ASTM D5315-04(2024)

Standard Test Method for Determination of N-Methyl-Carbamoyloximes and N-Methylcarbamates in Water by Direct Aqueous Injection HPLC with Post-Column Derivatization

SIGNIFICANCE AND USE
5.1 N-methylcarbamates and n-methylcarbomoyloximes are used in agriculture as insecticides and herbicides. They are sometimes found in both surface and ground waters and can be toxic to animals and plants at moderate to high concentrations. The manufacturing precursors and degradation products may be equally as hazardous to the environment.
SCOPE
1.1 This is a high-performance liquid chromatographic (HPLC) test method applicable to the determination of certain n-methylcarbamoyloximes and n-methylcarbamates in ground water and finished drinking water (1).2 This test method is applicable to any carbamate analyte that can be hydrolyzed to a primary amine. The following compounds have been validated using this test method:    
Analyte  
Chemical Abstract Services
Registry Number A  
Aldicarb  
116-06-3    
Aldicarb sulfone  
1646-88-4    
Aldicarb sulfoxide  
1646-87-3    
Baygon  
114-26-1    
Carbaryl  
63-25-2    
Carbofuran  
1563-66-2    
3-Hydroxycarbofuran  
16655-82-6    
Methiocarb  
2032-65-7    
Methomyl  
16752-77-5    
Oxamyl  
23135-22-0    
1.2 This test method has been validated in a collaborative round-robin study (2) and estimated detection limits (EDLs) have been determined for the analytes listed in 1.1 (Table 1). Observed detection limits may vary between ground waters, depending on the nature of interferences in the sample matrix and the specific instrumentation used.  
1.3 This test method is restricted to use by, or under the supervision of, analysts experienced in both the use of liquid chromatography and the interpretation of liquid chromatograms. Each analyst should demonstrate an ability to generate acceptable results with this test method using the procedure described in 12.3.  
1.4 When this test method is used to analyze unfamiliar samples for any or all of the analytes listed in 1.1, analyte identifications should be confirmed by at least one additional qualitative technique.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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. Additional guidance on laboratory safety is available and suitable references for the information are provided  (3-5).  
1.7 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: 31-Mar-2024
• ICS: 71.080.30 - Organic nitrogen compounds
• Technical Committee: D19 - Water
• Drafting Committee: D19.06 - Methods for Analysis for Organic Substances in Water

Relations

• Replaces: ASTM D5315-04(2017)e1 - Standard Test Method for Determination of N-Methyl-Carbamoyloximes and N-Methylcarbamates in Water by Direct Aqueous Injection HPLC with Post-Column Derivatization
• Effective Date: 01-Apr-2024
• Referred By: ASTM D3694-96(2024) - Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• Effective Date: 01-Apr-2024

Overview

ASTM D5315-04(2024) is an internationally recognized standard test method developed by ASTM International. It specifies procedures for the determination of N-methyl-carbamoyloximes and N-methylcarbamates in water using direct aqueous injection high-performance liquid chromatography (HPLC) with post-column derivatization. These analytes include certain widely used pesticides that are of environmental concern due to their toxicity and persistence in water sources. The standard is applicable for analyzing both groundwater and finished drinking water to identify potential contamination at trace levels.

Key Topics

• Target Compounds: The method is validated for a range of carbamate pesticides and related compounds common in agricultural practices. Confirmed analytes include:
  • Aldicarb and its sulfone/sulfoxide forms
  • Baygon (Propoxur)
  • Carbaryl
  • Carbofuran and 3-Hydroxycarbofuran
  • Methiocarb
  • Methomyl
  • Oxamyl
• Analytical Procedure: The standard requires direct aqueous sample injection onto a reverse-phase HPLC column. Following separation, analytes are hydrolyzed to primary amines, derivatized post-column, and detected by fluorescence for high sensitivity and selectivity.
• Quality Control: Robust quality control measures are integral, including:
  • Internal and external calibration with standard solutions
  • Use of laboratory reagent blanks, fortified samples, and quality control samples
  • Requirements for initial demonstration of laboratory capability and ongoing performance monitoring
  • Procedures to confirm analyte identification using supplementary qualitative techniques
• Sample Handling and Preservation: Strict sample preservation (acidification and cooling) is necessary to prevent degradation, particularly for unstable analytes.
• Analyst Competency: Only qualified analysts trained in HPLC and chromatogram interpretation should apply this method.

Applications

• Environmental Monitoring: Detection and quantification of pesticide residues in both surface and groundwater, supporting regulatory compliance and pollution control.
• Drinking Water Quality Assessment: Ensures public health by regularly checking for hazardous carbamate pesticide residues in municipal and private drinking water supplies.
• Agricultural Impact Studies: Evaluates the movement and persistence of common N-methylcarbamate pesticides from agricultural applications into wider water ecosystems.
• Industrial Effluent Analysis: Assesses manufacturing, processing, or remediation site discharges for compliance with environmental regulations.

By providing a sensitive, selective, and validated approach for the detection of N-methylcarbamate and related pesticides, ASTM D5315-04(2024) supports organizations in meeting water quality standards and protecting environmental and public health.

Related Standards

Users of ASTM D5315-04(2024) may also reference or require compliance with the following standards and guidelines:

• ASTM D1129 - Terminology Relating to Water
• ASTM D1193 - Specification for Reagent Water
• ASTM D3370 - Practices for Sampling Water from Flowing Process Streams
• ASTM D3694 - Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• ASTM E682 - Practice for Liquid Chromatography Terms and Relationships
• EPA Method 531.1 and 531.2 - US EPA methods for determination of carbamates in water

These documents collectively provide terminology, detailed procedures for sampling, reagent preparation, terminology definitions, and additional analytical techniques to ensure the accuracy and integrity of water quality testing for pesticide contamination.

---

Keywords: ASTM D5315-04(2024), N-methylcarbamoyloximes, N-methylcarbamates, water analysis, HPLC, post-column derivatization, carbamate pesticides in water, pesticide residue analysis, groundwater monitoring, drinking water safety, environmental testing standards, agricultural water quality.

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: D5315 − 04 (Reapproved 2024)
Standard Test Method for
Determination of N-Methyl-Carbamoyloximes and
N-Methylcarbamates in Water by Direct Aqueous Injection
HPLC with Post-Column Derivatization
This standard is issued under the fixed designation D5315; 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 1.4 When this test method is used to analyze unfamiliar
samples for any or all of the analytes listed in 1.1, analyte
1.1 This is a high-performance liquid chromatographic
identifications should be confirmed by at least one additional
(HPLC) test method applicable to the determination of certain
qualitative technique.
n-methylcarbamoyloximes and n-methylcarbamates in ground
1.5 The values stated in SI units are to be regarded as
water and finished drinking water (1). This test method is
standard. No other units of measurement are included in this
applicable to any carbamate analyte that can be hydrolyzed to
standard.
a primary amine. The following compounds have been vali-
dated using this test method: 1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
Chemical Abstract Services
A
Analyte Registry Number
responsibility of the user of this standard to establish appro-
Aldicarb 116-06-3
priate safety, health, and environmental practices and deter-
Aldicarb sulfone 1646-88-4
mine the applicability of regulatory limitations prior to use.
Aldicarb sulfoxide 1646-87-3
Baygon 114-26-1
Additional guidance on laboratory safety is available and
Carbaryl 63-25-2
suitable references for the information are provided (3-5).
Carbofuran 1563-66-2
1.7 This international standard was developed in accor-
3-Hydroxycarbofuran 16655-82-6
Methiocarb 2032-65-7
dance with internationally recognized principles on standard-
Methomyl 16752-77-5
ization established in the Decision on Principles for the
Oxamyl 23135-22-0
Development of International Standards, Guides and Recom-
A
mendations issued by the World Trade Organization Technical
Numbering system of Chemical Abstracts, Inc.
Barriers to Trade (TBT) Committee.
1.2 This test method has been validated in a collaborative
round-robin study (2) and estimated detection limits (EDLs) 2. Referenced Documents
have been determined for the analytes listed in 1.1 (Table 1). 3
2.1 ASTM Standards:
Observed detection limits may vary between ground waters,
D1129 Terminology Relating to Water
depending on the nature of interferences in the sample matrix
D1192 Guide for Equipment for Sampling Water and Steam
and the specific instrumentation used. 4
in Closed Conduits (Withdrawn 2003)
D1193 Specification for Reagent Water
1.3 This test method is restricted to use by, or under the
D2777 Practice for Determination of Precision and Bias of
supervision of, analysts experienced in both the use of liquid
Applicable Test Methods of Committee D19 on Water
chromatography and the interpretation of liquid chromato-
D3370 Practices for Sampling Water from Flowing Process
grams. Each analyst should demonstrate an ability to generate
Streams
acceptable results with this test method using the procedure
D3694 Practices for Preparation of Sample Containers and
described in 12.3.
for Preservation of Organic Constituents
E682 Practice for Liquid Chromatography Terms and Rela-
tionships
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. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 1, 2024. Published April 2024. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
ɛ1
approved in 1992. Last previous edition approved in 2017 as D5315 – 04 (2017) . Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/D5315-04R24. the ASTM website.
The boldface numbers in parentheses refer to the references at the end of this The last approved version of this historical standard is referenced on
test method. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5315 − 04 (2024)
TABLE 1 Relative Retention Times for the Primary and
of the same solution; the internal standard must be an analyte
Confirmation Columns and EDLs for the 10 Carbamate
that is not a sample component.
Pesticides
3.2.5 laboratory duplicates (LD1 and LD2), n—two sample
Retention Time (minutes)
Analyte
aliquots taken in the analytical laboratory and analyzed sepa-
A B C
Primary Confirmation EDL
rately with identical procedures; analyses of LD1 and LD2
Aldicarb 27.0 21.4 1.0
provide a measure of the precision associated with laboratory
Aldicarb sulfone 15.2 12.2 2.0
Aldicarb sulfoxide 15.0 17.5 2.0
procedures, but not with sample collection, preservation, or
Baygon (Propoxur) 29.6 23.4 1.0
storage procedures.
Carbaryl 30.8 25.4 2.0
Carbofuran 29.3 24.4 1.5
3.2.6 laboratory-fortified blank (LFB), n—an aliquot of
3-Hydroxycarbofuran 23.3 19.0 2.0
reagent water to which known quantities of the test method
Methiocarb 34.9 28.6 4.0
Methomyl 18.4 14.8 0.50 analytes are added in the laboratory; the LFB is analyzed
Oxamyl 17.4 14.6 2.0
exactly as a sample is; its purpose is to determine whether the
A
Primary column—250 mm by 4.6 mm inside diameter Altex Ultrasphere ODS, 5
methodology is in control and whether the laboratory is
μm.
capable of making accurate and precise methods at the required
B
Confirmation column—250 mm by 4.6 mm inside diameter Supelco LC-1, 5 μm.
C
test method detection limit.
Estimated method detection limit in micrograms per litre.
3.2.7 laboratory-fortified sample matrix (LFM), n—an ali-
quot of an environmental sample to which known quantities of
5 the test method analytes are added in the laboratory; the LFM
2.2 U.S. Environmental Protection Agency Standard:
is analyzed exactly as a sample is; its purpose is to determine
EPA Method 531.1 Revision 3.0, USEPA, EMSL-Cincinnati,
whether the sample matrix contributes bias to the analytical
results; the background concentrations of the analytes in the
EPA Method 531.2 Revision 1.0, USEPA, EMSL-Cincinnati,
sample matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background concen-
trations.
3. Terminology
3.2.8 laboratory performance check solution (LPC), n—a
3.1 Definitions:
solution of method analytes, surrogate compounds, and internal
3.1.1 For definitions of water terms used in this standard,
standards used to evaluate the performance of the instrument
refer to Terminology D1129.
system with respect to a defined set of method criteria.
3.1.2 For definitions of other terms used in this standard,
refer to Practice E682.
3.2.9 laboratory reagent blank (LRB), n—an aliquot of
reagent water treated exactly the same as a sample, including
3.2 Definitions of Terms Specific to This Standard:
being exposed to all glassware, equipment, solvents, reagents,
3.2.1 calibration standard (CAL), n—a solution prepared
internal standards, and surrogates that are used with other
from the primary dilution standard solution and stock standard
samples; the LRB is used to determine whether method
solutions of the internal standards and surrogate analytes; CAL
analytes or other interferences are present in the laboratory
solutions are used to calibrate the instrument response with
environment, the reagents, or the apparatus.
respect to analyte concentration.
3.2.10 primary dilution standard solution, n—a solution of
3.2.2 field duplicates (FD1 and FD2), n—two separate
several analytes prepared in the laboratory from stock standard
samples collected at the same time, placed under identical
solutions and diluted as necessary to prepare calibration
circumstances, and treated exactly the same throughout field
solutions and other necessary analyte solutions.
and laboratory procedures; analyses of FD1 and FD2 provide a
measure of the precision associated with sample collection,
3.2.11 quality control sample (QCS), n—a sample matrix
preservation, and storage, as well as with laboratory proce-
containing test method analytes or a solution of test method
dures.
analytes in a water miscible solvent that is used to fortify water
or environmental samples; the QCS is obtained from a source
3.2.3 field reagent blank (FRB), n—reagent water placed in
external to the laboratory and is used to check the laboratory
a sample container in the laboratory and treated in all respects
performance with externally prepared test materials.
as a sample, including being exposed to sampling site
conditions, storage, preservation, and all analytical procedures;
3.2.12 stock standard solution, n—a concentrated solution
the purpose of the FRB is to determine whether method
containing a single certified standard that is a method analyte,
analytes or other interferences are present in the field environ-
or a concentrated solution of a single analyte prepared in the
ment.
laboratory with an assayed reference compound; stock standard
3.2.4 internal standard, n—a pure analyte(s) added to a solutions are used to prepare primary dilution standards.
solution in known amount(s) and used to measure the relative
3.2.13 surrogate analyte, n—a pure analyte(s), which is
responses of other analytes and surrogates that are components
extremely unlikely to be found in any sample, and which is
added to a sample aliquot in known amount(s) before extrac-
tion; it is measured with the same procedures used to measure
Available from United States Environmental Protection Agency (EPA), William
other sample components; the purpose of a surrogate analyte is
Jefferson Clinton Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460,
http://www.epa.gov. to monitor the method performance with each sample.
D5315 − 04 (2024)
4. Summary of Test Method especially in TOC content. High reagent water TOC causes a
deterioration of column selectivity, baseline stability, and
4.1 The water sample is filtered, and a 200 μL to 400 μL
analyte sensitivity.
aliquot is injected onto a reverse phase HPLC column. Sepa-
6.5 Eliminate all sources of airborne primary amines, espe-
ration of the analytes is achieved using gradient elution
cially ammonia, which are absorbed into the mobile phases and
chromatography. After elution from the HPLC column, the
effect sensitivity.
analytes are hydrolyzed with sodium hydroxide (2.0 g/L
NaOH) at 95 °C. The methylamine formed during hydrolysis is
reacted with o-phthalaldehyde (OPA) and 2-mercaptoethanol 7. Apparatus
to form a highly fluorescent derivative that is detected by a
7.1 Sampling Equipment:
fluorescence detector (5).
6 7
7.1.1 Sample Bottle, 60 mL screw cap glass vials and caps
4.2 This test method is applicable to any carbamate analyte equipped with a PTFE-faced silicone septa. Prior to use, wash
the vials and septa as described in 6.1.1.
that can be hydrolyzed to a primary amine, not necessarily
methylamine.
7.2 Filtration Apparatus:
7.2.1 Macrofiltration Device, to filter derivatization solu-
5. Significance and Use
tions and mobile phases used in HPLC. It is recommended that
47 mm, 0.45 μm pore size filters be used.
5.1 N-methylcarbamates and n-methylcarbomoyloximes are
used in agriculture as insecticides and herbicides. They are 7.2.2 Microfiltration Device, to filter samples prior to HPLC
sometimes found in both surface and ground waters and can be analysis. Use a 13 mm filter holder and 13 mm diameter,
0.2 μm polyester filters.
toxic to animals and plants at moderate to high concentrations.
The manufacturing precursors and degradation products may
7.3 Syringes and Valves:
be equally as hazardous to the environment.
7.3.1 Hypodermic Syringe, 10 mL, glass, with Luer-Lok
tip.
6. Interferences
7.3.2 Syringe Valve, three-way.
6.1 Test method interferences may be caused by contami-
7.3.3 Syringe Needle, 7 cm to 10 cm long, 17 gauge, blunt
nants in solvents, reagents, glassware, and other sample pro- tip.
cessing apparatuses that lead to discrete artifacts or elevated
7.3.4 Micro Syringes, various sizes.
baselines in liquid chromatograms. Specific sources of con-
7.4 Miscellaneous:
tamination have not been identified. All reagents and apparatus
7.4.1 Solution Storage Bottles, amber glass, 10 mL to
must be routinely demonstrated to be free of interferences
15 mL capacity with TFE-fluorocarbon-lined screw cap.
under the analysis conditions by running laboratory reagent
blanks in accordance with 12.2.
7.5 High-Performance Liquid Chromatograph (HPLC):
6.1.1 Glassware must be cleaned scrupulously. Clean all 7.5.1 HPLC System, capable of injecting 200 μL to
1000 μL aliquots and performing ternary linear gradients at a
glassware as soon as possible after use by rinsing thoroughly
with the last solvent used in it. constant flow rate. A data system is recommended for measur-
ing peak areas. Table 2 lists the retention times observed for
6.1.2 After drying, store glassware in a clean environment
test method analytes using the columns and analytical condi-
to prevent any accumulation of dust or other contaminants.
tions described below.
Store the glassware inverted or capped with aluminum foil.
7.5.2 Column 1 (Primary Column), 250 mm long by
6.1.3 The use of high-purity reagents and solvents helps to
4.6 mm inside diameter, stainless steel, packed with 5 μm C-18
minimize interference problems.
material. Mobile phase is established at 1.0 mL/min as a
6.2 Interfering contamination may occur when a sample
containing low concentrations of analytes is analyzed imme-
diately after a sample containing relatively high concentrations
Sample bottle vial, Pierce No. 13075, available from Pierce Chemical Co., 3747
of analytes. A preventive technique is between-sample rinsing
N. Meridian Rd., Rockford, IL 61101, or equivalent, has been found suitable for use.
of the sample syringe and filter holder with two portions of 7
Sample bottle cap, Pierce No. 12722, available from Pierce Chemical Co., 3747
water. Analyze one or more laboratory method blanks after N. Meridian Rd., Rockford, IL 61101, or equivalent, has been found suitable for use.
Millipore Type HA, 0.45 μm for water, and Millipore Type FH, 0.5 μm for
analysis of a sample containing high concentrations of ana-
organics, available from Millipore Corp., 80 Ashby Rd., Bedford, MA 01730, or
lytes.
equivalent, has been found suitable for use.
Millipore stainless steel XX300/200, available from Millipore Corp., 80 Ashby
6.3 Matrix interference may be caused by contaminants
Rd., Bedford, MA 01730, or equivalent, has been found suitable for use.
present in the sample. The extent of matrix interference will 10
Nucleopore 180406, available from Costar Corp., 1 Alewife Center,
vary considerably from source to source, depending upon the Cambridge, MA 02140, or equivalent, has been found suitable for use.
Luer-Lok connectors are available from most laboratory suppliers.
water sampled. Positive analyte identifications must be con-
Hamilton HV3-3, available from Hamilton Co., P.O. Box 10030, Reno, NV
firmed using the alternative conformational columns, or LC/
89502, or equivalent, has been found suitable for use.
MS.
Consult HPLC manufacturer’s operation manuals for specific instructions
relating to the equipment.
6.4 The quality of the reagent water used to prepare stan-
Beckman Ultrasphere ODS, available from Beckman Instruments, 2500
dards and samples must conform to Specification D1193, Harbor Blvd., Fullerton, CA 92634, has been found suitable for use.
D5315 − 04 (2024)
A
TABLE 2 Retention Times for Method Analytes Retention Time
B C D
Analyte Primary Confirmation Confirmation
Minutes
Aldicarb sulfoxide 6.80 17.5
Aldicarb sulfone 7.77 12.2
Oxamyl 8.20 14.6
Methomyl 8.94 14.8
3-Hydroxycarbofuran 13.65 19
Aldicarb 16.35 21.4
Baygon (Propoxur) 18.86 24.4
Carbofuran 19.17 23.4
Carbaryl 20.29 25.4
Methiocarb 24.74 28.6
BDMC 25.28 .
A
Columns and analytical conditions are described in 7.5.2, 7.5.3.
B
Beckman Ultasphere ODS.
C
Supelco LC-1.
D
Waters Carbamate Analysis Column using ternary gradient conditions.
linear gradient from 15:85 methanol: water to 100 % methanol where such specifications are available. Other grades may be
in 32 min. Data presented in this test method were obtained used, provided it is first ascertained that the reagent is of
using this column. sufficiently high purity to permit its use without lessening the
7.5.3 Column 2 (Alternative Column), 250 mm long by accuracy of the determination. For trace analysis using organic
4.6 mm inside diameter, stainless steel, packed with 5 μm silica solvents for liquid-liquid extraction or elution from solid
beads coated with trimethylsilyl. Mobile phase is established sorbents, solvents specified as distilled-in-glass, nano-grade, or
at 1.0 mL/min as a linear gradient from 15:85 methanol: water pesticide-grade frequently have lower levels of interfering
to 100 % methanol in 32 min. impurities. In all cases, sufficient reagent blanks must be
7.5.4 Column 3 (Alternative Column, used for EPA 531.2 processed with the samples to ensure that all of the compounds
validation), 150 mm long by 3.9 mm inside diameter, stainless of interest are not present as blanks due to reagents or
steel, packed with 5 mm C . Mobile phase is a ternary glassware.
methanol, acetonitrile, water gradient over 24 min. See Annex
8.2 Purity of Water—Unless otherwise indicated, references
A1.
to water shall be understood to mean reagent water conforming
7.5.5 Post Column Reactor, capable of mixing reagents into
to Type I of Specification D1193. It must be shown that this
the mobile phase. The reactor should be constructed using
water does not contain contaminants at concentrations suffi-
PTFE tubing and should be equipped with pumps to deliver
cient to interfere with the analysis. The reagent water used to
0.1 mL ⁄min to 1.0 mL ⁄min of each reagent; mixing tees; and
generate the validation data in this test method was distilled
18,17
two 1.0 mL delay coils, with one thermostated at 90 °C. 21
water.
7.5.6 Fluorescence Detector, capable of excitation at
8.3 Buffer Solutions:
230 nm and detection of emission energies greater than
19 8.3.1 Monochloroacetic Acid (pH 3) (ClCH CO H) Buffer
3 2
418 nm, or variable wavelength fluorescence detector ca-
Solution—Prepare by mixing 156 mL of monochloroacetic acid
pable of 340 nm excitation, 465 nm emission with a 18 nm
(ClCH CO H) solution (236.2 g/L) and 100 mL of potassium
3 2
band width, and 16 mL flow cell.
acetate (KCH CO ) solution (245.4 g/L).
3 2
8.3.2 Buffered Water, to prepare 1 L, mix 10 mL of mono-
8. Reagents and Materials
chloroacetic acid buffer (pH 3) and 990 mL of water.
8.1 Purity of Reagents—Reagent-grade chemicals shall be
8.4 Helium, for degassing solutions and solvents.
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Commit-
8.5 HPLC Mobile Phase:
tee on Analytical Reagents of the American Chemical Society 8.5.1 Water, HPLC grade, or equivalent Type I Reagent
Water.
Newer manufactured columns have not been able to resolve aldicarb sulfone
from oxamyl. ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Supelco LC-1, available from Supelco, Inc., Supelco Park, Bellefonte, PA Standard-Grade Reference Materials, American Chemical Society, Washington,
16823, has been found suitable for use. DC. For suggestions on the testing of reagents not listed by the American Chemical
Waters Carbamate Analysis Column, available from Waters Corp., Milford, Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
MA, 01757, has been found suitable for use. U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
ABI URS 051 and URA 100, available from ABI Analytical, Inc., 170 copeial Convention, Inc. (USPC), Rockville, MD.
Williams Drive, Ramsey, NJ 07446, or equivalent, has been found suitable for use. Available from the Magnetic Springs Water Co., 1801 Lone Eagle St.,
A Schoffel Model 970 fluorescence detector was used to generate the Columbus, OH 43228.
validation data presented in this test method. Now available from Kratos Division Available from Burdick and Jackson. Distributed by Scientific Products, 1430
of ABI Analytical, Inc., 170 Williams Drive, Ramsey, NJ 07446. Waukegan Road, McGraw Park, IL 60085-6787.
D5315 − 04 (2024)
TABLE 3 Instrument Quality Control Standard
8.9.2 2-Mercaptoethanol (1 + 1)—Mix 10.0 mL of
Concen- 2-mercaptoethanol and 10.0 mL of acetonitrile. Cap and store
Test Analyte tration, Requirements
in hood. (Warning—Work in a hood due to reagent volatility
μg/L
and odor.)
Sensitivity 3-Hydroxycarbofuran 2 Detection of analyte;
S/N > 3 8.9.3 Sodium Borate Solution (19.1 g/L)—Dissolve 19.1 g
of sodium borate (Na B O × 10H O) in water. Dilute to 1.0 L
A 2 4 7 2
Chromatographic aldicarb sulfoxide 100 0.90 < PGF < 1.1
with water. The sodium borate will dissolve completely at
performance
A room temperature if prepared one day before use.
PGF = peak Gaussian factor
8.9.4 OPA Reaction Solution—Dissolve 100 mg 6 10 mg of
1.83 × W 1/2 o-phthalaldehyde (melting point range from 55 °C to 58 °C) in
~ !
PGF 5
10 mL of methanol. Add to 1.0 L of sodium borate solution
W~1/10!
(19.1 g/L). Mix, filter, and degas with helium. Add 100 μL of
2-mercaptoethanol (1 + 1) and mix. Make up fresh solutions
daily.
where:
W( ⁄2) = peak width at half height, and
8.10 Sodium thiosulfate (Na S O ).
2 2 3
W( ⁄10) = peak width at tenth height.
8.11 Stock Solutions, Standard (1.00 μg/μL)—Stock stan-
dard solutions may either be purchased as certified solutions or
prepared from pure standard materials by using the following
procedure:
8.5.2 Methanol, HPLC grade. Filter and degas before use. 8.11.1 Prepare stock standard solutions by weighing ap-
8.5.3 Acetonitrile, HPLC grade. Filter and degass before
proximately 0.0100 g of pure material. Dissolve the material in
use. methanol and dilute to volume in a 10 mL volumetric flask.
Larger volumes may be used at the convenience of the analyst.
8.6 Internal Standard Solution—Prepare an internal stan-
If the compound purity is certified at 96 % or greater, the
dard solution by weighing approximately 0.0010 g of pure
weight may be used without correction to calculate the
BDMC (4-Bromo-3,5-Dimethylphenyl N-Methylcarbamate,
concentration of the stock standard. Commercially prepared
98 % purity) to two significant figures. Dissolve the BDMC
stock standards may be used at any concentration if they are
in methanol and dilute to volume in a 10 mL volumetric flask.
certified by either the manufacturer or an independent source.
Transfer the internal standard solution to a TFE-fluorocarbon-
8.11.2 Transfer the stock standard solution into TFE-
sealed screw-cap bottle and store it at room temperature. The
fluorocarbon-sealed screw-cap vials. Store it at room tempera-
addition of 5 μL of the internal standard solution to 50 mL of
ture and protect it from light.
sample results in a final internal standard concentration of
8.11.3 Stock standard solutions should be replaced after two
10 μg ⁄L. Replace the solution when ongoing quality control
months, or sooner, if comparison with laboratory-fortified
indicates a problem.
blanks, or quality-control samples indicate a problem.
NOTE 1—BDMC has been shown to be an effective internal standard for
the method analytes (1), but other compounds may be used if the quality 9. Sample Collection and Handling
control requirements in Section 11 are met.
9.1 Collect the samples in accordance with Guide D1192,
8.7 Laboratory Performance Check Solution—Prepare the
Practices D3370, or Practices D3694.
concentrate by adding 20 μL of the 3-hydroxycarbofuran stock
9.2 Additionally, grab samples must be collected in glass
standard solution (8.11), 1.0 mL of the aldicarb sulfoxide stock
containers. Follow conventional sampling practices (6);
standard solution (8.11), and 1 mL of the internal standard
however, the bottle must not be prerinsed with sample before
fortification solution (8.7) to a 10 mL volumetric flask (Table
collection.
3). Dilute to volume with methanol. Mix concentrate thor-
oughly. Prepare a check solution by placing 100 μL of the
10. Preservation of Samples
concentrate solution into a 100 mL volumetric flask. Dilute to
10.1 Sample Preservation/pH Adjustment—Oxamyl,
volume with buffered water. Transfer to a TFE-fluorocarbon-
3-hydroxycarbofuran, aldicarb sulfoxide, and carbaryl can all
sealed screw-cap bottle and store it at room temperature. The
degrade rapidly in neutral and basic waters held at room
solution should be replaced when ongoing quality control
temperature (7, 8). This short-term degradation is of concern
indicates a problem.
during the periods of time that samples are being shipped and
8.8 Methanol, distilled-in-glass quality or equivalent.
that processed samples are held at room temperature in
autosampler trays. Samples targeted for the analysis of these
8.9 Post Column Derivatization Solutions:
three analytes must be preserved at a pH of 3, as shown as
8.9.1 Sodium Hydroxide (2 g/L)—Dissolve 2.0 g of sodium
follows. The pH adjustment also minimizes analyte biodegra-
hydroxide (NaOH) in water. Dilute to 1.0 L with water. Filter
dation.
and degas just before use.
10.1.1 Add 1.8 mL of monochloroacetic acid buffer solution
(pH 3) to the 60 mL sample bottle. Add buffer to the sample
bottle either at the sampling site or in the laboratory before
Available from Aldrich Chemical Co., Inc., 1001 West Saint Paul Ave.,
Milwaukee, WI 53233. shipping to the sampling site.
D5315 − 04 (2024)
10.1.2 If residual chlorine is present, add 80 mg of sodium a fresh calibration standard. If the repetition also fails, generate
thiosulfate per litre of sample to the sample bottle prior to a new calibration curve for that analyte using freshly prepared
collecting the sample.
standards.
10.1.3 After the sample is collected in a bottle containing
11.2.6 Single-point calibration is a viable alternative to a
buffer, seal the sample bottle and shake it vigorously for 1 min.
calibration curve. Prepare single-point standards from the
10.1.4 Samples must be iced or refrigerated at 4 °C from the
secondary dilution standards. Prepare the single-point stan-
time of collection until storage; they must be stored at −10 °C
dards at a concentration deviating from the sample extract
until analyzed. Preservation study results indicate that test
response by no more than 20 %.
method analytes are stable in water samples for at least 28 days
11.2.7 Verify calibration standards periodically (recom-
when adjusted to pH 3 and stored at −10 °C. However, analyte
mended at least quarterly) by analyzing a standard prepared
stability may be affected by the matrix; the analyst should
from reference material obtained from an independent source.
therefore verify that the preservation technique is applicable to
The results from these analyses must be within the limits used
the samples under study.
to check calibration routinely.
11. Calibration
11.3 External Standard Calibration Procedure:
11.1 Establish HPLC operating parameters equivalent to 11.3.1 Prepare calibration standards at a minimum of three
those indicated in 7.5. Calibrate the HPLC system using either (recommended five) concentration levels for each analyte of
the internal (11.2) or the external (11.3) standard technique.
interest by adding volumes of one or more stock standards to
a volumetric flask. Dilute to volume with buffered water. The
11.2 Internal Standard Calibration Procedure—The analyst
lowest standard should represent analyte concentrations near,
must select one or more internal standards similar in analytical
but above, the respective EDLs. The remaining standards
behavior to the analytes of interest. In addition, the analyst
should bracket the analyte concentrations expected in the
must demonstrate that the measurement of the internal standard
sample extracts, or they should define the working range of the
is not affected by method or matrix interferences. BDMC has
detector.
been identified as a suitable internal standard.
11.2.1 Prepare calibration standards at a minimum of three 11.3.2 Beginning with the standard of lowest concentration,
(recommended, five) concentration levels for each analyte of analyze each calibration standard in accordance with 13.2, and
interest by adding volumes of one or more of the stock
tabulate the response (peak height or area) versus the concen-
standards to a volumetric flask. Add a known constant amount tration in the standard. Use the results to prepare a calibration
of one or more internal standards to each calibration standard,
curve for each compound. Alternatively, if the ratio of response
and dilute to volume with buffered water. The lowest standard
to concentration (calibration factor) is a constant over the
should represent analyte concentration near, but above, their
working range <20 % relative standard deviation, assume
respective estimated detection limit (EDL) (Table 1). The
linearity through the origin and use the average ratio or
remaining standards should bracket the analyte concentrations
calibration factor in place of a calibration curve.
expected in the sample extracts, or they should define the
11.3.3 Verify the working calibration curve or calibration
working range of the detector.
factor on each working day by measuring a minimum of two
11.2.2 Analyze each calibration standard in accordance with
calibration check standards, one at the beginning and one at the
the procedure in 13.2. Tabulate the peak height or area
end of the analysis day. These check standards should be at two
responses against the concentration for each compound and
different concentration levels in order to verify the concentra-
internal standard.
tion curve. For extended analysis periods (longer than 8 h), it
11.2.3 Calculate response factors (RF) for each analyte,
is strongly recommended that check standards be interspersed
surrogate, and internal standard using Eq 1 as follows:
with the samples at regular intervals during the course of the
A C
~ ! ~ !
s is analyses. If the response for any analyte varies from the
RF 5 (1)
~A ! ~C !
is s
predicted response by more than 620 %, repeat the test using
a fresh calibration standard. If the results still do not agree,
where:
generate a new calibration curve or use a single-point calibra-
A = response for the analyte to be measured,
s
tion standard in accordance with 11.3.4.
A = response for the internal standard,
is
C = concentration of the internal standard, μg/L, and 11.3.4 Single-point calibration is a viable alternative to a
is
C = concentration of the analyte to be measured, μg/L.
calibration curve. Prepare single-point standards from the
s
secondary dilution standards. Prepare the single-point stan-
11.2.4 If the RF value over the working range is constant
dards at a concentration deviating from the sample extract
(20 % RSD or less) use the average response factor for
response by no more than 20 %.
calculations. Alternatively, use the results to plot a calibration
curve of response ratios (A /A ) versus C . 11.3.5 Verify the calibration standards periodically,
s is s
(recommended, at least quarterly), by analyzing a standard
11.2.5 Verify the working calibration curve or RF on each
prepared from reference material obtained from an independent
working shift by the measurement of one or more calibration
source. The results from these analyses must be within the
standards. If the response for any analyte varies from the
predicted response by more than 620 %, repeat the test using limits used to check calibration routinely.
D5315 − 04 (2024)
TABLE 4 Acceptance Limits for the Analysis of a Laboratory
12.4 The analyst is permitted to modify HPLC columns,
Quality Control Sample as Percent of Mean Recovery
HPLC conditions, internal standards, or detectors to improve
Overall
separations or lower analytical costs. The analyst must repeat
Concentration Mean Acceptance
Analyte Standard
A B C
Level Recovery Limits, %
the procedures described in 12.3 each time such test method
B
Deviation
modifications are made.
Aldicarb 10.0 9.46 0.58 81.6–118
Aldicarb sulfone 20.0 19.3 1.33 79.3–121
12.5 Assessing the Internal Standards:
Aldicarb sulfoxide 20.0 19.6 1.35 79.3–121
Baygon (Propoxur) 10.0 9.52 0.78 75.4–124
12.5.1 When using the internal standard calibration
Carbaryl 20.0 19.5 1.35 79.2–121
procedure, the analyst is expected to monitor the internal
Carbofuran 20.0 19.1 0.68 89.3–111
standard response (the peak area or peak height) of all samples
3-Hydroxycarbofuran 20.0 19.2 1.31 79.5–120
Methiocarb 50
...