ASTM D7644-23

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: D7644 − 16 D7644 − 23
Standard Test Method for
Determination of Bromadiolone, Brodifacoum, Diphacinone
and Warfarin in Water by Liquid Chromatography/Tandem
Mass Spectrometry (LC/MS/MS)
This standard is issued under the fixed designation D7644; 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 bromadiolone, brodifacoum, diphacinone and warfarin (referred to
collectively as rodenticides 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 rodenticides 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 rodenticides.
1.2.2 The reporting limit was calculated from the concentration of the Level 1 calibration standard, as shown in Table 4,
accounting for the dilution of a 40 mL water sample up to a final volume of 50 mL with methanol to ensure analyte solubility.
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.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent 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 2016June 2023. Originally approved in 2010. Last previous edition approved in 20102016 as
ɛ2
D7644D7644 – 16. – 10 . DOI: 10.1520/D7644-16.10.1520/D7644-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. 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
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TABLE 1 Detection Verification Level and Reporting Range
DVL Reporting Range
Analyte
(ng/L) (ng/L)
Bromadiolone 20 125-2500
Brodifacoum 20 125-2500
Diphacinone 20 125-2500
Warfarin 20 125-2500
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:
U.S. EPA publication SW-846 Test Methods for Evaluating Solid Waste, Physical/Chemical Methods
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 detection verification level, DVL, n—a concentration that has a signal/noise (S/N) ratio greater than 3:1 and is at least 3 times
below the Reporting Limit (RL).
3.2.2 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.
3.2.3 reporting limit, RL, n—the concentration of the lowest-level calibration standard used for quantification accounting for the
sample dilution.
3.2.3.1 Discussion—
In this test method, a 40 mL sample aliquot is diluted to a 50 mL final volume after thoroughly rinsing the collection vial with
methanol for quantitative transfer. In this case, the lowest calibration level of 100 ppt would allow a reporting limit of 125 ppt to
be achieved.
3.2.4 rodenticides, n—in this test method, bromadiolone, brodifacoum, diphacinone, and warfarin collectively.
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
-3
3.3.6 mM, n—millimolar, 1 × 10 moles/L
3.3.7 MRM, n—Multiple Reaction Monitoring
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.
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FIG. 1 Example Primary SRM Chromatograms Signal/Noise Ratios
FIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise Ratios
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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.
4.2 For rodenticide analysis, samples are shipped to the lab 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, quantitatively transferred to a graduated cylinder using
three methanol rinses, filtered using a syringe driven filter unit, and analyzed directly by LC/MS/MS.
4.3 Bromadiolone, brodifacoum, diphacinone, warfarin, warfarin-D (surrogate) and 2-bromo-4-(1,1,3,3-tetramethylbutyl)phenol
(brominated octylphenol, Br-OP, surrogate) are identified by retention time and two SRM transitions. The target analytes and
surrogates are quantitated using the primary SRM transitions utilizing an external calibration. The final report issued for each
sample lists the concentration of bromadiolone, brodifacoum, diphacinone, warfarin, and 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 Bromadiolone, brodifacoum, diphacinone and warfarin are rodenticides for controlling mice, rats, and other rodents that pose
D7644 − 23
a threat to public health, critical habitats, native plants and animals, crops, food and water supplies. These rodenticides also present
human and environmental safety concerns. Warfarin and diphacinone are first-generation anticoagulants, while bromadiolone and
brodifacoum are second-generation. The anticoagulants interfere with blood clotting, and death can result from excessive bleeding.
The second-generation anticoagulants are especially hazardous for several reasons. They are highly toxic and persist a long time
in body tissues. The second-generation anticoagulants are designed to be toxic in a single feeding, but time-to-death occurs in
several days. This allows rodents to feed multiple times before death, leading to carcasses containing residues that may be many
times the lethal dose.
5.3 This test method has been investigated for use with reagent, surface, and drinking water for the selected rodenticides.
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 of 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.
NOTE 1—Any column that can achieve baseline resolution of these analytes may be used. Baseline resolution simplifies data analysis and can reduce the
chance of ion suppression, leading to higher limits of detection.
7.1.3 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.
7.2 Filtration Device:
7.2.1 Hypodermic syringe—A Lock Tip Glass Syringe capable of holding a syringe-driven filter unit or similar may be used.
7.2.1.1 A 50-mL lock tip glass syringe size is recommended since a 50-mL sample size is used in this test method.
Additional information about rodenticides is available from United States Environmental Protection Agency (EPA), http://www.epa.gov.
A Waters ACQUITY UPLC (a trademark of the Waters Corporation, Milford, MA) BEH C18, 2.1 × 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 Waters Quattro Premier (a trademark of the Waters Corporation, Milford, MA) XE tandem quadrupole mass spectrometer, or equivalent, was found suitable for use.
All parameters in this test method are based on this system and may vary depending on your instrument.
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7.2.2 Filter Unit —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 Methanol (CAS # 67-56-1).
8.5 Acetonitrile (CAS # 75-05-8).
8.6 Acetone (CAS # 67-64-1).
8.7 Ammonium Hydroxide (Concentrated, CAS # 1336-21-6).
8.8 Ascorbic Acid (CAS # 50-81-7).
8.9 Bromadiolone (CAS # 28772-56-7).
8.10 Brodifacoum (CAS # 56073-10-0).
8.11 Diphacinone (CAS # 82-66-6).
8.12 Warfarin (CAS # 81-81-2).
8.13 Warfarin-D (Phenyl-D , CAS # (unlabeled) 81-81-2).
5 5
8.13.1 Discussion—Warfarin-D is used as the electrospray positive analyte surrogate in this standard.
8.14 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP).
8.14.1 Discussion—Br-OP is used as the electrospray negative analyte surrogate in this standard.
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 unit 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.
A source of Warfarin-D is Cambridge Isotope Laboratories, 50 Frontage Road, Andover, MA 01810-5413. If you are aware of an alternative source 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, which you may attend.
A source of Br-OP is Accustandard, Inc., 125 Market Street, New Haven CT 06513. If you are aware of an alternative source 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,
which you may attend.
Teflon is a trademark of The Chemours Company in Wilmington, DE.
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9. Hazards
9.1 Normal laboratory safety applies to this 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—Grab samples must be collected in 40 mL pre-cleaned amber glass vials with Tefloninert-lined-lined caps
demonstrated to be free of interferences. Surface water samples are collected unpreserved, shipped between 0°C and 6°C,above
freezing and 6 °C, and stored in the laboratory between 0°C and 6°C.above freezing and 6 °C. Chlorinated drinking water samples
are dechlorinated with ascorbic acid; 10 mg of ascorbic acid is added to each 40 mL 40 mL vial prior to water collection. This test
method requires a 40 mL 40 mL sample size per analysis. Conventional sampling practices should be followed. Refer to Guide
D3856 and Practices D3694.
10.1.1 Ammonium acetate was evaluated as an agent to bind free chlorine in drinking water and was found to be ineffective in
the preservation of the rodenticides in chlorinated drinking water. Ascorbic acid was effective as a dechlorinating agent in chlorine
fortified Chicago tap water, which contained 3.2 ppm free chlorine and was dechlorinated with 10 mg ascorbic acid per 40 mL
water sample.
10.2 The samples are collected using 40 mL glass vials. A 40 mL volume is collected directly into the sample collection vial
without using any other measuring devices. This is a requirement due to the rodenticides’ affinity for surfaces, which will lead to
biased low results if transferring between containers. Before collection, the vials must be evaluated to determine a 40 mL 40 mL
sample volume. For example, the vials used in this test method were calibrated before use to determine that filling the vial to
approximately 1.6 cm below the rim would result in a 40 mL 40 mL sample volume. The greatest amount of water held by the
40 mL 40 mL vials used in this test method was approximately 42 mL. Vials filled to 42 mL in the field would not allow the
laboratory to spike the samples before quantitatively transferring to the 50 mL 50 mL graduated cylinder. It is imperative that the
samplers do not overfill the vials.
10.3 Preservation—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. Chlorinated drinking water samples are dechlorinated with ascorbic acid; 10 mg
of ascorbic acid is added to each 40 mL vial prior to water collection.
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 2.
TABLE 2 Gradient Conditions for Liquid Chromatography
Percent Percent
Time Flow 95%95 % Water/ 95%95 % Methanol/
(min) (μL/min) 5%5 % Methanol, 5%5 % Water,
5 mM NH OH 5 mM NH OH
4 4
0.0 300 100 0
2.0 300 100 0
6.0 300 20 80
6.1 200 5 95
7.0 200 5 95
8.5 200 0 100
13.0 300 0 100
14.0 300 100 0
16.0 300 100 0
A Hach Pocket Colorimeter II (a trademark of Hach Company in Loveland, CO) Pocket Chlorine meter was used to measure free chlorine.
D7644 − 23
NOTE 2—If yourthe instrument does not have a 50 μL injection capability a different volume may be used. This is a performance-based method and
modifications are allowed as long as minimum performance criteria are met.
11.2 LC Sample Manager Conditions:
11.2.1 Wash Solvents—Weak wash is 4.0 mL of 95 % water/5 % methanol. Strong wash is 2.0 mL of methanol. The strong wash
solvent is needed to eliminate carry-over between injections of rodenticide samples. The weak wash is used to remove the strong
wash solvent. These rodenticides were shown to carry-over when acetonitrile was used for this analysis. The use of methanol
corrected this problem while providing separation and sensitivity. 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%50 % Acetonitrile/50 % Water; Time: 5 minutes.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 standard contains two surrogates and four target compounds that can be acquired in 5 MRM acquisition
functions. Variable parameters regarding retention times, SRM transitions, and cone and collision energies are shown in Table 3.
Mass spectrometer parameters used in the development of this test method are listed below:
The instrument is set in the Electrospray source setting.
Capillary Voltage: 3.5 kV
Cone: Variable depending on analyte (Table 3)
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: 14.0
High Mass Resolution 1: 14.0
Ion Energy 1: 0.5
Entrance Energy: -1
Collision Energy: Variable depending on analyte (Table 3)
Exit Energy: 0
Low Mass Resolution 2: 14.0
High Mass resolution 2: 14.0
Ion Energy 2: 0.7
Multiplier: 650
-3
Gas Cell Pirani Gauge: 7.0 × 10 Torr
Inter-Channel Delay: 0.02 seconds
Inter-Channel Delay: 0.02 s
Inter-Scan Delay: 0.02 seconds
TABLE 3 Retention Times, SRM Ions, and Analyte-Specific Mass Spectrometer Parameters
SRM Primary/
Primary/ Retention Time Cone Voltage Collision Energy
Analyte ESI Mass Transition Confirmatory
Confirmatory (min) (Volts) (eV)
(Parent > Product) SRM Area Ratio
Primary 7.45 and 30 22 509.1 > 251.1
Bromadiolone Positive 1.0
Confirmatory 7.59 30 22 511.1 > 251.1
Primary 42 20 523.1 > 335.1
Brodifacoum Positive 7.95 1.1
Confirmatory 42 33 523.1 > 178
Primary 32 25 339.1 > 166.8
Diphacinone Negative 6.36 3.0
Confirmatory 32 47 339.1 > 115.8
Primary 26 14 309.1 > 162.8
Warfarin Positive 5.38 1.7
Confirmatory 26 20 309.1 > 251.1
Warfarin-D Primary 26 14 314.2 > 162.8
Positive 5.37 1.7
(Surrogate) Confirmatory 26 19 314.2 > 256.1
Br-OP
Negative Primary 8.92 35 25 283.1 > 78.6 N/A
(Surrogate)
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Inter-Scan Delay: 0.02 s
Dwell: 0.1 seconds
Dwell: 0.1 s
12. Calibration and Standardization
12.1 The mass spectrometer must be calibrated per manufacturer specifications before analysis. In order to obtain valid and
accurate analytical values through 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 7 calibration standards containing the 7 concentration
levels of the rodenticides and surrogates prior to analysis as shown in Table 4. A calibration stock standard solution is prepared
from standard materials or they are purchased as certified solutions. Stock standard solution A containing bromadiolone,
brodifacoum, diphacinone, warfarin, warfarin-D (surrogate), and 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (surrogate) is
prepared at Level 7 concentration, and aliquots of that solution are diluted to prepare Levels 1 through 6. The following steps will
produce standards with the concentration values shown in Table 4. 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 7) by adding to a 100 mL volumetric flask individual methanol solutions of the
following: 50 μL of bromadiolone, brodifacoum, diphacinone, warfarin, warfarin-D (surrogate) each at 4.0 mg/L and 50 μL of
2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (surrogate) at 0.2 g/L, dilute to 100 mL with 80 % water/20 % methanol. The
preparation of the Level 7 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, solubility at that concentration will have
to be ensured.
12.2.2 Aliquots of solution A are then diluted with 80 % water/20 % methanol to prepare the desired calibration levels in 2 mL
amber glass LC vials. The calibration vials must be used within 24 hours 24 h 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 monitoring the primary and
confirmatory SRM transition 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
3 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 additional 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 properly use the software. 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 of the curve to be <0.98, this point must be re-injected or a new
TABLE 4 Concentrations of Calibration Standards (ppt)
Analyte/Surrogate LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7
Bromadiolone 100 200 500 750 1000 1500 2000
Brodifacoum 100 200 500 750 1000 1500 2000
Diphacinone 100 200 500 750 1000 1500 2000
Warfarin 100 200 500 750 1000 1500 2000
Warfarin-D (Surrogate) 100 200 500 750 1000 1500 2000
Br-OP (Surrogate) 5000 10 000 25 000 37 500 50 000 75 000 100 000
Management and Technical Requirements for Laboratories Performing Environmental Analysis; Module 4: Quality Systems for Chemical Testing; The NELAC Institute,
2017.
D7644 − 23
calibration curve must be regenerated. If the low or high point is excluded, minimally a five point curve is acceptable but the
reporting range must be modified to reflect this change.point used to generate the curve 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.
12.2.6.1 An initial seven point curve over the calibration range is encouraged in the event the low or high point 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 % 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 bromadiolone, brodifacoum, diphacinone, warfarin,
warfarin-D (surrogate) and Br-OP (surrogate) at a concentration in the calibration range of Levels 3 to 5. A 750 ppt spike for
bromadiolone, brodifacoum, diphacinone and warfarin, 1000 ppt spike for warfarin-D (surrogate), and 50 000 ppt spike for Br-OP
(surrogate) were 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
concentration other than the recommended concentration is used, refer to Practice D5847 for information on applying the F test
and t test F-test and t-test in evaluating the acceptability of the mean and standard deviation.
TABLE 5 QC Acceptance Criteria
Initial Demonstration of Performance Lab Control Sample
Test Conc. Recovery (%) Precision Recovery (%)
Analyte
(ng/L)
Lower Upper Maximum Lower Up
...