Document 6bQJbQn1JQmy5roLBDEQJ7er4

3M Environmental Laboratory AR116- ooz^ k) M ethod Analysis of Potassium Perfluorooctanesulfonate or O ther Fluorochemical Compounds in Urine E xtracts U sing H PL C -E lectrospray/M ass Spectrometry/M ass Spectrom etry Method Number: ETS-8-97.0 Adoption Date: Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Revision Date: Approved By: Laboratory Manager Group Leader ------ _ 7 / u /y 5 Date f/Z-37# Date - A C iu m -____________________ ______________________ Technical Reviewer Date 1.0 Scope and Application_________________________________________________ 1.1 Scope: This method describes the analysis of urine extracts for fluorochemicals using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemicals or other ionizable compounds. 1.3 Matrices: Human, rat, and monkey urine, or other fluids as designated in the validation report. Word 6/95 ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 1 o f 10 001059 2.Q Summary of Method 2.1 This method describes the analysis of fluorochemicals extracted from urine or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 Definitions________________________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has WindowsNT 4.0 versions. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx NT User's Guide). 4.0 Warnings and Cautions_____________ _________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 2 o f 10 001060 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_____________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment_________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials_____________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen regulated to approximately 100 psi. (House air system) 7.1.2 High purity grade argon regulated to approximately 6 psi. 7.1.3 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.4 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards____________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ASTM type I, or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 3 o f 10 001061 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and twenty matrix standards are prepared during the extraction procedure. See ETS-8-96.0. 9.0 Sample Handling_____________________________________________________ _ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control_________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a solvent blank, method blank, and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization__________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by regression (linear or otherwise, see 11.2), weighted 1/x, not forced through zero, with IS reference (surrogate is used as an internal standard) using MassLynx or other suitable software. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 4 o f 10 001062 11.2 Use the following parameters for determining a calibration curve for each compound^ Compound Weighting Regression Fit Response type PFOS 1/X Linear IS reference POAA 1/X Quadratic IS reference Suitability of curve tting parameters for other fluorochemical compounds will be addressed by the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 50 ppb of analyte, generate a calibration curve consisting of the standards from 25 ppb to 250 ppb rather than the full range of the curve (25 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures_______________________________________________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 5 o f 10 001063 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9.0 minutes 12.2.2.4 Solvent ramp = Time 0.00 min 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 min. MeOH 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Click on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 6 of 10 001064 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): (ne/mL of PFOS calc, from std. Curve x Dilution Factor) x 1 u.g______ Initial Volume of matrix (mL) 1000 ng Final Volume (mL) 14.0 Method Performance_____________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-96.0, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 7 o f 10 001065 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records__________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by a linear or quadratic fit, referenced to the internal standard (surrogate), weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software (Excel 7.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data________________________ 17.1 Attachment A: ETS-8-97.0 Data summary spreadsheet. 18.0 References_______________________________________________________________ 18.1 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.2 The validation report associated with this method is FACT-TOX-131, W2067 ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 8 o f 10 001066 19.0 Affected Documents 19.1 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 9 of 10 >1067