35 50 75 100
Carbofuran- C (Surrogate) 1 5 10 25 35 50 75 100
13 15
Methomyl- C , N (Surrogate) 1 5 10 25 35 50 75 100
6.2 All glassware is washed in hot water with detergent and 8. Reagents and Materials
rinsed in hot water followed by distilled water. The glassware
8.1 Purity of Reagents—High Performance Liquid Chroma-
is then dried and heated in an oven at 250 °C for 15 min to
tography (HPLC) pesticide residue analysis and spectropho-
30 min. All glassware is subsequently cleaned with acetone
tometry grade chemicals must be used in all tests. Unless
followed by methanol.
indicated otherwise, it is intended that all reagents must
6.3 All reagents and solvents should be pesticide residue
conform to the Committee on Analytical Reagents of the
purity or higher to minimize interference problems.
American Chemical Society. Other reagent grades may be
used provided they are first determined to be of sufficiently
6.4 Matrix interferences may be caused by contaminants in
high purity to permit their use without affecting the accuracy of
the sample. The extent of matrix interferences can vary
the measurements.
considerably from sample source depending on variations of
the sample matrix.
8.2 Purity of Water—Unless otherwise indicated, references
to water must be understood to mean reagent water conforming
7. Apparatus
to Type 1 of Specification D1193. It must be demonstrated that
7.1 LC/MS/MS System:
this water does not contain contaminants at concentrations
7.1.1 Liquid Chromatography (LC) System—A complete LC
sufficient to interfere with the analysis.
system is needed to analyze samples. This should include a
8.3 Gases—Ultrapure nitrogen and argon.
sample injection system, a solvent pumping system capable of
mixing solvents, a sample compartment capable of maintaining
8.4 Acetonitrile (CAS # 75-05-8).
required temperature and a temperature controlled column
8.5 Methanol (CAS # 67-56-1).
compartment. A system that is capable of performing at the
flows, pressures, controlled temperatures, sample volumes, and
8.6 Acetone (CAS # 67-64-1).
requirements of the standard may be used.
5 8.7 Ammonium Formate (CAS # 540-69-2).
7.1.2 Analytical Column —A C18 column was used to
develop this test method.
8.8 Acetic Acid (Glacial, CAS # 64-19-7).
7.2 Tandem Mass Spectrometer (MS/MS) System—A
8.9 Aldicarb (CAS # 116-06-3).
MS/MS system capable of MRM analysis. Any system that is
8.10 Aldicarb Sulfone (CAS # 1646-88-4).
capable of performing at the requirements in this standard may
be used.
8.11 Aldicarb Sulfoxide (CAS # 1646-87-3).
7.3 Filtration Device:
8.12 Carbofuran (CAS # 1563-66-2).
7.3.1 Hypodermic Syringe—A lock tip glass syringe capable
8.13 Oxamyl (CAS # 23135-22-0).
of holding a syringe-driven filter unit or similar may be used.
7.3.1.1 A 25-mL lock tip glass syringe size is recommended
8.14 Methomyl (CAS # 16752-77-5).
since a 25-mL sample size is used in this test method.
8.15 Thiofanox (CAS # 39196-18-4).
7.3.2 Filter Unit —PVDF filter units were used to filter the
samples. 13 15 13 15
8.16 Methomyl- C , N (acetohydroxamate- C , N,
2 2
CAS # (unlabeled) 16752-77-5).
A Waters ACQUITY UPLC (a trademark of Waters Technologies Corporation 13 13
8.17 Carbofuran- C (Ring- C , CAS # (unlabeled) 1563-
6 6
in Wilmington, DE) BEH C18, 2.1 mm × 100 mm, 1.7 μm particle size was used,
66-2).
if you are aware of an alternative column that meets the performance of the standard,
please provide this information to ASTM International Headquarters. Your com-
ments will receive careful consideration at a meeting of the responsible technical
committee, which you may attend.
6 7
A Millex HV Syringe Driven Filter Unit PVDF 0.22 μm (Millipore ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Corporation, Catalog #SLGV033NS; Millex is a trademark of Merck KGAA, Standard-Grade Reference Materials, American Chemical Society, Washington,
Darmstadt, Germany) was used, if you are aware of an alternative filter that meets DC. For suggestions on the testing of reagents not listed by the American Chemical
the performance of the standard, please provide this information to ASTM Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
International Headquarters. Your comments will receive careful consideration at a U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
meeting of the responsible technical committee, which you may attend. copeial Convention, Inc. (USPC), Rockville, MD.
D7645 − 23
TABLE 3 Gradient Conditions for Liquid Chromatography
9. Hazards
Percent Percent
9.1 Normal laboratory safety applies to this test method.
Time Flow 95 % Water/ 95 % Methanol/
Analysts should wear safety glasses, gloves, and lab coats
(min) (μL/min) 5 % Methanol, 5 % Water,
5 mM NH CO H 5 mM NH CO H
when working in the lab. Analysts should review the Safety 4 2 4 2
0.0 300 100 0
Data Sheets (SDS) for all reagents used in this test method.
2.0 300 100 0
3.0 300 95 5
10. Sampling
5.0 300 85 15
10.1 Sampling and Preservation—Grab samples should be 10.0 300 0 100
11.5 300 0 100
collected in ≥25 mL pre-cleaned amber glass bottles with
12.0 300 100 0
inert-lined caps demonstrated to be free of interferences. All
14.0 300 100 0
samples are acidified with glacial acetic acid to pH ≤3.8 upon
collection. A few drops or less of glacial acetic acid is required
per 40 mL water sample collected. Chlorinated drinking water
11.2.2 Temperatures—Column, 30 °C; Sample
samples are also dechlorinated with ascorbic acid; 10 mg of
compartment, 15 °C.
ascorbic acid is added to each 40 mL volume of water prior to
11.2.3 Seal Wash—Solvent: 50 Acetonitrile/50 Water; Time:
collection. Drinking water samples must be dechlorinated upon
5 min.
collection. Aldicarb oxidizes when residual chlorine is present
11.3 Mass Spectrometer Parameters:
in the sample. This test method is based on a 25 mL sample
11.3.1 To acquire the maximum number of data points per
size per analysis. If different sample sizes are used, spiking
SRM channel while maintaining adequate sensitivity, the tune
solution amounts and preservatives will need to be modified.
parameters may be optimized according to the instrument.
Conventional sampling practices should be followed. Refer to
Each peak requires at least 10 scans per peak for adequate
Guide D3856 and Practices D3694. Store samples between
quantitation. This test method contains two surrogates, which
above freezing and 6 °C from the time of collection until
are isotopically labeled methomyl and carbofuran, and seven
analysis. Analyze the sample within 14 days of collection.
carbamates, which are split up into seven MRM acquisition
NOTE 1—Less sample volume is acceptable, but the spike amounts and
functions to optimize sensitivity. Variable parameters regarding
sample preservatives must be adjusted accordingly.
retention times, SRM transitions, and cone and collision
10.1.1 EPA Method 531.2 demonstrated that carbamates are
energies are shown in Table 4. Mass spectrometer parameters
more stable under acidic conditions. Potassium dihydrogen
used in the development of this test method are listed below:
citrate buffer is used in Method 531.2 to bring the pH to ~3.8,
The instrument is set in the Electrospray positive source setting.
but this buffer is incompatible with LC/MS/MS. Therefore, the
Capillary Voltage: 3.5 kV
pH adjustment is accomplished with acetic acid in this test Cone: Variable depending on analyte (Table 4)
Extractor: 2 Volts
method. EPA Method 531.2 demonstrated that carbamates
RF Lens: 0.1 Volts
under acidic conditions are stable for at least 28 days. EPA
Source Temperature: 120 °C
Desolvation Temperature: 375 °C
Method 531 demonstrated that oxamyl and methomyl are
Desolvation Gas Flow: 800 L/h
stable for at least 70 days at pH 3 6 0.2. Holding time is
Cone Gas Flow: 25 L/h
dependent upon an individual matrix and will vary. Practice
Low Mass Resolution 1: 14.5
High Mass Resolution 1: 14.5
D4841 may be used to conduct a holding time study on an
Ion Energy 1: 0.5
individual matrix.
Entrance Energy: –1
Collision Energy: Variable depending on analyte (Table 4)
11. Preparation of LC/MS/MS
Exit Energy: 0
Low Mass Resolution 2: 14.5
11.1 LC Chromatograph Operating Conditions:
High Mass resolution 2: 14.5
11.1.1 Injection volumes of all calibration standards and
Ion Energy 2: 0.7
samples are made at 50 μL volume using a full loop injection.
Multiplier: 650
-3
Gas Cell Pirani Gauge: 7.0 × 10 Torr
If a 50 μL volume loop is installed in the LC, a “full loop”
Inter-Channel Delay: 0.005 s
mode is the preferred technique when performing fast, quali-
Inter-Scan Delay: 0.005 s
tative analyses. This mode should be used whenever accuracy Dwell: 0.075 s
and precision are the primary concerns. The first sample
12. Calibration and Standardization
analyzed after the calibration curve is a blank to ensure there is
no carry-over. The gradient conditions for the liquid chromato- 12.1 The mass spectrometer must be calibrated in accor-
dance with manufacturer specifications before analysis. In
graph are shown in Table 3.
order to obtain accurate analytical values through using this
11.2 LC Sample Manager Conditions:
test method within the confidence limits, the following proce-
11.2.1 Wash Solvents—Weak wash is 2.4 mL of 95 %
dures must be followed when performing the test method.
water/5 % methanol. Strong wash is 1.2 mL of methanol. The
Prepare all solutions in the lab using Class A volumetric
strong wash solvent is needed to eliminate carry-over between
glassware.
injections of carbamate samples. The weak wash is used to
remove the strong wash solvent. Instrument manufacturer 12.2 Calibration and Standardization—To calibrate the
specifications should be followed in order to eliminate sample instrument, analyze eight calibration standards containing the
carry-over. eight concentration levels of the carbamates, methomyl- C ,
D7645 − 23
TABLE 4 Retention Times, SRM Ions, and Analyte-Specific Mass Spectrometer Parameters
SRM Primary/
Primary/ Retention Time Cone Voltage Collision Energy
Analyte Mass Transition Confirmatory
Confirmatory (min) (Volts) (eV)
(Parent > Product) SRM Area Ratio
Aldicarb Primary 8.50 10 7 208.0 > 115.6 1.4
Confirmatory 10 16 208.0 > 88.7
Aldicarb Sulfone Primary 6.20 13 13 240.1 > 147.8 1.6
Confirmatory 13 11 240.1 > 165.8
Aldicarb Sulfoxide Primary 5.72 16 6 207.1 > 131.9 1.1
Confirmatory 16 14 207.1 > 88.8
Carbofuran Primary 8.96 22 12 222.1 > 164.9 1.2
Confirmatory 22 20 222.1 > 122.7
Methomyl Primary 6.68 15 10 162.9 > 87.7 1.8
Confirmatory 15 10 162.9 > 105.7
Oxamyl Primary 6.44 11 11 237.1 > 71.8 2.6
Confirmatory 11 7 237.1 > 89.7
Thiofanox Primary 9.32 12 8 219.1 > 56.8 4.8
Confirmatory 12 5 219.1 > 75.9
Carbofuran- C Primary 8.96 22 11 228.1 > 170.9 1.3
(Surrogate) Confirmatory 22 21 228.1 > 128.8
13 15
Methomyl- C , N Primary 6.68 18 8 165.8 > 90.7 1.7
(Surrogate) Confirmatory 18 9 165.8 > 108.7
15 13
N and carbofuran- C prior to analysis as shown in Table 2. SRM transition of each analyte is used for quantitation and the
A calibration stock standard solution is prepared from standard confirmatory SRM transition for confirmation. This gives
materials or they are purchased as certified solutions. Stock added confirmation by isolating the parent ion, forming two
Standard Solution A containing the carbamates and surrogates product ions via fragmentation, and relating it to the retention
is prepared at Level 8 concentration and aliquots of that time in the calibration standard.
solution are diluted to prepare Levels 1 through 7. The 12.2.4 The calibration software manual should be consulted
following steps will produce standards with the concentration to use the software correctly. The quantitation method is set as
values shown in Table 2. The analyst is responsible for an external calibration using the peak areas in ppt or ppb units,
recording initial component weights carefully when working as long as the analyst is consistent. Concentrations may be
with pure materials and correctly carrying the weights through calculated using the data system software to generate linear
the dilution calculations. regression or quadratic calibration curves. Forcing the calibra-
12.2.1 Prepare Stock Standard Solution A (Level 8) by tion curve through the origin is not recommended. Curves
adding to a 50 mL volumetric flask individual solutions of the should be evaluated using relative error or relative standard
following: 100 μL of aldicarb, aldicarb sulfone, aldicarb error.
sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox, each 12.2.5 Linear calibration may be used if the point of origin
13 15
at 50 ppm in methanol and 50 μL of methomyl- C , N in is excluded and a fit weighting of 1/X is used in order to give
methanol and carbofuran- C in 1,4-dioxane each at 100 ppm, more emphasis to the lower concentrations. Each calibration
dilute to 50 mL with water. The preparation of the Level 8 point used to generate the curve must have a calculated percent
standard can be accomplished using different volumes and deviation less than 25 % from the generated curve.
concentrations of stock solutions as is accustomed in the 12.2.6 Quadratic calibration may be used if the point of
individual laboratory. Depending on the prepared stock origin is excluded, and a fit weighting of 1/X is used in order
concentrations, the solubility at that concentration will have to to give more emphasis to the lower concentrations. Each
be ensured. calibration point used to generate the curve must have a
12.2.2 Aliquots of Solution A are then diluted with water to calculated percent deviation less than 25 % from the generated
prepare the desired calibration levels in 2-mL amber glass LC curve.
vials. The calibration vials must be used within 24 h to ensure 12.2.7 The retention time window of the SRM transitions
optimum results. Stock calibration standards are routinely must be within 5 % of the retention time of the analyte in a
replaced every 7 days if not previously discarded for quality midpoint calibration standard. If this is not the case, re-analyze
control failure. Calibration standards are not filtered. the calibration curve to determine if there was a shift in
12.2.3 Inject each standard and obtain its chromatogram. An retention time during the analysis and the sample needs to be
external calibration technique is used to monitor the primary re-injected. If the retention time is still incorrect in the sample,
and confirmatory SRM transitions of each analyte. Calibration refer to the analyte as an unknown.
software is utilized to conduct the quantitation of the target 12.2.8 A midpoint calibration check standard must be ana-
analytes and surrogates using the primary SRM transition. The lyzed at the end of each batch of 20 samples or within 24 h
ratios of the primary/confirmatory SRM transition area counts after the initial calibration curve was generated. This end
are given in Table 4 and will vary depending on the individual
tuning conditions. The primary/confirmatory SRM transition
Management and Technical Requirements for Laboratories Performing Envi-
area ratio must be within 35 % of the individual labs’ accepted
ronmental Analysis; Module 4: Quality Systems for Chemical Testing; The NELAC
primary/confirmatory SRM transition area ratio. The primary Institute, 2017.
D7645 − 23
calibration check should be the same calibration standard that 12.3.3 This study should be repeated until the single opera-
was used to generate the initial curve. The results from the end tor precision and mean recovery are within the limits in Table
calibration check standard must have a percent deviation less
5. If a concentration other than the recommended concentration
than 30 % from the calculated concentration for the target is used, refer to Practice D5847 for information on applying the
analytes and surrogates. If the results are not within these
F test and t test in evaluating the acceptability of the mean and
criteria, the problem must be corrected and either all samples in
standard deviation.
the batch must be re-analyzed against a new calibration curve
12.3.3.1 The QC acceptance criteria for the Initial Demon-
or the affected results must be qualified with an indication that
stration of Performance in Table 5 were generated from a
they do not fall within the performance criteria of the test
single-laboratory. Data from reagent, surface, and drinking
method. If the analyst inspects the vial containing the end
water matrices are shown in Section 16, Precision and Bias. It
calibration check standard and notices that the sample evapo-
is recommended that the laboratory generate their own in-
rated affecting the concentration, a new end calibration check
house QC acceptance criteria which meets or exceeds the
standard may be made and analyzed. If this new end calibration
criteria in this standard. References on how to generate QC
check standard has a percent deviation less than 30 % from the
acceptance criteria are Practices D2777, D5847, E2554, or
calculated concentration for the target analytes and surrogates,
Method 8000B in EPA Publication SW-846.
the results may be reported unqualified.
12.4 Surrogate Spiking Solution:
12.3 If a laboratory has not performed the test before or if
12.4.1 A surrogate spiking 50 % methanol/50 % 1,4-
there has been a major change in the measurement system, for
13 15
dioxane solution containing methomyl- C , N and
example, new analyst, new instrument, etc., a precision and
carbofuran- C is added to all samples. A stock surrogate
bias study must be performed to demonstrate laboratory
spiking solution is prepared at 50 ppm. Spiking 25 μL of this
capability.
spiking solution into a 25 mL water sample results in a
12.3.1 Analyze at least four replicates of a sample solution
concentration of 50 ppb of the surrogate in the sample. The
containing the carbamates and surrogates at a concentration in
result obtained for the surrogate recovery must fall within the
the calibration range of Levels 5 to 7. The Level 6 concentra-
limits of Table 5. If the limits are not met, the affected results
tion of the eight-point calibration curve was used to set the QC
must be qualified with an indication that they do not fall within
acceptance criteria in this test method. The matrix and chem-
the performance criteria o
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D7645 − 16 D7645 − 23
Standard Test Method for
Determination of Aldicarb, Aldicarb Sulfone, Aldicarb
Sulfoxide, Carbofuran, Methomyl, Oxamyl, and Thiofanox in
Water by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
This standard is issued under the fixed designation D7645; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This procedure test method covers the determination of aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl,
oxamyl, and thiofanox (referred to collectively as carbamates in this test method) in water by direct injection using liquid
chromatography (LC) and detected with tandem mass spectrometry (MS/MS). These analytes are qualitatively and quantitatively
determined by this test method. This test method adheres to multiple reaction monitoring (MRM) mass spectrometry.
1.2 The Detection Verification Level (DVL) and Reporting Range for the carbamates are listed in Table 1.
1.2.1 The DVL is required to be at a concentration at least 3 times below the Reporting Limit (RL) and have a signal/noise ratio
greater than 3:1. Fig. 1 displays the signal/noise ratios of the primary single reaction monitoring (SRM) transitions, and Fig. 2
displays the confirmatory SRM transitions at the DVLs for the carbamates.
1.2.2 The reporting limit is the concentration of the Level 1 calibration standard as shown in Table 2 for the carbamates.
1.3 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and healthsafety, health, and environmental practices and determine
the applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for
Organic Substances in Water.
Current edition approved Feb. 1, 2016April 15, 2023. Published May 2016May 2023. Originally approved in 2010. Last previous edition approved in 20142016 as
D7645D7645 – 16. – 14. DOI: 10.1520/D7645-16.DOI: 10.1520/D7645-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7645 − 23
TABLE 1 Detection Verification Level and Reporting Range
DVL Reporting Range
Analyte
(ng/L) (μg/L)
Aldicarb 250 1-100
Aldicarb Sulfone 250 1-100
Aldicarb Sulfoxide 250 1-100
Carbofuran 250 1-100
Methomyl 250 1-100
Oxamyl 250 1-100
Thiofanox 250 1-100
D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
D3694 Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
D3856 Guide for Management Systems in Laboratories Engaged in Analysis of Water
D4841 Practice for Estimation of Holding Time for Water Samples Containing Organic and Inorganic Constituents
D5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
E2554 Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method Using Control Chart Techniques
2.2 Other Documents:
EPA Publication SW-846 Test Methods for Evaluating Solid Waste, Physical/Chemical Methods
EPA Method 531 Measurement of N-Methyl Carbamoyloximes and N-Methyl Carbamates in Drinking Water by Direct Aqueous
Injection HPLC with Post Column Derivatization
EPA Method 531.2 Measurement of N-Methylcarbamoyloximes -Methyl Carbamoyloximes and N-Methylcarbamates -Methyl
Carbamates in Water by Direct Aqueous Injection HPLC with Postcolumn Post Column Derivatization
EPA Method 538 Determination of Selected Organic Contaminants in Drinking Water by Direct Aqueous Injection-Liquid
Chromatography/Tandem Mass Spectrometry (DAI-LC/MS/MS)
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 carbamates, n—in this test method, aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and
thiofanox collectively.
3.2.2 detection verification level, DVL, n—a concentration that has a signal/noise ratio greater than 3:1 and is at least 3 times below
the Reporting Limit (RL).
3.2.3 independent reference material, IRM, n—a material of known purity and concentration obtained either from the National
Institute of Standards and Technology (NIST) or other reputable supplier. The IRM shallsupplier; the IRM must be obtained from
a different lot of material than is used for calibration.
3.3 Acronyms:
3.3.1 CCC, n—Continuing Calibration Check
3.3.2 IC, n—Initial Calibration
3.3.3 LC, n—Liquid Chromatography
3.3.4 LCS/LCSD, n—Laboratory Control Sample/Laboratory Control Sample Duplicate
3.3.5 MeOH, n—Methanol
Available from National Technical Information Service (NTIS), U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA, 22161 or at http://www.epa.gov/
epawaste/hazard/testmethods/index.htm.
D7645 − 23
FIG. 1 Example Primary SRM Chromatograms Signal/Noise Ratios
FIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise Ratios
D7645 − 23
TABLE 2 Concentrations of Calibration Standards (PPB)
Analyte/Surrogate LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7 LV 8
Aldicarb 1 5 10 25 35 50 75 100
Aldicarb Sulfone 1 5 10 25 35 50 75 100
Aldicarb Sulfoxide 1 5 10 25 35 50 75 100
Carbofuran 1 5 10 25 35 50 75 100
Methomyl 1 5 10 25 35 50 75 100
Oxamyl 1 5 10 25 35 50 75 100
Thiofanox 1 5 10 25 35 50 75 100
Carbofuran- C (Surrogate) 1 5 10 25 35 50 75 100
13 15
Methomyl- C , N (Surrogate) 1 5 10 25 35 50 75 100
-3
3.3.6 mM, n—millimolar, 1 × 10 moles/L
3.3.7 MRM, n—Multiple Reaction Monitoring
3.3.8 MS/MSD, n—Matrix Spike/Matrix Spike Duplicate
3.3.9 NA, adj—Not Available
3.3.10 ND, n—non-detect
3.3.11 P&A, n—Precision and Accuracy
3.3.12 PPB, n—parts per billion
3.3.13 PPT, n—parts per trillion
3.3.14 QA, adj—Quality Assurance
3.3.15 QC, adj—Quality Control
3.3.16 RL, n—Reporting Limit
3.3.17 RSD, n—Relative Standard Deviation
3.3.18 RT, n—Retention Time
3.3.19 SDS, n—Safety Data Sheets
3.3.20 SRM, n—Single Reaction Monitoring
3.3.21 SS, n—Surrogate Standard
3.3.22 TC, n—Target Compound
-6
3.3.23 μM, n—micromolar, 1 × 10 moles/L
3.3.24 VOA, n—Volatile Organic Analysis
4. Summary of Test Method
4.1 This is a performance-based method, and modifications are allowed to improve performance.
D7645 − 23
4.2 For carbamate analysis, samples are shipped to the lab acidified between 0°C and 6°Cabove freezing and 6 °C and analyzed
within 14 days of collection. In the lab, the samples are spiked with surrogates, filtered using a syringe driven filter unit, and
analyzed directly by LC/MS/MS.
13 15 13
4.3 The carbamates, methomyl- C , N (surrogate) and carbofuran- C (surrogate) are identified by retention time and two SRM
2 6
transitions. The target analytes and surrogate are quantitated using the primary SRM transitions utilizing an external calibration.
The final report issued for each sample lists the concentration of carbamates and the surrogate recoveries.
5. Significance and Use
5.1 This test method has been developed by U.S. EPA Region 5 Chicago Regional Laboratory (CRL).
5.2 The N-methyl carbamate (NMC) pesticides: aldicarb, carbofuran, methomyl, oxamyl, and thiofanox have been identified by
EPA as working through a common mechanism. These affect the nervous system by reducing the ability of enzymes. Enzyme
inhibition was the primary toxicological effect of regulatory concern to EPA in assessing the NMC’s food, drinking water, and
residential risks. In most of the country, NMC residues in drinking water sources are at levels that are not likely to contribute
substantially to the multi-pathway cumulative exposure. Shallow private wells extending through highly permeable soils into
shallow, acidic ground water represent what the EPA believes to be the most vulnerable drinking water. Aldicarb sulfone and
aldicarb sulfoxide are breakdown products of aldicarb and should also be monitored due to their toxicological effects.
5.3 This test method has been investigated for use with reagent, surface, and drinking water for the selected carbamates: aldicarb,
aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox.
6. Interferences
6.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other apparatus producing discrete
artifacts or elevated baselines. All of these materials are demonstrated to be free from interferences by analyzing laboratory reagent
blanks under the same conditions as samples.
6.2 All glassware is washed in hot water with detergent and rinsed in hot water followed by distilled water. The glassware is then
dried and heated in an oven at 250°C for 15 to 30 minutes. 250 °C for 15 min to 30 min. All glassware is subsequently cleaned
with acetone followed by methanol.
6.3 All reagents and solvents should be pesticide residue purity or higher to minimize interference problems.
6.4 Matrix interferences may be caused by contaminants in the sample. The extent of matrix interferences can vary considerably
from sample source depending on variations of the sample matrix.
7. Apparatus
7.1 LC/MS/MS System:
7.1.1 Liquid Chromatography (LC) System—A complete LC system is needed to analyze samples. This should include a sample
injection system, a solvent pumping system capable of mixing solvents, a sample compartment capable of maintaining required
temperature and a temperature controlled column compartment. A system that is capable of performing at the flows, pressures,
controlled temperatures, sample volumes, and requirements of the standard may be used.
7.1.2 Analytical Column —A C18 column was used to develop this test method.
Additional information about Carbamate pesticides area available from United States Environmental Protection Agency (EPA), http://www.epa.gov.
A Waters ACQUITY UPLC (a trademark of Waters Technologies Corporation in Wilmington, DE) BEH C18, 2.12.1 mm × 100 mm, 1.7 μm particle size was used to
develop this test method. Any column that achieves adequate resolution may be used. The retention times and order of elution may change depending on the column used
andused, if you are aware of an alternative column that meets the performance of the standard, please provide this information to ASTM International Headquarters. Your
comments will receive careful consideration at a meeting of the responsible technical committee, need to be monitored.which you may attend.
A Quattro Premier XE (a trademark of Waters Technologies Corporation in Wilmington, DE) tandem quadrupole mass spectrometer was used to develop this test method.
All parameters in this test method are based on this system and may vary depending on your instrument.
D7645 − 23
7.2 Tandem Mass Spectrometer (MS/MS) System—A MS/MS system capable of MRM analysis. Any system that is capable of
performing at the requirements in this standard may be used.
7.3 Filtration Device:
7.3.1 Hypodermic syringe—Syringe—A lock tip glass syringe capable of holding a syringe-driven filter unit or similar may be
used.
7.3.1.1 A 25-mL lock tip glass syringe size is recommended since a 25-mL sample size is used in this test method.
7.3.2 Filter unitUnit —PVDF filter units were used to filter the samples.
8. Reagents and Materials
8.1 Purity of Reagents—High Performance Liquid Chromatography (HPLC) pesticide residue analysis and spectrophotometry
grade chemicals shallmust be used in all tests. Unless indicated otherwise, it is intended that all reagents shallmust conform to the
Committee on Analytical Reagents of the American Chemical Society. Other reagent grades may be used provided they are first
determined to be of sufficiently high purity to permit their use without affecting the accuracy of the measurements.
8.2 Purity of Water—Unless otherwise indicated, references to water shallmust be understood to mean reagent water conforming
to Type 1 of Specification D1193. It must be demonstrated that this water does not contain contaminants at concentrations sufficient
to interfere with the analysis.
8.3 Gases—Ultrapure nitrogen and argon.
8.4 Acetonitrile (CAS # 75-05-8).
8.5 Methanol (CAS # 67-56-1).
8.6 Acetone (CAS # 67-64-1).
8.7 Ammonium Formate (CAS # 540-69-2).
8.8 Acetic Acid (Glacial, CAS # 64-19-7).
8.9 Aldicarb (CAS # 116-06-3).
8.10 Aldicarb Sulfone (CAS # 1646-88-4).
8.11 Aldicarb Sulfoxide (CAS # 1646-87-3).
8.12 Carbofuran (CAS # 1563-66-2).
8.13 Oxamyl (CAS # 23135-22-0).
A Millex HV Syringe Driven Filter Unit PVDF 0.22 μm (Millipore Corporation, Catalog #SLGV033NS; Millex is a trademark of Merck KGAA, Darmstadt, Germany)
has been found suitable for use for this test method, any filter unit may be used was used, if you are aware of an alternative filter that meets the performance of this test method
the standard, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical
committee, may be used. which you may attend.
Reagent Chemicals, American Chemical Society Specifications, ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, D.C. For SuggestionsDC. For suggestions on the testing of reagents not listed by the American Chemical Society, see
AnnualAnalar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulators,Formulary, U.S.
Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
D7645 − 23
8.14 Methomyl (CAS # 16752-77-5).
8.15 Thiofanox (CAS # 39196-18-4).
13 15 13 15
8.16 Methomyl- C , N (acetohydroxamate- C , N, CAS # (unlabeled) 16752-77-5).
2 2
13 13
8.17 Carbofuran- C (Ring- C , CAS # (unlabeled) 1563-66-2).
6 6
9. Hazards
9.1 Normal laboratory safety applies to this test method. Analysts should wear safety glasses, gloves, and lab coats when working
in the lab. Analysts should review the Safety Data Sheets (SDS) for all reagents used in this test method.
10. Sampling
10.1 Sampling and Preservation—Grab samples should be collected in ≥25 mL pre-cleaned amber glass bottles with
Tefloninert-lined-lined caps demonstrated to be free of interferences. All samples are acidified with glacial acetic acid to pH ≤3.8
upon collection. A few drops or less of glacial acetic acid is required per 40 mL water sample collected. Chlorinated drinking water
samples are also dechlorinated with ascorbic acid; 10 mg of ascorbic acid is added to each 40 mL volume of water prior to
collection. Drinking water samples must be dechlorinated upon collection. Aldicarb oxidizes when residual chlorine is present in
the sample. This test method is based on a 25 mL sample size per analysis. If different sample sizes are used, spiking solution
amounts and preservatives will need to be modified. Conventional sampling practices should be followed. Refer to Guide D3856
and Practices D3694. Store samples between 0°C and 6°Cabove freezing and 6 °C from the time of collection until analysis.
Analyze the sample within 14 days of collection.
NOTE 1—Less sample volume is acceptable, but the spike amounts and sample preservatives must be adjusted accordingly.
10.1.1 EPA Method 531.2 demonstrated that carbamates are more stable under acidic conditions. Potassium dihydrogen citrate
buffer is used in Method 531.2 to bring the pH to ~3.8, but this buffer is incompatible with LC/MS/MS. Therefore, the pH
adjustment is accomplished with acetic acid in this test method. EPA Method 531.2 demonstrated that carbamates under acidic
conditions are stable for at least 28 days. EPA Method 531 demonstrated that oxamyl and methomyl are stable for at least 70 days
at pH 3 6 0.2. Holding time is dependent upon youran individual matrix and will vary. Practice D4841 may be used to conduct
a holding time study on youran individual matrix.
11. Preparation of LC/MS/MS
11.1 LC Chromatograph Operating Conditions:
11.1.1 Injection volumes of all calibration standards and samples are made at 50 μL volume using a full loop injection. If a 50
μL volume loop is installed in the LC, a “full loop” mode is the preferred technique when performing fast, qualitative analyses.
This mode should be used whenever accuracy and precision are the primary concerns. The first sample analyzed after the
calibration curve is a blank to ensure there is no carry-over. The gradient conditions for the liquid chromatograph are shown in
Table 3.
TABLE 3 Gradient Conditions for Liquid Chromatography
Percent Percent
Time Flow 95 % Water/ 95 % Methanol/
(min) (μL/min) 5 % Methanol, 5 % Water,
5 mM NH CO H 5 mM NH CO H
4 2 4 2
0.0 300 100 0
2.0 300 100 0
3.0 300 95 5
5.0 300 85 15
10.0 300 0 100
11.5 300 0 100
12.0 300 100 0
14.0 300 100 0
D7645 − 23
11.2 LC Sample Manager Conditions:
11.2.1 Wash Solvents—Weak wash is 2.4 mL of 95 % water/5 % methanol. Strong wash is 1.2 mL of methanol. The strong wash
solvent is needed to eliminate carry-over between injections of carbamate samples. The weak wash is used to remove the strong
wash solvent. Instrument manufacturer specifications should be followed in order to eliminate sample carry-over.
11.2.2 Temperatures—Column, 30°C;30 °C; Sample compartment, 15°C.15 °C.
11.2.3 Seal Wash—Solvent: 50 Acetonitrile/50 Water; Time: 5 minutes.5 min.
11.3 Mass Spectrometer Parameters:
11.3.1 To acquire the maximum number of data points per SRM channel while maintaining adequate sensitivity, the tune
parameters may be optimized according to yourthe instrument. Each peak requires at least 10 scans per peak for adequate
quantitation. This test method contains two surrogates, which are isotopically labeled methomyl and carbofuran, and seven
carbamates, which are split up into seven MRM acquisition functions to optimize sensitivity. Variable parameters regarding
retention times, SRM transitions, and cone and collision energies are shown in Table 4. Mass spectrometer parameters used in the
development of this test method are listed below:
The instrument is set in the Electrospray positive source setting.
Capillary Voltage: 3.5 kV
Cone: Variable depending on analyte (Table 4)
Extractor: 2 Volts
RF Lens: 0.1 Volts
Source Temperature: 120°C
Source Temperature: 120 °C
Desolvation Temperature: 375°C
Desolvation Temperature: 375 °C
Desolvation Gas Flow: 800 L/hr
Desolvation Gas Flow: 800 L/h
Cone Gas Flow: 25 L/hr
Cone Gas Flow: 25 L/h
Low Mass Resolution 1: 14.5
High Mass Resolution 1: 14.5
Ion Energy 1: 0.5
Entrance Energy: –1
Collision Energy: Variable depending on analyte (Table 4)
Exit Energy: 0
Low Mass Resolution 2: 14.5
High Mass resolution 2: 14.5
Ion Energy 2: 0.7
Multiplier: 650
-3
Gas Cell Pirani Gauge: 7.0 × 10 Torr
TABLE 4 Retention Times, SRM Ions, and Analyte-Specific Mass Spectrometer Parameters
SRM Primary/
Primary/ Retention Time Cone Voltage Collision Energy
Analyte Mass Transition Confirmatory
Confirmatory (min) (Volts) (eV)
(Parent > Product) SRM Area Ratio
Aldicarb Primary 8.50 10 7 208.0 > 115.6 1.4
Confirmatory 10 16 208.0 > 88.7
Aldicarb Sulfone Primary 6.20 13 13 240.1 > 147.8 1.6
Confirmatory 13 11 240.1 > 165.8
Aldicarb Sulfoxide Primary 5.72 16 6 207.1 > 131.9 1.1
Confirmatory 16 14 207.1 > 88.8
Carbofuran Primary 8.96 22 12 222.1 > 164.9 1.2
Confirmatory 22 20 222.1 > 122.7
Methomyl Primary 6.68 15 10 162.9 > 87.7 1.8
Confirmatory 15 10 162.9 > 105.7
Oxamyl Primary 6.44 11 11 237.1 > 71.8 2.6
Confirmatory 11 7 237.1 > 89.7
Thiofanox Primary 9.32 12 8 219.1 > 56.8 4.8
Confirmatory 12 5 219.1 > 75.9
Carbofuran- C Primary 8.96 22 11 228.1 > 170.9 1.3
(Surrogate) Confirmatory 22 21 228.1 > 128.8
13 15
Methomyl- C , N Primary 6.68 18 8 165.8 > 90.7 1.7
(Surrogate)
Confirmatory 18 9 165.8 > 108.7
D7645 − 23
Inter-Channel Delay: 0.005 seconds
Inter-Channel Delay: 0.005 s
Inter-Scan Delay: 0.005 seconds
Inter-Scan Delay: 0.005 s
Dwell: 0.075 seconds
Dwell: 0.075 s
12. Calibration and Standardization
12.1 The mass spectrometer must be calibrated in accordance with manufacturer specifications before analysis. In order to obtain
accurate analytical values through using this test method within the confidence limits, the following procedures must be followed
when performing the test method. Prepare all solutions in the lab using Class A volumetric glassware.
12.2 Calibration and Standardization—To calibrate the instrument, analyze eight calibration standards containing the eight
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concentration levels of the carbamates, methomyl- C , N and carbofuran- C prior to analysis as shown in Table 2. A calibration
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stock standard solution is prepared from standard materials or they are purchased as certified solutions. Stock Standard Solution
A containing the carbamates and surrogates is prepared at Level 8 concentration and aliquots of that solution are diluted to prepare
Levels 1 through 7. The following steps will produce standards with the concentration values shown in Table 2. The analyst is
responsible for recording initial component weights carefully when working with pure materials and correctly carrying the weights
through the dilution calculations.
12.2.1 Prepare Stock Standard Solution A (Level 8) by adding to a 50 mL volumetric flask individual solutions of the following:
100 μL of aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl, and thiofanox, each at 50 ppm in methanol
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and 50 μL of methomyl- C , N in methanol and carbofuran- C in 1,4-dioxane each at 100 ppm, dilute to 50 mL with water.
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The preparation of the Level 8 standard can be accomplished using different volumes and concentrations of stock solutions as is
accustomed in the individual laboratory. Depending on the prepared stock concentrations, the solubility at that concentration will
have to be ensured.
12.2.2 Aliquots of Solution A are then diluted with water to prepare the desired calibration levels in 2-mL amber glass LC vials.
The calibration vials must be used within 24 hoursh to ensure optimum results. Stock calibration standards are routinely replaced
every 7 days if not previously discarded for quality control failure. Calibration standards are not filtered.
12.2.3 Inject each standard and obtain its chromatogram. An external calibration technique is used to monitor the primary and
confirmatory SRM transitions of each analyte. Calibration software is utilized to conduct the quantitation of the target analytes and
surrogates using the primary SRM transition. The ratios of the primary/confirmatory SRM transition area counts are given in Table
4 and will vary depending on the individual tuning conditions. The primary/confirmatory SRM transition area ratio must be within
35 % of the individual labs’ accepted primary/confirmatory SRM transition area ratio. The primary SRM transition of each analyte
is used for quantitation and the confirmatory SRM transition for confirmation. This gives added confirmation by isolating the parent
ion, forming two product ions via fragmentation, and relating it to the retention time in the calibration standard.
12.2.4 The calibration software manual should be consulted to use the software correctly. The quantitation method is set as an
external calibration using the peak areas in ppt or ppb units, as long as the analyst is consistent. Concentrations may be calculated
using the data system software to generate linear regression or quadratic calibration curves. Forcing the calibration curve through
the origin is not recommended. Curves should be evaluated using relative error or relative standard error.
12.2.5 Linear calibration may be used if the coefficient of determination, r , is >0.98 for the analyte. The point of origin is excluded
and a fit weighting of 1/X is used in order to give more emphasis to the lower concentrations. If one of the calibration standards
other than the high or low point causes the rEach calibration point used to generate of the curve to be <0.98, this point must be
re-injected or a new calibration curve must be regenerated. If the low and/or high point is excluded, minimally a six point curve
is acceptable but the reporting range must be modified to reflect this change.must have a calculated percent deviation less than
25 % from the generated curve.
12.2.6 Quadratic calibration may be used if the coefficient of determination, r , is >0.99 for the analyte. The point of origin is
excluded, and a fit weighting of 1/X is used in order to give more emphasis to the lower concentrations. If one of the calibration
standards causes the curve to be <0.99, this point must be re-injected or a new calibration curve must be regenerated. Minimally
a six point curve is acceptable using a quadratic fit. Each calibration point used to generate the curve must have a calculated percent
deviation less than 25 % from the generated curve.
Teflon is a trademark of The Chemours Company, LLC, in Wilmington, DE.Management and Technical Requirements for Laboratories Performing Environmental
Analysis; Module 4: Quality Systems for Chemical Testing; The NELAC Institute, 2017.
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12.2.6.1 An initial eight point curve over the calibration range is an option in the event that the low or high point, or both, must
be excluded to obtain a coefficient of determination >0.99. In this event, the reporting range must be modified to reflect this change.
12.2.7 The retention time window of the SRM transitions must be within 5 % 5 % of the retention time of the analyte in a midpoint
calibration standard. If this is not the case, re-analyze the calibration curve to determine if there was a shift in retention time during
the analysis and the sample needs to be re-injected. If the retention time is still incorrect in the sample, refer to the analyte as an
unknown.
12.2.8 A midpoint calibration check standard must be analyzed at the end of each batch of 20 samples or within 24 hoursh after
the initial calibration curve was generated. This end calibration check should be the same calibration standard that was used to
generate the initial curve. The results from the end calibration check standard must have a percent deviation less than 30 % from
the calculated concentration for the target analytes and surrogates. If the results are not within these criteria, the problem must be
corrected and either all samples in the batch must be re-analyzed against a new calibration curve or the affected results must be
qualified with an indication that they do not fall within the performance criteria of the test method. If the analyst inspects the vial
containing the end calibration check standard and notices that the sample evaporated affecting the concentration, a new end
calibration check standard may be made and analyzed. If this new end calibration check standard has a percent deviation less than
30 % from the calculated concentration for the target analytes and surrogates, the results may be reported unqualified.
12.3 If a laboratory has not performed the test before or if there has been a major change in the measurement system, for example,
new analyst, new instrument, etc., a precision and bias study must be performed to demonstrate laboratory capability.
12.3.1 Analyze at least four replicates of a sample solution containing the carbamates and surrogates at a concentration in the
calibration range of Levels 5 to 7. The Level 6 concentration of the eight-point calibration curve was used to set the QC acceptance
criteria in this test method. The matrix and chemistry should be similar to the solution used in this test method. Each replicate must
be taken through the complete analytical test method including any sample preservation and pretreatment steps.
12.3.2 Calculate the mean (average) percent recovery and relative standard deviation (RSD) of the four values and compare to the
acceptable ranges of the QC acceptance criteria for the Initial Demonstration of Performance in Table 5.
12.3.3 This study should be repeated until the single operator precision and mean recovery are within the limits in Table 5. If a
concentra
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