ASTM D7644-10

Standard Test Method for Determination of Bromadiolone, Brodifacoum, Diphacinone and Warfarin in Water by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)

SIGNIFICANCE AND USE
This test method has been developed in support of the National Homeland Security Research Center, US EPA by Region 5 Chicago Regional Laboratory (CRL).
Bromadiolone, brodifacoum, diphacinone and warfarin are rodenticides for controlling mice, rats, and other rodents that pose 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.  
This method has been investigated for use with reagent, surface, and drinking water for the selected rodenticides.
SCOPE
1.1 This procedure 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 method. This 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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 31-May-2010
• 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

• Replaced By: ASTM D7644-10e1 - Standard Test Method for Determination of Bromadiolone, Brodifacoum, Diphacinone and Warfarin in Water by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
• Effective Date: 01-Jun-2010

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D7644 – 10
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.
TABLE 1 Detection Verification Level and Reporting Range
1. Scope
DVL Reporting Range
1.1 This procedure covers the determination of bromadi-
Analyte
(ng/L) (ng/L)
olone, brodifacoum, diphacinone and warfarin (referred to
Bromadiolone 20 125-2500
collectively as rodenticides in this test method) in water by
Brodifacoum 20 125-2500
directinjectionusingliquidchromatography(LC)anddetected
Diphacinone 20 125-2500
Warfarin 20 125-2500
with tandem mass spectrometry (MS/MS). These analytes are
qualitatively and quantitatively determined by this method.
This method adheres to multiple reaction monitoring (MRM)
2. Referenced Documents
mass spectrometry.
1.2 The Detection Verification Level (DVL) and Reporting
2.1 ASTM Standards:
Range for the rodenticides are listed in Table 1.
D1129 Terminology Relating to Water
1.2.1 The DVL is required to be at a concentration at least
D1193 Specification for Reagent Water
3 times below the Reporting Limit (RL) and have a signal/
D2777 Practice for Determination of Precision and Bias of
noise ratio greater than 3:1. Fig. 1 displays the signal/noise
Applicable Test Methods of Committee D19 on Water
ratios of the primary single reaction monitoring (SRM) transi-
D3694 Practices for Preparation of Sample Containers and
tions, and Fig. 2 displays the confirmatory SRM transitions at
for Preservation of Organic Constituents
the DVLs for the rodenticides.
D3856 Guide for Good Laboratory Practices in Laborato-
1.2.2 The reporting limit was calculated from the concen-
ries Engaged in Sampling and Analysis of Water
tration of the Level 1 calibration standard, as shown inTable 4,
D4841 Practice for Estimation of Holding Time for Water
accounting for the dilution of a 40 mL water sample up to a
Samples Containing Organic and Inorganic Constituents
final volume of 50 mL with methanol to ensure analyte
D5847 Practice for Writing Quality Control Specifications
solubility.
for Standard Test Methods for Water Analysis
1.3 Units—The values stated in SI units are to be regarded
E2554 Practice for Estimating and Monitoring the Uncer-
as standard. No other units of measurement are included in this
tainty of Test Results of a Test Method in a Single
standard.
Laboratory Using a Control Sample Program
1.4 This standard does not purport to address all of the
2.2 Other Documents:
safety concerns, if any, associated with its use. It is the
EPApublication SW-846 Test Methods for Evaluating Solid
responsibility of the user of this standard to establish appro-
Waste, Physical/Chemical Methods
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
This test method is under the jurisdiction of ASTM Committee D19 on Water Standards volume information, refer to the standard’s Document Summary page on
andisthedirectresponsibilityofSubcommitteeD19.06onMethodsforAnalysisfor the ASTM website.
Organic Substances in Water. Available from National Technical Information Service (NTIS), U.S. Depart-
Current edition approved June 1, 2010. Published July 2010. DOI: 10.1520/ ment of Commerce, 5285 Port Royal Road, Springfield, VA, 22161 or at http://
D7644-10. 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.
D7644 – 10
FIG. 1 Example Primary SRM Chromatograms Signal/Noise Ratios
FIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise Ratios
D7644 – 10
3. Terminology allows rodents to feed multiple times before death, leading to
carcassescontainingresiduesthatmaybemanytimesthelethal
3.1 Definitions:
dose.
3.1.1 detection verification level, DVL, n—a concentration
5.3 This method has been investigated for use with reagent,
thathasasignal/noise(S/N)ratiogreaterthan3:1andisatleast
surface, and drinking water for the selected rodenticides.
3 times below the Reporting Limit (RL).
3.1.2 reporting limit, RL, n—the concentration of the
6. Interferences
lowest-level calibration standard used for quantification ac-
6.1 Method interferences may be caused by contaminants in
counting for the sample dilution.
solvents, reagents, glassware and other apparatus producing
3.1.2.1 Discussion—In this test method, a 40 mL sample
discrete artifacts or elevated baselines. All of these materials
aliquot is diluted to a 50 mL final volume after thoroughly
are demonstrated to be free from interferences by analyzing
rinsing the collection vial with methanol for quantitative
laboratory reagent blanks under the same conditions as
transfer. In this case, the lowest calibration level of 100 ppt
samples.
would allow a reporting limit of 125 ppt to be achieved.
6.2 All glassware is washed in hot water with detergent and
3.1.3 rodenticides, n—in this test method, bromadiolone,
rinsed in hot water followed by distilled water. The glassware
brodifacoum, diphacinone, and warfarin collectively.
is then dried and heated in an oven at 250°C for 15 to 30
3.2 Abbreviations:
minutes. All glassware is subsequently cleaned with acetone
-3
3.2.1 mM—millimolar, 1 3 10 moles/L followed by methanol.
6.3 All reagents and solvents should be of pesticide residue
3.2.2 ND—non-detect
purity or higher to minimize interference problems.
3.2.3 ppt—parts per trillion, ng/L
6.4 Matrix interferences may be caused by contaminants in
the sample. The extent of matrix interferences can vary
4. Summary of Test Method
considerably from sample source depending on variations of
4.1 This is a performance based method, and modifications
the sample matrix.
are allowed to improve performance.
4.2 For rodenticide analysis, samples are shipped to the lab
7. Apparatus
between 0°C and 6°C and analyzed within 14 days of collec-
7.1 LC/MS/MS System:
tion. In the lab, the samples are spiked with surrogates,
7.1.1 Liquid Chromatography (LC) System—A complete
quantitatively transferred to a graduated cylinder using three 5
LC system is needed to analyze samples. Any system that is
methanol rinses, filtered using a syringe driven filter unit, and
capable of performing at the flows, pressures, controlled
analyzed directly by LC/MS/MS.
temperatures, sample volumes, and requirements of the stan-
4.3 Bromadiolone, brodifacoum, diphacinone, warfarin,
dard may be used.
warfarin-D (surrogate) and 2-bromo-4-(1,1,3,3-
5 7.1.2 AnalyticalColumn–Waters—ACQUITYUPLCtBEH
tetramethylbutyl)phenol (brominated octylphenol, Br-OP, sur-
C18, 2.1 3 100 mm, 1.7 mm particle size was used to develop
rogate) are identified by retention time and two SRM transi-
this test method.Any column that achieves adequate resolution
tions. The target analytes and surrogates are quantitated using
may be used. The retention times and order of elution may
the primary SRM transitions utilizing an external calibration.
change depending on the column used and need to be moni-
The final report issued for each sample lists the concentration
tored.
of bromadiolone, brodifacoum, diphacinone, warfarin, and
NOTE 1—Any column that can achieve baseline resolution of these
surrogate recoveries.
analytes may be used. Baseline resolution simplifies data analysis and can
reducethechanceofionsuppression,leadingtohigherlimitsofdetection.
5. Significance and Use
7.1.3 Tandem Mass Spectrometer (MS/MS) System—A
5.1 This test method has been developed in support of the 6
MS/MS system capable of MRM analysis. Any system that is
National Homeland Security Research Center, US EPA by
capable of performing at the requirements in this standard may
Region 5 Chicago Regional Laboratory (CRL).
be used.
5.2 Bromadiolone, brodifacoum, diphacinone and warfarin
7.2 Filtration Device:
are rodenticides for controlling mice, rats, and other rodents
7.2.1 Hypodermic Syringe—A Lock Tip Glass Syringe ca-
thatposeathreattopublichealth,criticalhabitats,nativeplants
pable of holding a Millext HV Syringe Driven Filter Unit
and animals, crops, food and water supplies. These rodenti-
PVDF 0.22 µm or similar may be used.
cides also present human and environmental safety concerns.
Warfarin and diphacinone are first-generation anticoagulants,
while bromadiolone and brodifacoum are second-generation.
Additional information about rodenticides can be found on the internet at
The anticoagulants interfere with blood clotting, and death can
http://www.epa.gov (2010).
result from excessive bleeding. The second-generation antico-
A Waters ACQUITY UltraPerformance Liquid Chromatography (UPLCt)
System was used to develop this test method.All parameters in this test method are
agulants are especially hazardous for several reasons. They are
based on this system and may vary depending on your instrument.
highly toxic and persist a long time in body tissues. The
A Waters Quattro Premiery XE tandem quadrupole mass spectrometer was
second-generation anticoagulants are designed to be toxic in a
usedtodevelopthistestmethod.Allparametersinthistestmethodarebasedonthis
single feeding, but time-to-death occurs in several days. This system and may vary depending on your instrument.
D7644 – 10
7.2.1.1 A 50 mL Lock Tip Glass Syringe size is recom- collected unpreserved, shipped between 0°C and 6°C, and
mended since a 50 mL sample size is used in this test method. stored in the laboratory between 0°C and 6°C. Chlorinated
7.2.2 Filter—MillextHVSyringeDrivenFilterUnitPVDF drinking water samples are dechlorinated with ascorbic acid;
0.22 µm (Millipore Corporation, Catalog # SLGV033NS) or 10 mg of ascorbic acid is added to each 40 mL vial prior to
similar may be used. watercollection.Thistestmethodrequiresa40mLsamplesize
per analysis. Conventional sampling practices should be fol-
8. Reagents and Materials
lowed. Refer to Guide D3856 and Practices D3694.
8.1 Purity of Reagents—High Performance Liquid Chroma- 10.1.1 Ammonium acetate was evaluated as an agent to
tography (HPLC) pesticide residue analysis and spectropho- bind free chlorine in drinking water and was found to be
tometry grade chemicals shall be used in all tests. Unless ineffectiveinthepreservationoftherodenticidesinchlorinated
indicated otherwise, it is intended that all reagents shall drinking water.Ascorbic acid was effective as a dechlorinating
conform to the Committee on Analytical Reagents of the agent in chlorine fortified Chicago tap water, which contained
American Chemical Society. Other reagent grades may be 3.2 ppm free chlorine and was dechlorinated with 10 mg
used provided they are first determined to be of sufficiently ascorbic acid per 40 mL water sample.
highpuritytopermittheirusewithoutaffectingtheaccuracyof 10.2 The samples are collected using 40 mL glass vials. A
40 mL volume is collected directly into the sample collection
the measurements.
8.2 Purity of Water—Unless otherwise indicated, references vial without using any other measuring devices. This is a
requirementduetotherodenticides’affinityforsurfaces,which
towatershallbeunderstoodtomeanreagentwaterconforming
toType 1 of Specification D1193. It must be demonstrated that will lead to biased low results if transferring between contain-
ers. Before collection, the vials must be evaluated to determine
this water does not contain contaminants at concentrations
sufficient to interfere with the analysis. a40mLsamplevolume.Forexample,thevialsusedinthistest
method were calibrated before use to determine that filling the
8.3 Gases—Ultrapure nitrogen and argon.
8.4 Methanol (CAS # 67-56-1). vialtoapproximately1.6cmbelowtherimwouldresultina40
8.5 Acetonitrile (CAS # 75-05-8). mL sample volume. The greatest amount of water held by the
8.6 Acetone (CAS # 67-64-1). 40mLvialsusedinthistestmethodwasapproximately42mL.
8.7 Ammonium Hydroxide (Concentrated, CAS # 1336-21- Vialsfilledto42mLinthefieldwouldnotallowthelaboratory
6). tospikethesamplesbeforequantitativelytransferringtothe50
8.8 Ascorbic Acid (CAS # 50-81-7). mLgraduatedcylinder.Itisimperativethatthesamplersdonot
8.9 Bromadiolone (CAS # 28772-56-7). overfill the vials.
8.10 Brodifacoum (CAS # 56073-10-0). 10.3 Preservation—Store samples between 0°C and 6°C
8.11 Diphacinone (CAS # 82-66-6). from the time of collection until analysis. Analyze the sample
8.12 Warfarin (CAS # 81-81-2). within 14 days of collection. Chlorinated drinking water
8.13 Warfarin-D (Phenyl-D ,CAS#(unlabeled)81-81-2). samples are dechlorinated with ascorbic acid; 10 mg of
5 5
8.13.1 Discussion—Warfarin-D is used as the electrospray ascorbic acid is added to each 40 mL vial prior to water
collection.
positive analyte surrogate in this standard.
8.14 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP).
11. Preparation of LC/MS/MS
8.14.1 Discussion—Br-OPis used as the electrospray nega-
11.1 LC Chromatograph Operating Conditions:
tive analyte surrogate in this standard.
11.1.1 Injection volumes of all calibration standards and
9. Hazards
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”
9.1 Normal laboratory safety applies to this method. Ana-
mode is the preferred technique when performing fast, quali-
lysts should wear safety glasses, gloves, and lab coats when
tative analyses. This mode should be used whenever accuracy
working in the lab.Analysts should review the Material Safety
and precision are the primary concerns. The first sample
Data Sheets (MSDS) for all reagents used in this method.
analyzed after the calibration curve is a blank to ensure there is
10. Sampling
no carry-over.The gradient conditions for the liquid chromato-
graph are shown in Table 2.
10.1 Sampling—Grab samples must be collected in 40 mL
pre-cleaned amber glass vials with Teflont lined caps demon-
NOTE 2—If your instrument does not have a 50 µL injection capability
strated to be free of interferences. Surface water samples are
a different volume may be used. This is a performance-based method and
modifications are allowed as long as minimum performance criteria are
met.
Reagent Chemicals, American Chemical Society Specifications, American
...