1、Designation: D7644 102Standard Test Method forDetermination of Bromadiolone, Brodifacoum, Diphacinoneand Warfarin in Water by Liquid Chromatography/TandemMass Spectrometry (LC/MS/MS)1This standard is issued under the fixed designation D7644; the number immediately following the designation indicates
2、 the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTETest Method was corrected editorially in 20121. Scope1
3、.1 This procedure covers the determination of bromadi-olone, brodifacoum, diphacinone and warfarin (referred tocollectively as rodenticides in this test method) in water bydirect injection using liquid chromatography (LC) and detectedwith tandem mass spectrometry (MS/MS). These analytes arequalitati
4、vely and quantitatively determined by this method.This method adheres to multiple reaction monitoring (MRM)mass spectrometry.1.2 The Detection Verification Level (DVL) and ReportingRange for the rodenticides are listed in Table 1.1.2.1 The DVL is required to be at a concentration at least3 times bel
5、ow the Reporting Limit (RL) and have a signal/noise ratio greater than 3:1. Fig. 1 displays the signal/noiseratios of the primary single reaction monitoring (SRM) transi-tions, and Fig. 2 displays the confirmatory SRM transitions atthe DVLs for the rodenticides.1.2.2 The reporting limit was calculat
6、ed from the concen-tration of the Level 1 calibration standard, as shown in Table 4,accounting for the dilution of a 40 mL water sample up to afinal volume of 50 mL with methanol to ensure analytesolubility.1.3 UnitsThe values stated in SI units are to be regardedas standard. No other units of measu
7、rement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limi
8、tations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committee D19 on WaterD3694 Practices for Preparation of Sample Containers andfo
9、r Preservation of Organic ConstituentsD3856 Guide for Management Systems in LaboratoriesEngaged in Analysis of WaterD4841 Practice for Estimation of Holding Time for WaterSamples Containing Organic and Inorganic ConstituentsD5847 Practice for Writing Quality Control Specificationsfor Standard Test M
10、ethods for Water AnalysisE2554 Practice for Estimating and Monitoring the Uncer-tainty of Test Results of a Test Method in a SingleLaboratory Using a Control Sample Program2.2 Other Documents:EPApublication SW-846 Test Methods for Evaluating SolidWaste, Physical/Chemical Methods31This test method is
11、 under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.06 on Methods forAnalysis forOrganic Substances in Water.Current edition approved June 1, 2010. Published February 2011 Last previousedition published in 2011 as D764410E01. DOI: 10.1520/D7644-
12、10E02.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from National Technical Information Service
13、(NTIS), U.S. Depart-ment of Commerce, 5285 Port Royal Road, Springfield, VA, 22161 or at http:/www.epa.gov/epawaste/hazard/testmethods/index.htm.TABLE 1 Detection Verification Level and Reporting RangeAnalyteDVL(ng/L)Reporting Range(ng/L)Bromadiolone 20 125-2500Brodifacoum 20 125-2500Diphacinone 20
14、125-2500Warfarin 20 125-25001Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.FIG. 1 Example Primary SRM Chromatograms Signal/Noise RatiosFIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise RatiosD7644 10223. Terminology3.1 Defi
15、nitions:3.1.1 detection verification level, DVL, na concentrationthat has a signal/noise (S/N) ratio greater than 3:1 and is at least3 times below the Reporting Limit (RL).3.1.2 reporting limit, RL, nthe concentration of thelowest-level calibration standard used for quantification ac-counting for th
16、e sample dilution.3.1.2.1 DiscussionIn this test method, a 40 mL samplealiquot is diluted to a 50 mL final volume after thoroughlyrinsing the collection vial with methanol for quantitativetransfer. In this case, the lowest calibration level of 100 pptwould allow a reporting limit of 125 ppt to be ac
17、hieved.3.1.3 rodenticides, nin this test method, bromadiolone,brodifacoum, diphacinone, and warfarin collectively.3.2 Abbreviations:3.2.1 mMmillimolar, 1 3 10-3moles/L3.2.2 NDnon-detect3.2.3 pptparts per trillion, ng/L4. Summary of Test Method4.1 This is a performance based method, and modifications
18、are allowed to improve performance.4.2 For rodenticide analysis, samples are shipped to the labbetween 0C and 6C and analyzed within 14 days of collec-tion. In the lab, the samples are spiked with surrogates,quantitatively transferred to a graduated cylinder using threemethanol rinses, filtered usin
19、g a syringe driven filter unit, andanalyzed directly by LC/MS/MS.4.3 Bromadiolone, brodifacoum, diphacinone, warfarin,warfarin-D5(surrogate) and 2-bromo-4-(1,1,3,3-tetramethylbutyl)phenol (brominated octylphenol, Br-OP, sur-rogate) are identified by retention time and two SRM transi-tions. The targe
20、t analytes and surrogates are quantitated usingthe primary SRM transitions utilizing an external calibration.The final report issued for each sample lists the concentrationof bromadiolone, brodifacoum, diphacinone, warfarin, andsurrogate recoveries.5. Significance and Use5.1 This test method has bee
21、n developed by US EPARegion5 Chicago Regional Laboratory (CRL).5.2 Bromadiolone, brodifacoum, diphacinone and warfarinare rodenticides for controlling mice, rats, and other rodentsthat pose a threat to public health, critical habitats, native plantsand animals, crops, food and water supplies. These
22、rodenti-cides 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 canresult from excessive bleeding. The second-generatio
23、n antico-agulants are especially hazardous for several reasons. They arehighly toxic and persist a long time in body tissues. Thesecond-generation anticoagulants are designed to be toxic in asingle feeding, but time-to-death occurs in several days. Thisallows rodents to feed multiple times before de
24、ath, leading tocarcasses containing residues that may be many times the lethaldose.45.3 This method has been investigated for use with reagent,surface, and drinking water for the selected rodenticides.6. Interferences6.1 Method interferences may be caused by contaminants insolvents, reagents, glassw
25、are and other apparatus producingdiscrete artifacts or elevated baselines. All of these materialsare demonstrated to be free from interferences by analyzinglaboratory reagent blanks under the same conditions assamples.6.2 All glassware is washed in hot water with detergent andrinsed in hot water fol
26、lowed by distilled water. The glasswareis then dried and heated in an oven at 250C for 15 to 30minutes. All glassware is subsequently cleaned with acetonefollowed by methanol.6.3 All reagents and solvents should be of pesticide residuepurity or higher to minimize interference problems.6.4 Matrix int
27、erferences may be caused by contaminants inthe sample. The extent of matrix interferences can varyconsiderably from sample source depending on variations ofthe sample matrix.7. Apparatus7.1 LC/MS/MS System:7.1.1 Liquid Chromatography (LC) SystemA completeLC system is needed to analyze samples.5Any s
28、ystem that iscapable of performing at the flows, pressures, controlledtemperatures, sample volumes, and requirements of the stan-dard may be used.7.1.2 Analytical ColumnWatersACQUITYUPLCt BEHC18, 2.1 3 100 mm, 1.7 m particle size was used to developthis test method.Any column that achieves adequate
29、resolutionmay be used. The retention times and order of elution maychange depending on the column used and need to be moni-tored.NOTE 1Any column that can achieve baseline resolution of theseanalytes may be used. Baseline resolution simplifies data analysis and canreduce the chance of ion suppressio
30、n, leading to higher limits of detection.7.1.3 Tandem Mass Spectrometer (MS/MS) SystemAMS/MS system capable of MRM analysis.6Any system that iscapable of performing at the requirements in this standard maybe used.7.2 Filtration Device:7.2.1 Hypodermic SyringeA Lock Tip Glass Syringe ca-pable of hold
31、ing a Millext HV Syringe Driven Filter UnitPVDF 0.22 m or similar may be used.4Additional information about rodenticides can be found on the internet athttp:/www.epa.gov (2010).5A Waters ACQUITY UltraPerformance Liquid Chromatography (UPLCt)System was used to develop this test method. All parameters
32、 in this test method arebased on this system and may vary depending on your instrument.6A Waters Quattro Premiery XE tandem quadrupole mass spectrometer wasused to develop this test method.All parameters in this test method are based on thissystem and may vary depending on your instrument.D7644 1023
33、7.2.1.1 A 50 mL Lock Tip Glass Syringe size is recom-mended since a 50 mL sample size is used in this test method.7.2.2 FilterMillext HV Syringe Driven Filter Unit PVDF0.22 m (Millipore Corporation, Catalog # SLGV033NS) orsimilar may be used.8. Reagents and Materials8.1 Purity of ReagentsHigh Perfor
34、mance Liquid Chroma-tography (HPLC) pesticide residue analysis and spectropho-tometry grade chemicals shall be used in all tests. Unlessindicated otherwise, it is intended that all reagents shallconform to the Committee on Analytical Reagents of theAmerican Chemical Society.7Other reagent grades may
35、 beused provided they are first determined to be of sufficientlyhigh purity to permit their use without affecting the accuracy ofthe measurements.8.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Type 1 of Specification D1193. It
36、 must be demonstrated thatthis water does not contain contaminants at concentrationssufficient to interfere with the analysis.8.3 GasesUltrapure 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 # 13
37、36-21-6).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-D5(Phenyl-D5, CAS # (unlabeled) 81-81-2).88.13.1 DiscussionWarfarin-D5is used as the electrospraypositive an
38、alyte surrogate in this standard.8.14 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP).98.14.1 DiscussionBr-OP is used as the electrospray nega-tive analyte surrogate in this standard.9. Hazards9.1 Normal laboratory safety applies to this method. Ana-lysts should wear safety glasses, gloves, and l
39、ab coats whenworking in the lab. Analysts should review the Material SafetyData Sheets (MSDS) for all reagents used in this method.10. Sampling10.1 SamplingGrab samples must be collected in 40 mLpre-cleaned amber glass vials with Teflont lined caps demon-strated to be free of interferences. Surface
40、water samples arecollected unpreserved, shipped between 0C and 6C, andstored in the laboratory between 0C and 6C. Chlorinateddrinking water samples are dechlorinated with ascorbic acid;10 mg of ascorbic acid is added to each 40 mL vial prior towater collection. This test method requires a 40 mLsampl
41、e sizeper analysis. Conventional sampling practices should be fol-lowed. Refer to Guide D3856 and Practices D3694.10.1.1 Ammonium acetate was evaluated as an agent tobind free chlorine in drinking water and was found to beineffective in the preservation of the rodenticides in chlorinateddrinking wat
42、er. Ascorbic acid was effective as a dechlorinatingagent in chlorine fortified Chicago tap water, which contained3.2 ppm free chlorine and was dechlorinated with 10 mgascorbic acid per 40 mL water sample.1010.2 The samples are collected using 40 mL glass vials. A40 mL volume is collected directly in
43、to the sample collectionvial without using any other measuring devices. This is arequirement due to the rodenticidesaffinity for surfaces, whichwill lead to biased low results if transferring between contain-ers. Before collection, the vials must be evaluated to determinea 40 mLsample volume. For ex
44、ample, the vials used in this testmethod were calibrated before use to determine that filling thevial to approximately 1.6 cm below the rim would result in a 40mL sample volume. The greatest amount of water held by the40 mL vials used in this test method was approximately 42 mL.Vials filled to 42 mL
45、 in the field would not allow the laboratoryto spike the samples before quantitatively transferring to the 50mL graduated cylinder. It is imperative that the samplers do notoverfill the vials.10.3 PreservationStore samples between 0C and 6Cfrom the time of collection until analysis. Analyze the samp
46、lewithin 14 days of collection. Chlorinated drinking watersamples are dechlorinated with ascorbic acid; 10 mg ofascorbic acid is added to each 40 mL vial prior to watercollection.11. Preparation of LC/MS/MS11.1 LC Chromatograph Operating Conditions:511.1.1 Injection volumes of all calibration standa
47、rds andsamples are made at 50 L volume using a full loop injection.If a 50 L volume loop is installed in the LC, a “full loop”mode is the preferred technique when performing fast, quali-tative analyses. This mode should be used whenever accuracyand precision are the primary concerns. The first sampl
48、eanalyzed after the calibration curve is a blank to ensure there isno carry-over. The gradient conditions for the liquid chromato-graph are shown in Table 2.NOTE 2If your instrument does not have a 50 L injection capabilitya different volume may be used. This is a performance-based method andmodific
49、ations are allowed as long as minimum performance criteria aremet.11.2 LC Sample Manager Conditions:11.2.1 Wash SolventsWeak wash is 4.0 mL of 95%water/5% methanol. Strong wash is 2.0 mL of methanol. Thestrong wash solvent is needed to eliminate carry-over betweeninjections of rodenticide samples. The weak wash is used to7Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, D.C. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for L
