1、Designation: D7644 102D7644 16Standard 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 i
2、ndicates 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.1 NOTEThis test method was corrected editorially in
3、 April 2011.2 NOTEThis test method was corrected editorially in February 2012.1. Scope1.1 This procedure covers the determination of bromadiolone, brodifacoum, diphacinone and warfarin (referred to collectivelyas rodenticides in this test method) in water by direct injection using liquid chromatogra
4、phy (LC) and detected with tandem massspectrometry (MS/MS). These analytes are qualitatively and quantitatively determined by this test method. This test methodadheres to multiple reaction monitoring (MRM) mass spectrometry.1.2 The Detection Verification Level (DVL) and Reporting Range for the roden
5、ticides are listed in Table 1.1.2.1 The DVLis required to be at a concentration at least 3 times below the Reporting Limit (RL) and have a signal/noise ratiogreater than 3:1. Fig. 1 displays the signal/noise ratios of the primary single reaction monitoring (SRM) transitions, and Fig. 2displays the c
6、onfirmatory 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 solu
7、bility.1.3 UnitsThe values stated in SI units are to be regarded as standard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to
8、establish appropriate safety and health practices and determine the applicability of regulatorylimitations 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 of Applica
9、ble Test Methods of Committee D19 on WaterD3694 Practices for Preparation of Sample Containers and for Preservation of Organic ConstituentsD3856 Guide for Management Systems in Laboratories Engaged in Analysis of Water1 This test method is under the jurisdiction of ASTM Committee D19 on Water and is
10、 the direct responsibility of Subcommittee D19.06 on Methods for Analysis forOrganic Substances in Water.Current edition approved June 1, 2010Feb. 1, 2016. Published February 2011May 2016. Originally approved in 2010. Last previous edition approved in 2010 as D7644 102. DOI: 10.1520/D7644-10E02.10.1
11、520/D7644-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.TABLE 1 Detection Verification Level and Reporti
12、ng RangeAnalyte DVL(ng/L) Reporting Range(ng/L)Bromadiolone 20 125-2500Brodifacoum 20 125-2500Diphacinone 20 125-2500Warfarin 20 125-2500This 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 ver
13、sion. Becauseit 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 versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM Internatio
14、nal, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1FIG. 1 Example Primary SRM Chromatograms Signal/Noise RatiosFIG. 2 Example Confirmatory SRM Chromatograms Signal/Noise RatiosD7644 162D4841 Practice for Estimation of Holding Time for Water Samples Containing Or
15、ganic and Inorganic ConstituentsD5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water AnalysisE2554 Practice for Estimating and Monitoring the Uncertainty of Test Results of a Test Method Using Control Chart Techniques2.2 Other Documents:3U.S. EPA publication
16、SW-846 Test Methods for Evaluating Solid Waste, Physical/Chemical Methods3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.3.2 Definitions:Definitions of Terms Specific to This Standard:3.2.1 detection verification level, DVL, na concentra
17、tion that has a signal/noise (S/N) ratio greater than 3:1 and is at least 3times below the Reporting Limit (RL).3.2.2 independent reference material, IRM, na material of known purity and concentration obtained either from the NationalInstitute of Standards andTechnology (NIST) or other reputable sup
18、plier.The IRM shall be obtained from a different lot of materialthan is used for calibration.3.2.3 reporting limit, RL, nthe concentration of the lowest-level calibration standard used for quantification accounting for thesample dilution.3.2.3.1 DiscussionIn this test method, a 40 mL sample aliquot
19、is diluted to a 50 mL final volume after thoroughly rinsing the collection vial withmethanol for quantitative transfer. In this case, the lowest calibration level of 100 ppt would allow a reporting limit of 125 ppt tobe achieved.3.2.4 rodenticides, nin this test method, bromadiolone, brodifacoum, di
20、phacinone, and warfarin collectively.3.3 Abbreviations:Acronyms:3.3.1 CCC, nContinuing Calibration Check3.3.2 IC, nInitial Calibration3.3.3 LC, nLiquid Chromatography3.3.4 LCS/LCSD, nLaboratory Control Sample/Laboratory Control Sample Duplicate3.3.5 MeOH, nMethanol3.3.6 mMmM, nmillimolar, 1 10-3 mol
21、es/L3.3.7 MRM, nMultiple Reaction Monitoring3.3.8 MS/MSD, nMatrix Spike/Matrix Spike Duplicate3.3.9 NA, adjNot Available3.3.10 NDND, nnon-detect3.3.11 P 10 mgof ascorbic acid is added to each 40 mL vial prior to water collection. This test method requires a 40 mL sample size per analysis.Conventiona
22、l sampling practices should be followed. Refer to Guide D3856 and Practices D3694.8 AWaters Quattro Premier XE tandem quadrupole mass spectrometer was used to develop this test method.All parameters in this test method are based on this systemand may vary depending on your instrument. Millex HV Syri
23、nge Driven Filter Unit PVDF 0.22 m (Millipore Corporation, Catalog #SLGV033NS; Millex is a trademarkof Merck KGAA, Darmstadt, Germany) has been found suitable for use for this test method, any filter unit may be used that meets the performance of this test method maybe used.9 Reagent Chemicals, Amer
24、ican Chemical Society Specifications, American Chemical Society, Washington, D.C. For Suggestions on the testing of reagents not listed bythe American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and NationalFormul
25、ators, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.10 A source of Warfarin-D5 is Cambridge Isotope Laboratories, 50 Frontage Road, Andover, MA 01810-5413.11 A source of Br-OP is Accustandard, Inc., 125 Market Street, New Haven CT 06513.12 Teflon is a trademark of The Chemours Company i
26、n Wilmington, DE.D7644 16510.1.1 Ammonium acetate was evaluated as an agent to bind free chlorine in drinking water and was found to be ineffectivein the preservation of the rodenticides in chlorinated drinking water. Ascorbic acid was effective as a dechlorinating agent inchlorine fortified Chicago
27、 tap water, which contained 3.2 ppm free chlorine and was dechlorinated with 10 mg ascorbic acid per40 mL water sample.1310.2 The samples are collected using 40 mL glass vials. A 40 mL volume is collected directly into the sample collection vialwithout using any other measuring devices. This is a re
28、quirement due to the rodenticides affinity for surfaces, which will lead tobiased low results if transferring between containers. Before collection, the vials must be evaluated to determine a 40 mL samplevolume. For example, the vials used in this test method were calibrated before use to determine
29、that filling the vial to approximately1.6 cm below the rim would result in a 40 mL sample volume. The greatest amount of water held by the 40 mL vials used in thistest method was approximately 42 mL. Vials filled to 42 mLin the field would not allow the laboratory to spike the samples beforequantita
30、tively transferring to the 50 mL graduated cylinder. It is imperative that the samplers do not overfill the vials.10.3 PreservationStore samples between 0C and 6C from the time of collection until analysis. Analyze the sample within14 days of collection. Chlorinated drinking water samples are dechlo
31、rinated with ascorbic acid; 10 mg of ascorbic acid is addedto each 40 mL vial prior to water collection.11. Preparation of LC/MS/MS11.1 LC Chromatograph Operating Conditions:511.1.1 Injection volumes of all calibration standards and samples are made at 50 L volume using a full loop injection. If a 5
32、0L 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 thecalibration curve is a blank to ensure there is no car
33、ry-over. The gradient conditions for the liquid chromatograph are shown inTable 2.NOTE 2If your instrument does not have a 50 L injection capability a different volume may be used. This is a performance-based method andmodifications are allowed as long as minimum performance criteria are met.11.2 LC
34、 Sample Manager Conditions:11.2.1 Wash SolventsWeak wash is 4.0 mLof 95 % water/5 % methanol. Strong wash is 2.0 mLof methanol. The strong washsolvent is needed to eliminate carry-over between injections of rodenticide samples. The weak wash is used to remove the strongwash solvent. These rodenticid
35、es were shown to carry-over when acetonitrile was used for this analysis. The use of methanolcorrected this problem while providing separation and sensitivity. Instrument manufacturer specifications should be followed inorder to eliminate sample carry-over.11.2.2 TemperaturesColumn, 30C; Sample comp
36、artment, 15C.11.2.3 Seal WashSolvent: 50% Acetonitrile/50% Water; Time: 5 minutes.11.3 Mass Spectrometer Parameters:711.3.1 To acquire the maximum number of data points per SRM channel while maintaining adequate sensitivity, the tuneparameters may be optimized according to your instrument. Each peak
37、 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. Variableparameters regarding retention times, SRM transitions, and cone and collision energies are shown in Table 3. Mas
38、s spectrometerparameters used in the development of this test method are listed below:The instrument is set in the Electrospray source setting.Capillary Voltage: 3.5 kVCone: Variable depending on analyte (Table 3)Extractor: 2 Volts13 A Hach Pocket Colorimeter II Colorimeter II (a trademark of Hach C
39、ompany in Loveland, CO) was used to measure free chlorine.TABLE 2 Gradient Conditions for Liquid ChromatographyTime(min)Flow(L/min)Percent95% Water/5% Methanol,5 mM NH4OHPercent95% Methanol/5% Water,5 mM NH4OH0.0 300 100 02.0 300 100 06.0 300 20 806.1 200 5 957.0 200 5 958.5 200 0 10013.0 300 0 1001
40、4.0 300 100 016.0 300 100 0D7644 166RF Lens: 0.1 VoltsSource Temperature: 120CDesolvation Temperature: 375CDesolvation Gas Flow: 800 L/hrCone Gas Flow: 25 L/hrLow Mass Resolution 1: 14.0High Mass Resolution 1: 14.0Ion Energy 1: 0.5Entrance Energy: -1Collision Energy: Variable depending on analyte (T
41、able 3)Exit Energy: 0Low Mass Resolution 2: 14.0High Mass resolution 2: 14.0Ion Energy 2: 0.7Multiplier: 650Gas Cell Pirani Gauge: 7.0 10-3 TorrInter-Channel Delay: 0.02 secondsInter-Scan Delay: 0.02 secondsDwell: 0.1 seconds12. Calibration and Standardization12.1 The mass spectrometer must be calib
42、rated per manufacturer specifications before analysis. In order to obtain valid andaccurate analytical values through this test method within the confidence limits, the following procedures must be followed whenperforming the test method. Prepare all solutions in the lab using Class A volumetric gla
43、ssware.12.2 Calibration and StandardizationTo calibrate the instrument, analyze 7 calibration standards containing the 7concentration levels of the rodenticides and surrogates prior to analysis as shown in Table 4. A calibration stock standard solutionis prepared from standard materials or they are
44、purchased as certified solutions. Stock standard solution A containingbromadiolone, brodifacoum, diphacinone, warfarin, warfarin-D5 (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
45、 1 through 6. Thefollowing steps will produce standards with the concentration values shown in Table 4. The analyst is responsible for recordinginitial component weights carefully when working with pure materials and correctly carrying the weights through the dilutioncalculations.12.2.1 Prepare stoc
46、k standard solutionA(Level 7) by adding to a 100 mL volumetric flask individual methanol solutions of thefollowing: 50 L of bromadiolone, brodifacoum, diphacinone, warfarin, warfarin-D5 (surrogate) each at 4.0 mg/L and 50 L of2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (surrogate) at 0.2 g/L, dilute
47、to 100 mL with 80 % water/20 % methanol. Thepreparation of the Level 7 standard can be accomplished using different volumes and concentrations of stock solutions as isaccustomed in the individual laboratory. Depending on the prepared stock concentrations, solubility at that concentration will haveto
48、 be ensured.12.2.2 Aliquots of solutionAare then diluted with 80 % water/20 % methanol to prepare the desired calibration levels in 2 mLamber glass LC vials. The calibration vials must be used within 24 hours to ensure optimum results. Stock calibration standardsare routinely replaced every 7 days i
49、f 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 andconfirmatory SRM transition of each analyte. Calibration software is utilized to conduct the quantitation of the target analytes andsurrogates using the primary SRM transition. The ratios of the primary/confirmatory SRM transition area counts are given in Table3 and will vary depending on the individual tuning co
