SOLVENT EXTRACTABLE NONVOLATILE COMPOUNDS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/PARTICLE BEAM/MASSSPECTROMETRY (HPLC/PB/MS)1.0
SCOPE AND APPLICATION
1.1This method describes the use of high performance liquid chromatography (HPLC),coupled with particle beam (PB) mass spectrometry (MS), for the determination of benzidines andnitrogen-containing pesticides in water and wastewater. The following compounds can bedetermined by this method:
CAS No.a92-87-533878-50-1
63-25-25344-82-191-94-1119-90-4612-82-8330-54-1330-55-2150-68-583-79-41982-49-6
Compound
Benzidine
Benzoylprop ethylCarbaryl
o-Chlorophenyl thiourea3,3'-Dichlorobenzidine3,3'-Dimethoxybenzidine3,3'-DimethylbenzidineDiuron
Linuron (Lorox)MonuronRotenoneSiduron
a
Chemical Abstract Service Registry Number
1.2The method also may be appropriate for the analysis of benzidines andnitrogen-containing pesticides in non-aqueous matrices. The method may be applicable to othercompounds that can be extracted from a sample with methylene chloride and are amenable toseparation on a reverse phase liquid chromatography column and transferable to the massspectrometer with a particle beam interface.
1.3Preliminary investigation indicates that the following compounds also may be amenableto this method: Aldicarb sulfone, Carbofuran, Methiocarb, Methomyl (Lannate), Mexacarbate(Zectran), and N-(1-Naphthyl)thiourea. Ethylene thiourea and o-Chlorophenyl thiourea have beensuccessfully analyzed by HPLC/PB/MS, but have not been successfully extracted from a watermatrix.
1.4Tables 4 - 6 present method detection limits (MDLs) for the target compounds, rangingfrom 2 to 25 µg/L. The MDLs are compound- and matrix-dependent.
1.5This method is restricted to use by, or under the supervision of, analysts experienced inthe use of HPLC and skilled in the interpretation of particle beam mass spectrometry. Each analystmust demonstrate the ability to generate acceptable results with this method.CD-ROM
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2.0SUMMARY OF METHOD
2.1The target compounds for this method must be extracted from the sample matrix priorto analysis.
2.1.1Benzidines and nitrogen-containing pesticides are extracted from aqueousmatrices at a neutral pH with methylene chloride, using a separatory funnel (Method 3510), acontinuous liquid-liquid extractor (Method 3520), or other suitable technique.
2.1.2Solid samples are extracted using Methods 3540 (Soxhlet), 3541 (AutomatedSoxhlet), 3550 (Ultrasonic extraction), or other suitable technique.
2.2An aliquot of the sample extract is introduced into the HPLC instrument and a gradientelution program is used to chromatographically separate the target analytes, using reverse-phaseliquid chromatography.
2.3Once separated, the analytes are transferred to the mass spectrometer via a particlebeam HPLC/MS interface. Quantitation is performed using an external standard approach. 2.4An optional internal standard quantitation procedure is included for samples whichcontain coeluting compounds or where matrix interferences preclude the use of the external standardprocedure.
2.5The use of ultraviolet/visible (UV/VIS) detection is an appropriate option for the analysisof routine samples, whose general composition has been previously determined. 3.0
INTERFERENCES
3.1Refer to Methods 3500 and 8000 for general discussions of interferences with the sampleextraction and chromatographic separation procedures.
3.2Although this method relies on mass spectrometric detection, which can distinguishbetween chromatographically co-eluting compounds on the basis of their masses, co-elution of twoor more compounds will adversely affect method performance. When two compounds coelute, thetransport efficiency of both compounds through the particle beam interface generally improves, andthe ion abundances observed in the mass spectrometer increase. The degree of signalenhancement by coelution is compound-dependent.
3.2.1This coelution effect invalidates the calibration curve and, if not recognized, willresult in incorrect quantitative measurements. Procedures are given in this method to checkfor co-eluting compounds, and must be followed to preclude inaccurate measurements. 3.2.2An optional internal standard calibration procedure has been included for use ininstances of severe co-elution or matrix interferences.
3.3A major source of potential contamination is HPLC columns which may contain siliconcompounds and other contaminants that could prevent the determination of method analytes.Generally, contaminants will be leached from the columns into mobile phase and produce a variablebackground. Figure 1 shows unacceptable background contamination from a column with stationaryphase bleed.CD-ROM
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3.4Contamination may occur when a sample containing low analyte concentrations isanalyzed immediately after a sample containing relatively high analyte concentrations. After analysisof a sample containing high analyte concentrations, one or more method blanks should be analyzed.Normally, with HPLC, this is not a problem unless the sample concentrations are at the percent level.4.0
APPARATUS AND MATERIALS
4.1High performance liquid chromatograph (HPLC) - An analytical system withprogrammable solvent delivery system and all necessary accessories including 5 µL injection loop,analytical columns, purging gases, etc. The solvent delivery system must be capable, at a minimum,of handling a binary solvent system, and must be able to accurately deliver flow rates between 0.20- 0.40 mL/min. Pulse dampening is recommended, but not required. The chromatographic systemmust be able to be interfaced with a mass spectrometer (MS). An autoinjector is recommended andshould be capable of accurately delivering 1 - 10 µL injections without affecting the chromatography.
4.1.1HPLC Columns - An analytical column is needed, and a guard column is highlyrecommended.
4.1.1.1Analytical Column - Reverse phase column, C18 chemically bonded to
4-10 µm silica particles, 150 - 200 mm x 2 mm, (Waters C-18 Novapak or equivalent).Residual acidic sites should be blocked (endcapped) with methyl or other non-polargroups and the stationary phase must be bonded to the solid support to minimize columnbleed. Select a column that exhibits minimal bleeding. New columns must beconditioned overnight before use by pumping a 75 - 100% v/v acetonitrile:water solutionthrough the column at a rate of about 0.05 mL/min. Other packings and column sizesmay be used if appropriate performance can be achieved.
4.1.1.2
Guard Column - Packing similar to that used in analytical column.
4.1.2HPLC/MS interface - The particle beam HPLC/MS interface must reduce the ionsource pressure to a level compatible with the generation of classical electron ionization (EI)
-6 Torr, while delivering sufficient quantities of analytesmass spectra, i.e., about 1 x 10-4 - 1 x 10
to the conventional EI source to meet sensitivity, accuracy, and precision requirements. Theconcentrations of background components with masses greater than 62 Daltons should bereduced to levels that do not produce ions greater than a relative abundance of 10% in themass spectra of the analytes.
4.2Mass spectrometer system - The mass spectrometer must be capable of electronionization at a nominal electron energy of 70 eV. The spectrometer should be capable of scanningfrom 45 to 500 amu in 1.5 seconds or less (including scan overhead). The spectrometer shouldproduce a mass spectrum that meets the criteria in Table 1 when 500 ng or less of DFTPPO areintroduced into the HPLC.
4.3Data system - A computer system must be interfaced to the mass spectrometer, andmust be capable of the continuous acquisition and storage on machine-readable media of all massspectra obtained throughout the duration of the chromatographic program. The computer softwaremust be capable of searching any HPLC/MS data file for ions of a specified mass and plotting suchabundance data versus time or scan number.
4.4
Volumetric flasks - Class A, in various sizes, for preparation of standards.
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4.5Vials - 10-mL amber glass vials with polytetrafluororethylene (PTFE)-lined screw capsor crimp tops.
4.6 Analytical balance - capable of weighing 0.0001 g.4.7
Extract filtration apparatus4.7.1
Syringe - 10-mL, with Luer-Lok fitting.
4.7.2Syringe filter assembly, disposable - 0.45 µm pore size PTFE filter in filterassembly with Luer-Lok fitting (Gelman Acrodisc, or equivalent).5.0
REAGENTS
5.1Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it isintended that all reagents shall conform to the specifications of the Committee on AnalyticalReagents of the American Chemical Society, where such specifications are available. Other gradesmay be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit itsuse without lessening the accuracy of the determination.
5.2Organic-free reagent water - All references to water in this method refer to organic-freereagent water, as defined in Chapter One.
5.3
Solvents - All solvents must be HPLC-grade or equivalent.5.3.15.3.25.3.3
Acetonitrile, CH3CNMethanol, CH3OH
Ammonium acetate, NH4OOCCH3, (0.01M in water).
5.4Mobile phase - Two mobile phase solutions are needed, and are designated Solvent Aand Solvent B. Degas both solvents in an ultrasonic bath under reduced pressure and maintain bypurging with a low flow of helium.
5.4.1Solvent A is a water:acetonitrile solution (75/25, v/v) containing ammoniumacetate at a concentration of 0.01M.
5.4.2
Solvent B is 100 % acetonitrile.
5.5Stock standard solutions - Stock solutions may be prepared from pure standard materialsor purchased as certified solutions. Commercially-prepared stock standards may be used at anyconcentration if they are certified by the manufacturer.
5.5.1Prepare stock standard solutions by accurately weighing 0.0100 g of pure materialin a volumetric flask. Dilute to known volume in a volumetric flask. If compound purity iscertified at 96% or greater, the weight may be used without correction to calculate theconcentration of the stock standard. Commercially-prepared stock standards may be used atany concentration if they are certified by the manufacturer or by an independent source.
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5.5.1.1Dissolve benzidines and nitrogen-containing pesticides in methanol,
acetonitrile, or organic-free reagent water.
5.5.1.2Certain analytes, such as 3,3'-dimethoxybenzidine, may require dilution
in 50% (v/v) acetonitrile:water or methanol:water solution.
5.5.1.3Benzidines may be used for calibration purposes in the free base or acid
chlorides forms. However, the concentration of the standard should be calculated as thefree base.
5.5.2Transfer the stock standard solutions into amber bottles with PTFE-linedscrew-caps or crimp tops. Store at -10EC or less and protect from light. Stock standardsolutions should be checked frequently for signs of degradation or evaporation, especially justprior to preparing calibration standards from them.
5.6Surrogate spiking solution - The recommended surrogates are benzidine-D8,caffeine-15N2, 3,3'-dichlorobenzidine-D 6, and bis(perfluorophenyl)-phenylphosphine oxide. Preparea solution of the surrogates in methanol or acetonitrile at a concentration of 5 mg/mL of each. Othersurrogates may be included in this solution as needed. (A 10-µL aliquot of this solution added to 1L of water gives a concentration of 50 µg/L of each surrogate). Store the surrogate spiking solutionin an amber vial in a freezer at -10EC or less.
5.7MS performance check solution - Prepare a 100 ng/µL solution of DFTPPO in acetonitrile.Store this solution in an amber vial in a freezer at -10EC or less.
5.8
Calibration solutions
This method describes two types of calibration procedures that may be applied to the targetcompounds: external standard calibration, and internal standard calibration. Each procedurerequires separate calibration standards. In addition, the performance characteristics of theHPLC/PB/MS system indicate that it may be necessary to employ a second order regression forcalibration purposes, unless a very narrow calibration range is chosen. See Method 8000 foradditional information on non-linear calibration techniques.
5.8.1For external standard calibration, prepare calibration standards for all targetcompounds and surrogates in acetonitrile. DFTPPO may be added to one or more calibrationsolutions to verify MS tune (see Sec. 7.3). Store these solutions in amber vials at -10EC orless. Check these solutions at least quarterly for signs of deterioration.
5.8.2Internal standard calibration requires the use of suitable internal standards (seeMethod 8000). Ideally, stable, isotopically-labeled, analogs of the target compounds shouldbe used. These labeled compounds are included in the calibration standards and must alsobe added to each sample extract immediately prior to analysis. Prepare the calibrationstandards in a fashion similar to that for external standard calibration, but include each internalstandard in each of the calibration standards.
The concentration of the internal standards should be 50 - 100 times the lowestconcentration of the unlabeled target compounds. In addition, the concentration of the internalstandards does not vary with the concentrations of the target compounds, but is held constant.Store these solutions in amber vials at -10EC or less. Check these solutions at least quarterlyfor signs of deterioration.
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5.9Internal standard spiking solution - This solution is required when internal standardquantitation is used. Prepare a solution containing each of the internal standards that will be usedfor quantitation of target compounds (see Sec. 5.8.2) in methanol. The concentration of this solutionmust be such that a 1-µL volume of the spiking solution added to a 1-mL final extract will result ina concentration of each internal standard that is equal to the concentration of the internal standardin the calibration standards in Sec. 5.8.2. Store this solution in an amber vial at -10EC or less.Check this solution at least quarterly for signs of deterioration. This solution is not necessary if onlyexternal standard calibration will be used.
5.10Sodium chloride, NaCl - granular, used during sample extraction.6.0
SAMPLE COLLECTION, PRESERVATION, AND HANDLING6.1
See the introductory material to this chapter, Organic Analytes, Sec. 4.1.
6.2Samples should be extracted within 7 days and analyzed within 30 days of extraction.Extracts should be stored in amber vials at -10EC or less.7.0
PROCEDURE
7.1Samples may be extracted by Method 3510 (separatory funnel), Method 3520 (continuousextractor), Method 3535 (solid-phrase extraction), or other appropriate technique. Prior to extraction,add a 10-µL aliquot of the surrogate spiking solution and 100 g of sodium chloride to the sample, andadjust the pH of the sample to 7.0. Samples of other matrices should be extracted by an appropriatesample preparation technique. The concentration of surrogates in the sample should be 20-50 timesthe method detection limit. Concentrate the extract to 1 mL, and exchange the solvent to methanol,following the procedures in the extraction method.
7.2Establish chromatographic, particle beam interface, and mass spectrometer conditions,using the following conditions as guidance.
Mobile phase purge:Mobile phase flow rate:
Gradient elution:
Helium at 30 mL/min, continuous
0.25 - 0.3 mL/min through the column
Hold for 1 min at 25% acetonitrile (Solvent A), thenprogram linearly to about 70% acetonitrile (60%Solvent B) in 29 min. Start data acquisitionimmediately.
45 - 80EC250 - 290EC70 eV
62 to 465 amu, at #1.5 sec/scan
Desolvation chamber temperature:
Ion source temperature:
Electron energy:
Scan range:NOTE:Post-column addition is an option that improves system precision and, thereby,may improve sensitivity. Post-column flow rates depend on the requirements ofthe interface and may range from 0.1 to 0.7 mL/min of acetonitrile. Maintain aminimum of 30% acetonitrile in the interface.
Analyte-specific chromatographic conditions are also shown in Table 2. (The particle beaminterface conditions will depend on the type of nebulizer). CD-ROM
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7.2.1The analyst should follow the manufacturer's recommended conditions for theirinterface's optimum performance. The interface is usually optimized during initial installationby flow injection with caffeine or benzidine, and should utilize a mobile phase ofacetonitrile/water (50/50, v/v). Major maintenance may require re-optimization.
7.2.2Fine tune the interface by making a series of injections into the HPLC column ofa medium concentration calibration standard and adjusting the operating conditions (Sec. 7.2)until optimum sensitivity and precision are obtained for the maximum number of targetcompounds. 7.3
Initial calibration
7.3.1Once the operating conditions have been established, calibrate the MS mass andabundance scales using DFTPPO to meet the recommended criteria in Table 1.
7.3.2Inject a medium concentration standard containing DFTPPO, or separately injectinto the HPLC a 5-µL aliquot of the 100 ng/µL DFTPPO solution and acquire a mass spectrum.Use HPLC conditions that produce a narrow (at least ten scans per peak) symmetrical peak.If the spectrum does not meet the criteria (Table 1), the MS ion source must be retuned andadjusted to meet all criteria before proceeding with calibration. An average spectrum acrossthe HPLC peak may be used to evaluate the performance of the system.
Manual (not automated) ion source tuning procedures specified by the manufacturershould be employed during tuning. Mass calibration should be accomplished while anacetonitrile/water (50/50, v/v) mixture is pumped through the HPLC column and the optimizedparticle beam interface. For optimum long-term stability and precision, interface and ionsource parameters should be set near the center of a broad signal plateau rather than at thepeak of a sharp maximum (sharp maxima exhibit short-term variations with particle beaminterfaces and gradient elution conditions).
7.3.3System performance criteria for the medium concentration standard - Evaluatethe stored HPLC/MS data with the data system software and verify that the HPLC/PB/MSsystem meets the following performance criteria.
7.3.3.1HPLC performance - 3,3'-dimethylbenzidine and
3,3'-dimethoxybenzidine should be separated by a valley whose height is less than 25%of the average peak height of these two compounds. If the valley between them exceeds25%, modify the gradient. If this fails, the HPLC column requires maintenance. SeeSec. 7.4.6.
7.3.3.2Peak tailing - Examine a total ion chromatogram and examine the
degree of peak tailing. Severe tailing indicates a major problem and systemmaintenance is required to correct the problem. See Sec. 7.4.6
7.3.3.3MS sensitivity - The signal-to-noise ratio for any compound's spectrum
should be at least 3:1.
7.3.3.4Column bleed - Figure 1 shows an unacceptable chromatogram with
column bleed. Figure 2 shows an acceptable ion chromatogram. Figure 3 is the massspectrum of dimethyloctadecyl-silanol, a common stationary phase bleed product. Ifunacceptable column bleed is present, the column must be changed or conditioned toproduce an acceptable background.
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7.3.3.5Coeluting compounds - Compounds which coelute cannot be measured
accurately because of carrier effects in the particle beam interface. Peaks must beexamined carefully for coeluting substances and if coeluting compounds are present atgreater than 10% of the concentration of the target compound, either conditions must beadjusted to resolve the components, or internal standard calibration must be used.7.3.4Once optimized, the same instrument operating conditions must be used for theanalysis of all calibration standards, samples, blanks, etc.
7.3.5Once all the performance criteria are met, inject a 5-µL aliquot of each of theother calibration standards using the same HPLC/MS conditions.
7.3.5.1The general method of calibration is a second order regression of
integrated ion abundances of the quantitation ions (Table 3) as a function of amountinjected. For second order regression, a sufficient number of calibration points must beobtained to accurately determine the equation of the curve. (See Method 8000 for theappropriate number of standards to be employed for a non-linear calibration). Non-linearcalibration models can be applied to either the external standard or the internal standardcalibration approaches described here.
7.3.5.2For some analytes the instrument response may be linear over a narrow
concentration range. In these instances, an average calibration factor (externalstandard) or average response factor (internal standard) may be employed for samplequantitation (see Method 8000).
7.3.6If a linear calibration model is used, calculate the mean calibration factor orresponse factor for each analyte, including the surrogates, as described in Method 8000.Calculate the standard deviation (SD) and the relative standard deviation (RSD) as well. TheRSD of an analyte or surrogate must be less than or equal to 20%, if the linear model is to beapplied. Otherwise, proceed as described in Method 8000.7.4
Calibration verification
Prior to sample analysis, verify the MS tune and initial calibration at the beginning of each8-hour analysis shift using the following procedure:
7.4.1Inject a 5-µL aliquot of the DFTPPO solution or a mid-level calibration standardcontaining 500 ng of DFTPPO, and acquire a mass spectrum that includes data for m/z62-465. If the spectrum does not meet the criteria in Table 1, the MS must be retuned to meetthe criteria before proceeding with the continuing calibration check.
7.4.2Inject a 5-µL aliquot of a medium concentration calibration solution and analyzewith the same conditions used during the initial calibration.
7.4.3
Demonstrate acceptable performance for the criteria shown in Sec. 7.3.3.
7.4.4Using the initial calibration (either linear or non-linear, external standard or internalstandard), calculate the concentrations in the medium concentration calibration solution andcompare the results to the known values in the calibration solution. If calculatedconcentrations deviate by more than 20% from known values, adjust the instrument and injectthe standard again. If the calibration cannot be verified with the second injection, then a new
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initial calibration must be performed after taking corrective actions such as those described inSec. 7.9. 7.5
Sample Analysis
7.5.1The column should be conditioned overnight before each use by pumping aacetonitrile:water (70% v/v) solution through it at a rate of about 0.05 mL/min.
7.5.2Filter the extract through a 0.45 µm filter. If internal standard calibration isemployed, add 10 µL of the internal standard spiking solution to the 1-mL final extractimmediately before injection.
7.5.3Analyze a 5-µL aliquot of the extract, using the operating conditions establishedin Secs. 7.2 and 7.3. 7.6
Qualitative identification
The qualitative identification of compounds determined by this method is based on retentiontime and on comparison of the sample mass spectrum, after background correction, withcharacteristic ions in a reference mass spectrum. The reference mass spectrum must be generatedby the laboratory using the conditions of this method. The characteristic ions from the referencemass spectrum are defined as the three ions of greatest relative intensity, or any ions over 30%relative intensity, if less than three such ions occur in the reference spectrum. Compounds areidentified when the following criteria are met.
7.6.1The intensities of the characteristic ions of a compound must maximize in thesame scan or within one scan of each other. Selection of a peak by a data system targetcompound search routine where the search is based on the presence of a targetchromatographic peak containing ions specific for the target compound at a compound-specificretention time will be accepted as meeting this criterion.
7.6.2The retention time of the sample component is within ± 10% of the retention timeof the standard.
7.6.3The relative intensities of the characteristic ions agree within 20% of the relativeintensities of these ions in the reference spectrum. (Example: For an ion with an abundanceof 50% in the reference spectrum, the corresponding abundance in a sample spectrum canrange between 30% and 70%.)
7.6.4Structural isomers that produce very similar mass spectra should be identified asindividual isomers if they have sufficiently different HPLC retention times. Sufficient GCresolution is achieved if the height of the valley between two isomer peaks is less than 25%of the sum of the two peak heights. Otherwise, structural isomers are identified as isomericpairs.
7.6.5Identification is hampered when sample components are not resolvedchromatographically and produce mass spectra containing ions contributed by more than oneanalyte. When HPLC peaks obviously represent more than one sample component (i.e., abroadened peak with shoulder(s) or a valley between two or more maxima), appropriateselection of analyte spectra and background spectra is important.
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7.6.6Examination of extracted ion current profiles of appropriate ions can aid in theselection of spectra, and in qualitative identification of compounds. When analytes coelute(i.e., only one chromatographic peak is apparent), the identification criteria may be met, buteach analyte spectrum will contain extraneous ions contributed by the coeluting compound.7.7
Quantitative Analysis
7.7.1Complete chromatographic resolution is necessary for accurate and precisemeasurements of analyte concentrations. Compounds which coelute cannot be measuredaccurately because of carrier effects in the particle beam interface. Peaks must be examinedcarefully for coeluting substances and if coeluting compounds are present at greater than 10%of the concentration of the target compound, either conditions must be adjusted to resolve thecomponents, or the results for the target compound must be flagged as potentially positivelybiased.
7.7.2Calculate the concentration of each analyte, using either the external standardor internal standard calibration. See Method 8000 for the specific equations to be employedfor either the non-linear or linear calibration models.
7.7.3If the response for any quantitation ion exceeds the initial calibration range of theHPLC/PB/MS system, the sample extract must be diluted and reanalyzed. When internalstandard calibration is employed, additional internal standard must be added to the dilutedextract to maintain the same concentration as in the calibration standards.7.8
HPLC-UV/VIS Detection (optional)7.8.1
Prepare calibration solutions as outlined in Sec. 5.8.
7.8.2Inject 5 µL of each calibration solution onto the HPLC, using the chromatographicconditions outlined in Secs. 7.2.1 and 7.2.2. Integrate the area under the full chromatographicpeak at the optimum wavelength (or at 230 nm if that option is not available) for each targetcompound at each concentration.
7.8.3The retention time of the chromatographic peak is an important criterion foranalyte identification. Therefore, the ratio of the retention time of the sample analyte to thestandard analyte should be 1.0 ± 0.1.
7.8.4Calculate calibration factors or response factors as described in Method 8000,for either external standard or internal standard calibration, and evaluate the calibrationlinearity as described in Method 8000.7.8.5above.
Verify the calibration at the beginning of each 8-hour analytical shift, as described
7.8.6Once the calibration has been verified, inject a 5-µL aliquot of the sample extract,start the HPLC gradient elution, load and inject the sample aliquot, and begin data acquisition.Refer to Method 8000 for guidance on calculation of concentration.7.9
Corrective Actions
When the initial calibration cannot be verified, one or more of the following corrective actionsmay be necessary.CD-ROM
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7.9.1Major maintenance such as cleaning an ion source, cleaning the entrance lens,quadrapole rods, etc., will require a new initial calibration.
7.9.2Check and adjust HPLC and/or MS operating conditions; check the MS resolution,and calibrate the mass scale.
7.9.3Replace the mobile phases with fresh solvents. Verify that the flow rate from theHPLC pump is constant.
7.9.47.9.57.9.67.9.77.9.8
Flush the HPLC column with acetonitrile.
Replace the HPLC column. This action will cause a change in retention times.Prepare fresh calibration solutions, and repeat the initial calibration step.Replace any components that leak.
Replace the MS electron multiplier, or any other faulty components.
7.9.9Clean the interface to eliminate plugged components and/or replace skimmersaccording to the manufacturer's instructions.
7.9.10If peak areas are determined by the instrument software, verify values by manualintegration.
7.9.11Increasing ion source temperature can reduce peak tailing, but excessive ionsource temperature can affect the quality of the spectra for some compounds.
7.9.12Air leaks into the interface may effect the quality of the spectra (e.g., DFTPPO),especially when the ion source is operated at temperatures in excess of 280EC.8.0
QUALITY CONTROL
8.1Refer to Chapter One and Method 8000 for specific quality control (QC) procedures.Quailty control procedures to ensure the proper operation of the various sample preparationtechniques can be found in Method 3500. Each laboratory should maintain a formal qualityassurance program. The laboratory should also maintain records to document the quality of the datagenerated.
8.2Quality control procedures necessary to evaluate the HPLC system operation are foundin Method 8000, Sec. 7.0 and includes evaluation of retention time windows, calibration verificationand chromatographic analysis of samples. Necessary instrument QC is found in the followingsections.
8.2.1The HPLC/PB/MS system should be tuned to meet the DFTPPO criteria in Secs.7.3.1 and 7.4.1.
8.2.2There should be an initial calibration of the HPLC/PB/MS system as describedin Sec. 7.3.
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8.2.3The HPLC/PB/MS system should meet the system performance criteria in Sec.7.3.3, each 8 hours.
8.3Initial Demonstration of Proficiency - Each laboratory must demonstrate initial proficiencywith each sample preparation and determinative method combination it utilizes, by generating dataof acceptable accuracy and precision for target analytes in a clean matrix. The laboratory must alsorepeat the following operations whenever new staff are trained or significant changes ininstrumentation are made. See Method 8000, Sec. 8.0 for information on how to accomplish thisdemonstration.
8.4Sample Quality Control for Preparation and Analysis - The laboratory must also haveprocedures for documenting the effect of the matrix on method performance (precision, accuracy,and detection limit). At a minimum, this includes the analysis of QC samples including a methodblank, a matrix spike, a duplicate, and a laboratory control sample (LCS) in each analytical batch andthe addition of surrogates to each field sample and QC sample.
8.4.1Documenting the effect of the matrix should include the analysis of at least onematrix spike and one duplicate unspiked sample or one matrix spike/matrix spike duplicate pair.The decision on whether to prepare and analyze duplicate samples or a matrix spike/matrixspike duplicate must be based on a knowledge of the samples in the sample batch. If samplesare expected to contain target analytes, then laboratories may use one matrix spike and aduplicate analysis of an unspiked field sample. If samples are not expected to contain targetanalytes, laboratories should use a matrix spike and matrix spike duplicate pair.
8.4.2A Laboratory Control Sample (LCS) should be included with each analytical batch.The LCS consists of an aliquot of a clean (control) matrix similar to the sample matrix and ofthe same weight or volume. The LCS is spiked with the same analytes at the sameconcentrations as the matrix spike. When the results of the matrix spike analysis indicate apotential problem due to the sample matrix itself, the LCS results are used to verify that thelaboratory can perform the analysis in a clean matrix.
8.4.3See Method 8000, Sec. 8.0 for the details on carrying out sample quality controlprocedures for preparation and analysis.
8.5Surrogate recoveries - The laboratory must evaluate surrogate recovery data fromindividual samples versus the surrogate control limits developed by the laboratory. See Method8000, Sec. 8.0 for information on evaluating surrogate data and developing and updating surrogatelimits.
8.6It is recommended that the laboratory adopt additional quality assurance practices for usewith this method. The specific practices that are most productive depend upon the needs of thelaboratory and the nature of the samples. Whenever possible, the laboratory should analyzestandard reference materials and participate in relevant performance evaluation studies.9.0
METHOD PERFORMANCE
9.1Single laboratory accuracy and precision data for the benzidines and nitrogen-containingpesticides are presented in Tables 4 - 6. Five to seven 1-L aliquots of organic-free reagent water,containing approximately five times the MDL of each analyte, were analyzed with this procedure(Reference 1). The final extract volume was 0.5 mL for these determinations.
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9.1.1
Method detection limits (MDLs) are presented in Tables 4 - 6.
9.1.2A multi-laboratory (12 laboratories) validation of the determinative step was donefor four of the analytes (benzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, 3,3'-dichlorobenzidine). Table 7 provides the results from this study for single laboratory precision,overall laboratory precision, and overall laboratory accuracy. The two concentration levelsshown represent the two extremes of the concentration range studied. 10.0REFERENCES1.
Bellar, T.A., Behymer, T.D., Ho, J.S., Budde, W.L., \"Method 553: Determination of Benzidinesand Nitrogen-Containing Pesticides in Water by Liquid-Liquid Extraction or Liquid-SolidExtraction and Reverse Phase High Performance Liquid Chromatography/Particle Beam/MassSpectrometry\1992.
Bellar, T.A., Behymer, T.D., Budde, W.L., \"Investigation of Enhanced Ion Abundances froma Carrier Process in High-Performance Liquid Chromatography Particle Beam MassSpectrometry\Behymer, T.D., Bellar, T.A., and Budde, W.L., \"Liquid Chromatography/Particle Beam/MassSpectrometry of Polar Compounds of Environmental Interest\1686-1690.
Ho, J.S., Behymer, T.D., Budde, W.L., and Bellar, T.A., \"Mass Transport and Calibration inLiquid Chromatography/ Particle Beam/ Mass Spectrometry\1992, 3, 662-671.
2.
3.
4.
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TABLE 1
ION ABUNDANCE CRITERIA FOR BIS(PERFLUOROPHENYL)PHENYLPHOSPHINE
(DECAFLUOROTRIPHENYLPHOSPHINE OXIDE, DFTPPO)
Purpose of Specification1
Low mass sensitivityMid-mass sensitivity
Mid-mass resolution and isotope ratioBase peak
Baseline threshold check
Important high mass fragmentMolecular ion
High mass resolution and isotope ratio
m/z77168169271365438458459
Relative AbundancePresent, major ionPresent, major ion4 - 10% of 168Present, major ion5 - 10% of base peakPresentPresent
15 - 24% of mass 458
1
The primary use of all the ions is to check the mass calibration of the mass spectrometer.The second use of these ions are the mass resolution checks, including the natural isotopeabundance ratios. The correct setting of the baseline threshold is indicated by the presenceof low intensity ions, and is the third use of this test. Finally, the ion abundance ranges mayprovide some standardization to fragmentation patterns of the target compounds.
TABLE 2
RECOMMENDED HPLC CHROMATOGRAPHIC CONDITIONSFOR BENZIDINES AND NITROGEN-CONTAINING PESTICIDES
Initial MobilePhase (v/v %)InitialTime (min)GradientTimeFinal MobilePhase (v/v %)
75/25
(water1/CH3CN)
129
30/70
(water1/CH3CN)
1 Water contains 0.01M ammonium acetate.
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TABLE 3
RETENTION TIME DATA AND QUANTITATION IONS FOR TARGET COMPOUNDS
Compound
Benzidine
Benzoylprop ethylCaffeineCarbaryl
O-Chlorophenyl thiourea3,3'-Dichlorobenzidine3,3'-Dimethoxybenzidine3,3'-DimethylbenzidineDiuron
Ethylene thioureaLinuronRotenoneSiduronSurrogates:c
Benzidine-d8Caffeine-15N2
3,3'-Dichlorobenzidine-d6Bis(perfluorophenyl)- phenylphosphine oxide
RetentionTimeSystem 1a
4.324.81.410.12.716.68.18.511.01.216.021.114.8
RetentionTime
b
System 24.931.31.614.73.022.711.512.416.11.421.927.420.6
Quantitation
Ion
1841051941441512522442127210216119293
4.21.316.522.0
4.81.622.628.9
192196258271
a b c
These retention times were obtained on a Hewlett-Packard 1090 liquid chromatograph witha Waters C18 Novapak 15 cm x 2 mm column using gradient conditions given in Table 1.These retention times were obtained on a Waters 600 MS liquid chromatograph with aWaters C18 Novapak 15 cm x 2 mm column using gradient conditions given in Sec. 7.2.These compounds cannot be used as surrogates if their unlabeled analogs are present (seeSec. 3.2).
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TABLE 4
ACCURACY AND PRECISION DATA FROM SIX DETERMINATIONS OF THE TARGETCOMPOUNDS IN ORGANIC-FREE REAGENT WATER USING LIQUID-LIQUID EXTRACTION
Compound
Benzidine
Benzoylprop ethylCaffeine Carbaryl
o-Chlorophenyl thiourea3,3'-Dichlorobenzidine3,3'-Dimethoxybenzidine3,3'-Dimethylbenzidine Diuron
Ethylene thiourea Linuron MonuronRotenone Siduron
TrueConc.(µg/L)22.932.514.456.632.624.831.631.725.032.095.031.250.327.9
MeanObservedConc.(µg/L)20.533.010.552.215.321.729.231.826.20.089.531.844.929.6
Std.Dev.(µg/L)0.81.10.92.92.20.72.31.01.30.03.91.29.41.4
RSD(%)3.33.36.35.16.82.97.33.15.10.04.13.818.85.2
MeanAccuracy(% ofTrue)89.6101.672.692.347.089.692.3100.4104.80.094.2101.989.3106.3
MDL(µg/L)2.53.73.19.87.4*2.47.73.34.4 *13.14.031.64.7
* Not recovered
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TABLE 5
ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS OF THE TARGET
COMPOUNDS IN ORGANIC-FREE REAGENT WATER USING SOLID-PHASE
EXTRACTION (C18 CARTRIDGE)a
Compound
Benzidine
Benzoylprop ethylCaffeine Carbaryl
o-Chlorophenyl thiourea3,3'-Dichlorobenzidine3,3'-Dimethoxybenzidine3,3'-Dimethylbenzidine Diuron
Ethylene thioureaLinuron MonuronRotenoneSiduron
a
TrueConc.(µg/L)22.932.514.456.632.65.031.631.725.032.095.031.250.327.9
MeanObservedConc.(µg/L)12.229.36.453.90.04.425.531.424.40.088.930.545.024.8
Std.Dev.(µg/L)1.72.01.41.80.00.41.81.01.40.04.82.92.42.0
RSD(%)13.76.921.43.30.010.07.13.15.60.05.49.65.47.9
MeanAccuracy(% ofTrue)53.290.244.295.20.089.680.899.097.60.093.697.889.688.9
MDL(µg/L)5.36.34.45.7*1.45.73.04.4*15.19.17.56.3
Reagent water contained 0.01 M ammonium acetate.
* Not recovered.
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TABLE 6
ACCURACY AND PRECISION DATA FROM SIX DETERMINATIONS OF THE TARGETCOMPOUNDS IN ORGANIC-FREE REAGENT WATER USING SOLID-PHASE EXTRACTION
(NEUTRAL POLYSTYRENE/DIVINYLBENZENE POLYMER DISK)
Compound
Benzidine
Benzoylprop ethylCaffeine Carbaryl
o-Chlorophenyl thiourea3,3'-Dichlorobenzidine3,3'-Dimethoxybenzidine3,3'-DimethylbenzidineDiuron
Ethylene thioureaLinuronMonuronRotenoneSiduron
TrueConc.(µg/L)22.932.514.456.632.65.031.631.725.032.095.031.250.327.9
MeanObservedConc.(µg/L)24.731.10.759.50.05.032.831.526.10.097.934.440.526.8
Std.Dev.(µg/L)2.43.00.54.70.00.52.22.11.80.08.72.56.01.0
RSD(%)9.89.672.57.90.09.46.76.77.00.09.07.314.83.6
MeanAccuracy(% ofTrue)108.095.85.2105.10.0101.7103.899.4104.50.0103.0110.480.596.1
MDL(µg/L)8.110.11.815.8*1.67.47.16.1*29.38.420.23.4
* Not recovered.
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TABLE 7
MEAN RECOVERIES, MULTI-LABORATORY PRECISION AND ESTIMATES OF SINGLEANALYST PRECISION FOR THE MEASUREMENTS OF FOUR BENZIDINES BY LC/PB/MS
10 µg/L Test Conc.Recovery(%)961049896
RSDMulti-lab10201418
RSDSingleAnalyst5.618109.4
100 µg/L Test Conc.Recovery(%)
97959797
RSDMulti-lab10108.69.1
RSDSingleAnalyst9.17.04.94.6
Compound
Benzidine
3,3'-Dimethoxybenzidine3,3'-Dimethylbenzidine 3,3'-Dichlorobenzidine
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FIGURE 1
AN UNACCEPTABLE CHROMATOGRAM WITH COLUMN BLEED
FIGURE 2
AN ACCEPTABLE CHROMATOGRAM FOLLOWING COLUMN FLUSHING
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FIGURE 3
MASS SPECTRUM OF DIMETHYLOCTADECYL-SILANOL, A COMMON STATIONARY PHASE BLEED PRODUCT
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FIGURE 4
TOTAL ION CHROMATOGRAM OF ANALYTES AND SURROGATES
(140-950 ng Injected)
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METHOD 8325
SOLVENT EXTRACTABLE NONVOLATILE COMPOUNDS BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/PARTICLE BEAM/MASS
SPECTROMETRY (HPLC/PB/MS)
CD-ROM8325 - 23
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