ALLOWAY STANDARD OPERATING PROCEDURES FOR METHOD 525.2

Transcription

1 ALLOWAY STANDARD OPERATING PROCEDURES FOR METHOD DETERMINATION OF ORGANIC COMPOUNDS IN DRINKING WATER BY LIQUID-SOLID EXTRACTION AND CAPILLARY COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY Marion, OHIO Originator: Section Supervisor: QA Manager: Date: Date: Date: Page 1 of 43

2 Section 1.0: Scope and Application This is a general purpose method that provides procedures for determination of organic compounds in finished drinking water, raw source water, or drinking water in any treatment stage. The method is applicable to a wide range of organic compounds that are efficiently partitioned from the water sample onto a C 18 organic phase chemically bonded to a solid inorganic matrix and sufficiently volatile and thermally stable for gas chromatography. Singlelaboratory accuracy and precision data have been determined at two concentrations for a number of different compounds. The method is used to determine the concentrations of the following compounds: ANALYTE CAS REGISTRY NUMBER Nominal MRL (g/l) Acetochlor Alachlor Atrazine Benzo(a) pyrene Bis (2-Ethylhexyl) adipate Bis (2-Ethylhexyl) phthalate Butachlor Hexachlorobenzene Hexachlorocyclopentadiene Metolachlor Metribuzin Propachlor Simazine Acenaphthene Acenaphthylene Anthracene Benzo(a) anthracene Benzo(b) fluoranthene Benzo(k) fluoranthene Benzo(g,h,i) perylene Chrysene Dibenz[a,h] anthracene Fluoranthene Fluorene Indeno[1,2,3-cd] pyrene Naphthalene Phenanthrene Pyrene Page 2 of 43

3 Section 2.0: Summary of Method Organic compound analytes, internal standards, and surrogates are extracted from a water sample by passing one liter of sample water through a cartridge containing about one gram of a solid inorganic matrix coated with a chemically bonded C 18 organic phase (solid-phase extraction, SPE). The organic compounds are eluted from the SPE cartridge with a small quantity of Methylene Chloride and Ethyl Acetate, and concentrated to 1.0 ml. The sample components are separated, identified, and measured by injecting a 1.0 µl aliquot of Ethyl Acetate extract onto a high resolution fused silica capillary column of a GC/MS unit. Compounds eluting from the GC column are identified by comparing their measured mass spectra and retention times to reference spectra and retention times in a data base. Reference spectra and retention times for analytes are obtained by the measurement of calibration standards under the same conditions used for samples. The concentration of each identified component is measured by relating the MS response of the quantitation ion produced by that compound to the MS response of the quantitation ion produced by a compound that is used as an internal standard. Section 3.0: Definitions 3.1 Acceptance Criteria Specified limits placed on characteristics of an item, process, or service defined in required documents. 3.2 Analytical Batch Composed of prepared environmental samples which are analyzed together as a group. It can include prepared samples originating from various environmental matrices and can not exceed 20 samples. 3.3 Calibration To determine, by measurement or comparison with a standard, the correct value of each scale reading on a meter, instrument, or other device. The levels of the applied calibration standard should bracket the range of planned or expected sample measurements. 3.4 Calibration Curve The graphical relationship between the known values, such as concentrations, of a series of calibration standards and their instrument response. 3.5 Calibration Standard (CAL) -- A solution prepared from the primary dilution standard solution. The CAL is used to calibrate an instrument. 3.6 Field Blank or Trip Blank A blank prepared in the field or laboratory by filling a clean container with pure de-ionized water and appropriate preservative, if any, for the specific sampling activity being undertaken. 3.7 High Quality Pure Reagent Water Water in which no target analytes or interferences are detected as required by the analytical method. Page 3 of 43

4 3.8 Holding Times (Maximum Allowable Holding Times) The maximum times that samples may be held prior to analysis and still be considered valid or not compromised. 3.9 Internal Standard A known amount of standard which is added to a test portion of a sample as a reference for evaluating and controlling the precision and bias of the applied analytical method Method Blank (MB) -- An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with other samples. The MB is used to determine if method analytes or other interferences are present in the laboratory environment, the reagents, or the apparatus Laboratory Fortified Blank (LFB) -- An aliquot of reagent water to which a known quantity of the method analyte is added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether the method is in control, and whether the laboratory is capable of making accurate and precise measurements at the required method detection limit Matrix The component or substrate that contains the analyte of interest. 3. Matrix Spike (MS) or Laboratory Fortified Sample Matrix (LFSM) An aliquot of a field sample to which known quantities of the method analytes and the preservation compounds are added. The LFSM is processed and analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix must be determined in a separate aliquot and the measured values in the LFSM corrected for background concentrations Matrix Spike Duplicate (MSD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD) A second aliquot of the field sample used to prepare the LFSM, which is fortified, extracted and analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate to access method precision and accuracy when the occurrence of a method analyte is infrequent Field Duplicates (FD1 and FD2) Two separate samples collected at the same time and place under identical circumstances, and treated the same throughout field and laboratory procedures. Field Duplicates are used to estimate the precision associated with sample collection, preservation, and storage, as well as with laboratory procedures Procedural Standard Calibration - A calibration method where aqueous calibration standards are prepared and processed (e.g., purged, extracted, and/or derivatized) in Page 4 of 43

5 exactly the same manner as a sample. All steps in the process from addition of sampling preservatives through instrumental analyses compensates for any inefficiencies in the processing procedure Quality Control The overall system of technical activities whose purpose is to measure and control the quality of a product or service so that it meets the needs of users Raw Data Any original factual information from a measurement activity or study recorded in a laboratory notebook, worksheets, records, memoranda, notes, or exact copies thereof that are necessary for the reconstruction and evaluation of the report activity Reference Method A method of known and documented accuracy and precision issued by an organization recognized as competent to do so Reporting Limit Check standard (RLC) A check standard analyzed at the reporting level of the compounds of interest Standard Operating Procedures (SOPs) A written document which details the method of an operation, analysis or action whose techniques and procedures are thoroughly prescribed and which is accepted as the method for performing certain routine or repetitive tasks Stock Standard Solution (SSS) A concentrated solution containing a single certified standard that is a method analyte, or a concentrated solution of a single analyte prepared in the laboratory with an assayed reference compound. Stock standard solutions are used to prepare primary dilution standards Surrogate A substance with properties that mimic the analyte of interest. It is unlikely to be found in environmental samples and is added to them for quality control purposes. Calculated as percent recovery QCS / Calibration validation -- A sample prepared using a PDS of method analytes that is obtained from a source external to the laboratory and different from the source of calibration standards. The QCS is used to check calibration standard integrity. Section 4.0: Interferences 4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing apparatus that lead to anomalous peaks or elevated baselines in liquid chromatograms. The Method Blank is used as a tool to monitor for the presence of interferences. Page 5 of 43

6 4.2 Glassware must be carefully cleaned as detailed in the laboratory s Standard Operating Procedure for Glassware Preparation. 4.3 Interfering contamination may occur when a sample containing low concentrations of compounds is analyzed immediately after a sample containing relatively high concentrations of compounds. Syringes and injection port liners must be cleaned carefully or replaced as needed. After analysis of a sample containing high concentrations of compounds, a solvent blank or a Method Blank is analyzed to ensure that accurate values are obtained for the next sample. If suspicion of carryover exists, reanalysis of the affected samples is performed. 4.4 Samples and standards must be contained in the same solvent to ensure chromatographic comparability. Section 5.0: Safety 5.1 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been precisely defined. However, each one must be treated as a potential health hazard, and exposure to these chemicals should be minimized. Each analyst is responsible for adherence to the procedures outlined in the Chemical Hygiene Plan. Material Safety Data Sheets are stored in the Quality Assurance section of the laboratory. 5.2 Some method analytes have been tentatively classified as known or suspected human or mammalian carcinogens. Pure standard materials and stock standard solutions of these compounds should be handled with suitable protection to skin, eyes, etc. 5.3 Safety glasses, gloves, and lab coats must be worn when handling samples and solvents. Safety glasses must be worn when handling glassware and chromatographic columns. Safety glasses must be worn when handling equipment within the laboratory. Section 6.0: Equipment and Supplies 6.1 Sample Bottles: 1-L amber glass bottles fitted with Teflon-lined screw caps. 6.2 Glassware Syringes, gas tight (Hamilton or equivalents) dedicated for standard preparation µL µL µL µL µL Page 6 of 43

7 6.2.2 Volumetric Flasks (Class A,TC) 1. Pyrex 1.0 ml 2. Pyrex 5.0 ml 3. Pyrex 10.0 ml 4. Pyrex 1000 ml Glass funnels Test Tubes/Centrifuge Tubes 6.3 Clear GC vials 2 ml used for sample extracts 6.4 Amber vials 15 ml used for standards 6.5 Agilent 250µL silanized flat bottom inserts 6.6 Kimble vial caps 11mm, PTFF/Rubber 6.7 Balance Analytical, capable of accurately weighing to the nearest 0.01 g. 6.8 Muffle Furnace 6.9 N-EVAP 6.10 Thermometer 6.11 Permanent ink marker 6.12 Prep sheet 6. Crimper 6.14 Rubber pipette bulb 6.15 Disposable Pasteur pipettes 6.16 ph paper 6.17 Bottle top dispenser for solvent bottles 6.18 SPE cartridge extraction manifold system - Phenomenex or equivalent, 12-position, rotary vane vacuum pump, with water collection vessel. Page 7 of 43

8 6.19 Gas Chromatograph/Mass Spectrometer (1) VARIAN 3900 Gas Chromatograph, VARIAN CP-8400 autosampler, Saturn 2100T GC/MS ion trap detector, and Workstation 5.52 software Column Restek Rxi-5ms (or equivalent), 30 m, 0.25 mm ID fused silica capillary column coated with a 0.25 µm bonded film of Polyphenyl-methyl silicone. The temperature range is from -60 ºC to 325 ºC. The column is conditioned at 320 ºC for 90 minutes Solid-Phase Extraction (SPE) Cartridges inert non-leaching plastic that are packed with 1 g of Ocetadecyl (C 18 ) groups chemically bonded to silica substrates. Thermo Scientific (product # ) or equivalent. Section 7.0: Reagents and Calibration Standards 7.1 High Quality Reagent Water 7.2 Methylene Chloride - high purity pesticide quality 7.3 Methanol - high purity pesticide quality 7.4 Ethyl Acetate - high purity pesticide quality 7.5 Helium - carrier gas 7.6 Hexane high purity pesticide quality 7.7 Hydrochloric acid, (37%) 7.8 Ascorbic Acid Solution See Alloway s reagent traceability software for the preparation of this solution. 7.9 Stock Standard Solutions (SSS) Solutions of surrogates, internal standards and target analytes are purchased from various commercial vendors at appropriate concentrations. The lab does not prepare any compounds for Method analyses from pure materials All stock standard solutions, when received, are logged into the reagent traceability software program. Page 8 of 43

9 7.9.3 All ampules for standards are of amber glass and stored at 6 ºC. Stock standard solution that is not immediately used for standard preparation is saved in an amber vial and also stored at 6 ºC. Appropriate information is written and/or referenced onto this vial Compound purity of all stock standard solutions purchased from commercial vendors for Method is greater than 99.9% Fortification Solution of Internal Standard and Surrogates The lab purchases SSS of internal standard (Acenaphthene-d10, Phenanthrened10, and Chrysene-d12) / surrogate (1,3-Dimethyl-2-nitrobenzene, Triphenylphosphate, and Perylene-d12) mix at a concentration of 500 µg/ml in Acetone. Note: the fortification solution must be made with Acetone and cannot contain the eluting solvents Methylene Chloride and/or Ethyl Acetate This mix is diluted by a factor of ten by adding 1.0 ml of each standard to 7.0 ml of acetone in a 10.0 ml volumetric flask. This solution is then brought to a final volume of 10.0 ml with Acetone. The final concentration, for all six compounds, is 50.0 µg/ml This solution is given a standard preparation ID number in the Reagent Traceability Program and stored in a 15-mL amber vial at 6 ºC. A laboratory technician will add 100 µl of this solution to each sample, which assuming a 100% extraction efficiency, will give a concentration of 5.0 µg/ml in the 1.0 ml final extract Tuning Solution Standard A stock standard solution is purchased from a commercial vendor, with the compound and concentrations as found in the following table, for the purpose of checking the performance of the GC/MS system. Page 9 of 43

10 Concentrations in purchased Stock Standard Mix Compound Concentration (µg/ml) Anthracene 100 Benzo(a)anthracene 100 Chrysene 100 Phenanthrene 100 4,4 -DDT 1000 Endrin 1000 DFTPP See Alloway s reagent traceability software for the preparation of this standard Calibration Solutions There are four SSS used to make the solutions for the initial calibration at ten different levels, 0.01, 0.05, 0.10, 0.20, 0.50, 0.75, 1.00, 1.25, 1.50, and g/ml. The levels for Bis(2-Ethylhexyl)phthalate are 0.05, 0.25, 0.50, 1.00, 2.50, 3.75, 5.00, 6.25, 7.50, and 10.0-g/mL The PAH calibration curves are generated from a separate set of standards at 7 different levels: 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0-g/mL. See the Reagent traceability software for details on the preparation of these standards A custom mix is obtained that contains all analytes at 100-g/mL in Acetone except for Metribuzin. Metribuzin is obtained separately in Methanol at a concentration of 100-g/mL. Bis(2-ethylhexyl) phthalate is at 500-g/mL. Note: Metribuzin is unstable in Acetone and must be ordered as a separate mix The IS/Surrogate solution described in Section 7.10 is also used. Note that the IS/Surrogate concentration in all calibration solutions must be the same at 5.0 µg/ml Recovery Standard p-terphenyl-d14 at 500-g/mL is used to monitor the recovery of the internal standards See Alloway s reagent traceability database for the preparation of the calibration standards. Page 10 of 43

11 7. LFB / Matrix Spike Fortification Solution mL of the SSS custom mix (referenced in Section at 100 g/ml), 1-mL Metribuzin (at 100 g/ml in Methanol), is added to 5-mL of Acetone in a 10-mL volumetric flask. This solution is then brought to a final volume of 10-mL with Acetone. The final concentration for all compounds in this solution is 10.0 g/ml with the exception of Bis(2-Ethylhexy) phthalate which is 50-g/mL This solution is given a standard preparation ID number in the Reagent Traceability Software Program and stored in a 10-mL amber vial at 6 C. The laboratory technician will add this solution to the LFB s and Matrix Spike / Matrix Spike Duplicates as proscribed in Section and This will give a final concentration of 1.0-g/mL for LFB-1, LFB-2, and the Matrix Spike / Matrix Spike Duplicate For the PAH Spiking solution add 0.5-mL of the 500-g/mL stock standard in 5- ml Acetone. Take to volume with acetone. This yields a stock solution with a concentration of 50-g/mL For the LFB-3 Fortification Solution, this solution corresponds to a spike at the RL check for each of the target analytes, Refer to the reagent tractability software for instructions on the preparation of the LFB-3 fortification solution Report Limit Check Standard - This standard is used to evaluate the sensitivity of the instrument with respect to the PQL levels for each of the analytes. The concentration of this standard corresponds to the PQL for each of the analytes. Refer to the Reagent Traceability software for the preparation of this standard. Section 8.0: Sample Collection, Preservation, Shipment, and Storage 8.1 When sampling from a water tap, open the tap and allow the system to flush until the water temperature has stabilized. 8.2 All samples should be iced or refrigerated to 6 ºC from the time of collection until the time of extraction. 8.3 Residual chlorine should be reduced by the addition of Sodium sulfite to each water sample. Sodium sulfite is added by the lab before sending the bottles to clients. Hydrochloric acid is shipped with the sample bottles and is added at the sampling site to retard the microbiological degradation of some analytes in unchlorinated water. Page 11 of 43

12 8.4 The samples must be extracted within 14 days of collection. The extracts must be analyzed within 30 days of sample extraction. Section 9.0: Calibration and Standardization 9.1 Before sample analysis can begin, three general conditions must be met First, the analysis of DFTPP and several other compounds is done to assure that the GC/MS system meets the various method requirements described in Sections 9.1 through 9.6. This analysis is often called a Tune and is performed using the solution described in Section No analysis of calibration and/or samples is considered valid if the tuning criteria are not met first. If all tune criteria are met, the tune is considered valid for a maximum of 12.0 hours. After 12 hours, the tune must be repeated and meet all criteria before any analysis can continue. The criteria for the DFTPP are as follows: Mass (M/z) Relative Abundance Criteria Purpose of Checkpoint 51 10% - 80% of the Base Peak Low-mass Sensitivity 68 <2% of Mass 69 Low-mass Sensitivity 70 <2% of Mass 69 Low-mass Sensitivity % - 80% of the Base Peak Low to Mid-mass Sensitivity 197 <2% of Mass 198 Mid-mass Resolution 198 Base Peak of >50% of Mass 442 Mid-mass Resolution and Sensitivity 199 5% - 9% of Mass 198 Mid-mass Resolution and Isotope Ratio % - 60% of the Base Peak Mid to High-mass Sensitivity 365 >1% of the Base Peak Baseline Threshold 441 Present and < Mass 443 High-mass Resolution 442 Base Peak of >50% of Mass 198 High-mass Resolution and Sensitivity % - 24% of Mass 442 High-mass Resolution and Isotope Ratio Note: The Tune Criteria are generated from the DFTPP peak at the apex or an average of 3 or 5 consecutive scans across the DFTPP peak and must be centered on the peak apex with background subtraction The Mass Selective Detector (MSD) must be tuned if: the detector has just been cleaned, a filament has broken and the back up filament will be used, or the tuning check has failed twice in a row Second, demonstration and documentation of an acceptable initial calibration is required. This initial calibration consists of either the ten calibration standards described in Section or the seven calibration standards described in Section Page 12 of 43

13 All calibration solutions must be analyzed within the same 12-hour window of a valid tune. The primary consideration for an initial calibration is linearity of each compound through the range of calibration. Calibration is required at least every three months or if the continuing calibration check fails repeatedly, whichever occurs first. Calibration is also required whenever extensive maintenance is required on the instrument, i.e. changing the column, filament, detector, etc Third, demonstration and documentation of acceptable continuing calibration is required. This continuing calibration must include at least one analysis of a known concentration preferably at or near the mid-point of the initial calibration. The exact concentration chosen should vary for each 12 hour tune clock window. This continuing calibration should follow immediately after an acceptable tune to assure maximum use of the 12 hour tune clock window The lab uses 0.5-µg/mL through 1.25-µg/mL calibration standards to verify the calibration curve An additional continuing calibration standard must be analyzed to demonstrate system sensitivity at the minimum reporting level. 9.2 A calibration standard is analyzed at the end of each tune clock window to bracket the samples with a standard. 9.3 In addition to the criteria listed in Section 9.1, the degradation of Endrin and 4,4 -DDT into their respective breakdown products must not exceed 20%. If the breakdown for Endrin and/or 4,4 -DDT exceeds 20%, then GC port maintenance must be done. This maintenance usually includes but is not limited to changing the injection port septa, replacing the injection port liner (silanized glass gooseneck with 2 mm i.d.), and replacing the gold seal. Clipping 6 to 12 inches off the capillary column is also recommended To calculate Endrin and 4,4 -DDT breakdown, first locate them and their breakdown components at their retention times (RT) and confirm using the mass spectra ions in Section Then use the following calculations to ascertain that the breakdown(s) do not exceed 20%: ,4 -DDT breakdown is calculated as follows: Page of 43

14 Sum of TIC area of degradation peaks (DDEDDD) % 4,4' DDT Breakdown 100 Sum of TIC areas of all three peaks (DDT DDE DDD) Endrin breakdown is calculated as follows: Sum of TIC area of degradatio n peaks (EAEK) % Endrin Breakdown 100 Sum of TIC areas of all three peaks (Endrin EA EK) 9.4 Phenanthrene and Anthracene should be separated by baseline. Their RTs are approximately 9 minutes +/- 0.2 minutes. If their respective peaks do not resolve to the baseline, column replacement is probably necessary. 9.5 Benzo(a)anthracene and Chrysene should be separated by a valley whose height is less than 25% of the average peak height of these two compounds. The RT for these two compounds is approximately 15 minutes +/- 0.2 minutes. If the valley exceeds 25%, column replacement is probably necessary. 9.6 The required conditions for the GC/MS are listed in Section Initial Calibration Once all conditions are met for the tune, an initial calibration can be performed within the 12 hour tune clock. The lab utilizes ten calibration levels of 0.01 µg/ml through 2.0 µg/ml, as described in Section For PAH calibration, the lab utilizes seven calibration levels of 0.1 µg/ml through 10.0 µg/ml. The chromatographic and mass spectrometer conditions are the same as the tune. Depending on the curve, these analyses are then used to obtain a response factor (RF) for each compound and surrogate. The RF calculation is as follows and is performed by the Saturn software: RF Q Ax is A Qx is Where: A x = integrated abundance of the quantitation ion of the analyte. A is = integrated abundance of the quantitation ion internal standard. Q x = quantity of analyte injected in ng or concentration units. Q is = quantity of internal standard injected in ng or concentration units. Page 14 of 43

15 9.7.2 From these RFs, a mean RF for each target compound and surrogate is obtained and used to calculate the relative standard deviation (RSD) from the mean. If the RSD of any analyte or surrogate exceeds 30%, a linear regression (A x /A is versus Q x ) may be used if the correlation coefficient (r) is greater than or equal to with the coefficient of determination (r2) from the instrument being greater than or equal to A quadratic regression model is not to be used QCS / Calibration Validation Standard Once the curve meets the criteria specified in Section A Quality Control Standard is analyzed immediately following the generation of the calibration curve and for each daily run. This standard is obtained from a source that is separate from that used to prepare the calibration standard, and is prepared at a level that corresponds to the midpoint of the calibration curve. This standard is used to validate the calibration standards. The recovery limits are identical to that of the calibration Check Standard (70-0%). 9.8 Continuing Calibration Before the continuing calibration standard is analyzed, perform a tune check to meet the conditions described in Sections 9.1 through 9.6. If the tune check meets the criteria, using the same GC/MS conditions as the initial calibration. The concentration is varied over the course of time. Alloway then analyzes a calibration verification/qcs/ccv (second source) standard The CCV must meet the following conditions before sample analysis can begin The surrogate recoveries must be within 70% - 0% If all the analytes in the CCV meet the limits of 70-0% then analysis may proceed and results reported If any analyte in the CCV fails to meet acceptable recovery (high) then the analyst must ascertain that the target analyte is not detected in the sample. If the compound is not detected the result is reported without qualification If any analyte in the CCV fails to meet acceptable recovery (high) and the results of the samples are detected at or above the reporting level the CCV and the samples need to be re-analyzed once acceptable recovery is achieved either by acceptance of a new calibration curve or corrective action. Page 15 of 43

16 If the recovery is low, indicating a low bias and/or the compound is detected, then the result must be considered suspect and the CCV and sample re-analyzed. Results for re-analyzed samples that exceed regulatory limits may be reported with the following qualification: The CCV was below the lower limit indicating a low bias. This result exceeds the regulatory limit and is considered a reportable value under the TNI standards If the re-analyzed CCV is exceeded low <70%, this indicates a low bias. Results for samples that do not exceed a regulatory limit need to be reanalyzed following the acceptance of a new calibration curve or following corrective action The internal standard and surrogate abundances must be checked against the initial calibration and/or the immediately previous CCV. These abundances must be 50% against the initial calibration curve mean abundances or 30% against the immediate previous continuing calibration check The concentration of the continuing calibration should be within the midrange of the initial calibration and vary for each 12 hour work shift If the response if a target analyte(s) is above that of the highest standard in the Initial Calibration curve, then the sample must be diluted so that the response of the sample falls within the range of the Initial Calibration. 9.9 Reporting Limit Check Standard (RLC) If all of the continuing calibration criteria are met, then the Reporting Limit Check standard (RLC) must be analyzed prior to the analysis of field samples The analysis of these analytes should give recoveries of 50% - 150% of the actual concentration. The analyst must ascertain how any target compounds outside the recovery criteria will impact continued sample analysis. Of particular concern would be the ability to see these low concentrations in the samples if the corresponding recovery is very low in the RLC The recoveries of the analytes should meet the criteria above (9.9.2); if the recovery is high and no target analytes are detected in the sample then the results can be reported. If the recovery is low then the system does not meet the required sensitivity and analysis cannot proceed until corrective action is taken to restore system sensitivity. If the recovery is high and Page 16 of 43

17 there is detection then two conditions must be considered. Is the detection greater than the level in the CCV? If so and the CCV is within limits the result can be reported. If the level in the sample less than the level in the CCV and greater than in the RL checks, then the sample must be reanalyzed when system sensitivity meets proper MRL QC requirements Closing CCV A midlevel calibration standard is analyzed at the end of the 12-hour analytical run and serves to bracket the samples and should meet the requirements of Section Section 10.0: Quality Control 10.1 Minimum quality control (QC) requirements are: initial demonstration of laboratory capability and method detection limit study, followed by regular analyses of method blanks (MBs), laboratory fortified blanks (LFBs), and laboratory fortified matrix samples (LFMs). The recoveries of surrogates and internal standards in all quality control and client samples must also be monitored Initial Demonstration of Capability (IDOC) Four laboratory fortified blanks (LFBs) must be analyzed at a concentration at the middle of the calibration range (1.0 µl). The LFBs must be extracted and analyzed according to Sections 11.0 through Calculate the measured concentration of each analyte in each replicate, the mean concentration of each analyte in all replicates, and mean accuracy (expressed as percentage of true value), and the precision (as relative standard deviation, RSD) of the measurements for each analyte For each analyte and surrogate, the mean accuracy should be 70% - 0%, and the RSD should be < 30%. If these criteria are not met, the source of the problem must be located, and fresh LFBs must be analyzed The initial demonstration of capability is used primarily to preclude a laboratory from analyzing unknown samples via a new, unfamiliar method prior to obtaining some experience with it. It is expected that as laboratory personnel gain experience with this method the quality of data will improve beyond those required here. Page 17 of 43

18 10.3 Method Detection Limit Study (MDL) Seven laboratory fortified blanks (LFBs) must be analyzed at a low concentration level (at the PQL of each analyte i.e. LFB-3). The analyses should be performed over a period of several days to produce realistic method detection limits. The LFBs must be extracted and analyzed according to Sections 11.0 through.0. The MDL is calculated using the equation in section As an alternative, the results of seven LFB-3 extracts (from 7 consecutive extraction batches) may be used to generate an MDL study. Refer to Alloway Method 5546 Determination of Method Detection Limits for a more thorough explanation of study requirements Method Blank (MB) -- Before processing any samples, the analyst must demonstrate that all glassware and reagent interferences are under control. Each time a set of samples is extracted within a 12 hour shift or reagents are changed, an MB must be included An MB must also be analyzed any time a new supply of cartridges is received from a supplier. It must be demonstrated that the MB is reasonably free of contamination that would prevent the determination of any analyte of concern. It must also be demonstrated that the particle size and packing of the SPE cartridges are acceptable. Consistent flow rate with all samples is an indication of acceptable particle size distribution, packing, and proper preparation If the MB falls below the reporting levels, samples can be reported If the MB is detected at or above the reporting level, samples that are below the reporting level can be reported with a qualification statement If the MB is detected at or above the reporting level, instrument results that are 10 times higher than the method blank instrument result can be reported. Use the following qualification statement: Analyte detected in the method blank. Analyte detected in the method blank is of the amount detected in this sample. Evaluate data accordingly If the method blank is detected at or above the reporting level, instrument results that are not 10 times higher than the method blank result should be re-analyzed or if reported the result must be qualified in the LIMS with the following statement: Analyte detected in the method blank >1/10 of the amount detected in this sample. Evaluate data accordingly. Page 18 of 43

19 The MB is considered to the clean if the analytes are below their respective Reporting Limits for the samples Assessing Laboratory Performance -- Laboratory Fortified Blank (LFB) Three LFBs are analyzed with every analytical batch of samples. A batch of samples must contain 20 or fewer client samples. Two LFBs are analyzed at a concentration of 1.0-µg/mL to demonstrate precision. The third LFB is an LFB at a concentration that corresponds to the PQL for each of the analytes The recoveries of the analytes should meet the criteria of 70% - 0%, except for LFB-3 which should meet % of expected value If the LFB meets limits results can be reported If the initial LFB fails (70-0% or % for LFB3) the LFB can be reanalyzed a second time If the re-analyzed LFB meets requirements 70-0% (50-150% for LFB3) analysis may proceed If the re-analyzed LFB is exceeded high >0% (150% for LFB3) and results of samples are below the reporting level the results of samples can be reported. Use the following qualification statement in the LIMS: (The LFB was above the upper limit indicating a high bias. This non-detect result is not affected by the high bias of the LFB) If the re-analyzed LFB is exceeded high >0% (150% for LFB3) and results of samples are detected at or above the reporting level the samples need to be re-analyzed following acceptance of a new calibration curve or following corrective action (unless the marginal Exceedance criteria is met) If the re-analyzed LFB is exceeded low < 70% (50% for LFB3) this indicates a low bias. Results for samples that exceed a regulatory limit may be reported. Use the following qualification Page 19 of 43

20 statement in the LIMS: The LFB was below the lower limit indicating a low bias. This result exceeds the regulatory limit and is considered a reportable value under the TNI standards If the re-analyzed LFB is exceeded low < 70% (50% for LFB3) this indicates a low bias. Results for samples that do not exceed a regulatory limit need to be re-analyzed following acceptance of a new calibration curve or following corrective action (unless the marginal Exceedance criteria is met) LFB Marginal Exceedance (ME) between 3 and 4 standard deviations around the mean (upper and lower). The following table documents the number of Marginal Exceedances (ME) that is acceptable for the specific number of analytes present in the LFB. Use the following qualification statement in the LIMS for each analyte that is determined to be a marginal Exceedance: The LFB was (above the upper limit or below the lower limit) but met the Marginal Exceedance requirement therefore data is reportable. Number of Analytes in LFB Number of Allowed Marginal Exceedances > <11 0 NOTE: The State of Ohio does not recognize Marginal Exceedance Assessing Method Performance -- Laboratory Fortified Sample Matrix The laboratory must add a known concentration to a minimum of at least one sample per analytical batch. A batch of samples must contain 20 or fewer client samples. LFMs are analyzed at a concentration of 1.0 µg/ml. The LFMs must be extracted and analyzed according to Sections 11.0 through The recoveries of the compounds should be within the control limits established in Section 10.5 for LFBs. However, if the recovery of any analyte is not within the limits and the LFBs for the batch demonstrate that the laboratory is in control, Page 20 of 43

21 the sample is reported with the following qualification statement: The matrix spike fell outside method limits (biased high) or (biased low). The result for this sample should be evaluated accordingly Assessing Method Performance -- Field Duplicate or Laboratory Fortified Sample Matrix Duplicate (FD or LFSMD) Within each extraction batch, a minimum of one field duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD) must be analyzed. Duplicates check the precision associated with sample collection, preservation, storage, and laboratory procedures. If target analytes are not routinely observed in Field samples, then a LFSMD should be analyzed rather than an FD Calculate the relative percent difference (%RSD) for duplicate measurements (FD1 and FD2) using the equation, Standard Deviation of FD1and FD2 % RSD 100 FD1 FD %RSDs for Field duplicates should be 30%.If the %RSD for any analyte falls outside the range, and the laboratory performance for that analyte is shown to be in control in the LFB, the recovery is judged to be matrix biased. The result should then be flagged on the report as suspect due to matrix effect If an LFSMD is analyzed instead of a Field Duplicate, calculate the %RSD for the duplicate LFSMs (LFSM and LFSMD) using the equation, Standard deviation of LFSM and LFSMD % RSD 100 LFSM LFSMD %RSDs for the duplicate LFSMs should be 30%. If the %RSD / Recovery falls outside the acceptance range, and the laboratory performance for that analyte is shown to be in control in the LFB, the recovery is judged to be matrix biased. The result for that analyte in the unfortified sample is labeled suspect/matrix and flagged as such on the report with the following qualification statement: The matrix spike duplicate fell outside method limits and the result for this sample should be evaluated accordingly. Page 21 of 43

22 10.8 Assessing Surrogate Recovery The following surrogates are monitored by the laboratory for this method: 1,3- dimethyl-2-nitrobenzene, triphenylphosphate, and perylene-d12. The recoveries of all three surrogates must meet the acceptance criteria of 70% - 0% in both QC samples and field samples Assessing Internal Standard Recovery The following internal standards are monitored by the laboratory for this method: Acenaphthene-d10, Phenanthrene-d10, and Chrysene-d12. The recoveries of all three internal standards must be in excess of 70% of the immediately previous CCC or in excess of 50% of the recoveries of the Initial Calibration. Note that p- Terphenyl-d14 is added to extract to assess surrogate recovery Generation of Reference Spectra Reference Spectra are generated from the analysis of a mid-point standard. The reference spectra are loaded into the data analysis software (Varian Saturn Method Editor, see Saturn software manual or help files for details on how to do this). Reference spectrum should be updated every six months Identification of Analytes Identify a sample component by comparison of its mass spectrum (after background subtraction) to a reference spectrum in the user-created data base. The GC retention time of the sample component should be within five seconds of the retention time observed for that same compound in the most recently analyzed continuing calibration check standard In general, all ions that are present above 10% relative abundance in the mass spectrum of the standard should be present in the mass spectrum of the sample component and should agree within absolute 20%. For example, if an ion has a relative abundance of 30% in the standard spectrum, its abundance in the sample spectrum should be in the range of 10% - 50%. Some ions, particularly the molecular ion, are of special importance, and should be evaluated even if they are below 10% relative abundance Identification is hampered when sample components are not resolved chromatographically and produce mass spectra containing ions contributed by more than one analyte. When GC peaks obviously represent more than one sample component (i.e., broadened peak with shoulder(s) or valley between two or more maxima), appropriate analyte spectra and background spectra can be selected by examining plots of characteristic ions for tentatively identified components. When analytes co-elute (i.e., only one GC peak is apparent), the Page 22 of 43

23 identification criteria can be met but each analyte spectrum will contain extraneous ions contributed by the co-eluting compound Structural isomers that produce very similar mass spectra can be explicitly identified only if they have sufficiently different GC retention times. Acceptable resolution is achieved if the height of the valley between two isomer peaks is less than 25% of the average height of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs. Benzo[b]fluoranthene and Benzo[k]fluoranthene may be measured as an isomeric pair Each multi-component analyte can be identified by the presence of its individual components in a characteristic pattern based on the relative amounts of each component present. Chromatograms of standard materials of multi-component analytes should be carefully evaluated, so that these patterns can be recognized by the analyst If any detection(s) are noted in a client sample, the spectra for the detection(s) are printed out along with the appropriate reference spectra. This is done for comparison purposes and will also help facilitate secondary review due to all of the appropriate spectra being present in hard copy form The Primary and Secondary ions used for qualitative identification of analytes are as follows: Page 23 of 43

24 Analyte Primary ion Secondary ion Tune Compounds 4,4 -DDT , 165 4,4 -DDE , 318 4,4 -DDD , 165 Endrin , 81 Endrin Aldehyde , 345 Endrin Ketone , 1 Target Compounds Hexachlorocyclopentadiene , 235, 0 Propachlor , 77, 169 Hexachlorobenzene , 282, 142 Simazine , 8, 201 Atrazine , 172, 8 Acetochlor , 174, 2 Metribuzin , 182, 199 Alachlor , 146, 2 Metolachlor , 163, 146 Butachlor , 188, 146 Di(2-Ethylhexyl)adipate , 101, 55 Bis(2-Ethylhexyl)phthalate , 150, 105 Benzo(a)pyrene , 253, 1 Propachlor , 77, 169 Acenaphthene ,152,151 Acenaphthylene ,150,126 Anthracene ,177,152 Benzo(a) anthracene ,226,114 Benzo(b) fluoranthene ,250,126 Benzo(k) fluoranthene ,126,250 Benzo(g,h,i) perylene 276 8,277,7 Chrysene ,229,1 Dibenz[a,h] anthracene , 9, 8 Fluoranthene ,101,201 Fluorene ,164,163 Indeno[1,2,3-cd] pyrene 276 8, 277, 7 Naphthalene ,127,129 Phenanthrene ,179,152 Pyrene ,200,203 Internal Standards Acenaphthene-d , 160, 163 Phenanthrene-d , 189, 184 Chrysene-d , 236, 241 Surrogates 1,3-dimethyl-2-nitrobenzene 4 77, 106, 79 Triphenylphosphate , 327, 169 Perylene-d , 265, 2 Page 24 of 43

25 Section 11.0: Analytical Procedures 11.1 Extraction The overall extraction procedure of a sample for EPA Method is a four step process: sample prep, column conditioning, drawing sample through SPE column, and drying and concentration of the sample extract Mark bottle with a marking pen or grease pen to indicate the level of the sample in the bottle for future determination of sample volume used in the extraction. Do the same for all the QC sample bottles Sample Prep: Add 1000 ml of reagent water to four amber bottles. Mark the bottles as Blank, LFB-1, LFB-2, and LFB Add hydrochloric acid to each bottle until a ph of <2 is reached Allow samples to sit for approximately 10 minutes before adding Methanol and Ascorbic Acid solution Add 5 ml of Methanol to each sample Add 2 ml of Ascorbic Acid solution to each sample Allow sample to sit for approximately 10 minutes before adding fortification solutions Add 100 μl of the Internal Standard/Surrogate Fortification Standard to every sample and QC samples Add 100 μl of the Target Analyte Standard to LFB-1, LFB-2, and Matrix Spike Sample, and add 10 μl of the LFB-3 Spike Standard to LFB-3 sample If the sample involves PAHs, add 20 μl of the PAH Spiking Solution to PAH LFB-1, LFB-2, and PAH Matrix Spike sample. Add 2 μl of the PAH Spike Solution to PAH LFB-3 sample If the sample is an effluent or does not look like it will draw through the column, check with the analyst or Lab Manager a 1:10 dilution Page 25 of 43

26 Column Conditioning: may be able to be performed on the sample prior to extraction (only if client detection limits allow) Place SPE columns in extraction manifold Turn on the vacuum pump and set to approximately 10 psi Fill the column with Ethyl Acetate and draw through the column Fill the column with Methylene Chloride and draw through the column Make sure that all the Methylene Chloride is completely drawn through the column and column is dry before proceeding. Note: ensures that the column is free from potential contamination from column manufacture Fill the column with Methanol and draw through sorbant until about 0.25 inch remains above the sorbant. DO NOT allow the Methanol to be drawn completely through the sorbant nor allow the sorbant to go dry from this point on until the sample is completely drawn through the SPE column. Note: The Methanol activates the sorbant material for sample extraction Repeat except with dechlorinated reagent water twice to equilabrate column Drawing samples through the SPE columns: Place samples around the extraction manifold Connect Teflon tube with stopper into the open end of column and the other end of the Teflon tube into the sample. Use caution to not allow the Teflon tubing to come into contact with lab benches, walls, counters, lab coats, etc. Page 26 of 43

27 Turn on the valve below the SPE column on the manifold and completely draw the sample through the SPE column Once the sample is completely drawn through the SPE column, turn the valve off. Note: DO NOT allow a vacuum to remain on (after the sample has drawn through the column) for more than 10 minutes. Doing so results in the loss of analytes With a Kimwipe, tamp out any remaining moisture from the column above the sorbant. Turn on vacuum pump for another couple of minutes to finish drying the sorbant out Prepare test tubes to collect the sample elutant. Immediately prior to use, rinse out the test tubes with Methylene Chloride, and place the elution tubes in the extraction manifold directly beneath the SPE columns Rinse the sample bottle with 6 ml Ethyl Acetate and then add the Ethyl Acetate to the SPE cartridge. Elute the Ethyl Acetate through the cartridge until about 0.25 inch remains above the frit. Let soak for approximately one minute Rinse the sample bottle with 6 ml Methylene Chloride and then add the Methylene Chloride to the SPE cartridge. Elute the Methylene Chloride through the cartridge until approximately 0.25 inch remains above the frit. Let soak for approximately one minute. Draw he remaining solvent through the cartridge into the test tube. Save the sample bottles to determine sample volume determination Elute only enough Methylene Chloride through the SPE cartridge to fill the remainder of the test tube Completely draw the solvent through the sorbant into the collection tubes. Add 10 µl of p-terphenyl-d14 recovery standard to each tube. Sample elutant is now ready for drying and concentration Sample drying and concentration: Prepare funnels and centrifuge tubes. Place a small amount of glass wool in a funnel, and fill the funnel with 5-7 grams of Baked Page 27 of 43

28 Sodium Sulfate. Immediately prior to use, rinse the centrifuge tubes with Methylene Chloride Wet the Baked Sodium Sulfate with Methylene Chloride until it is loaded with Methylene Chloride and the Methylene Chloride is flowing through the funnel into a waste beaker. Also rinse the funnel stem with Methylene Chloride Pour the sample elutant through the Baked Sodium Sulfate into a centrifuge tube. Rinse the elutant collection tube with a small amount of Methylene Chloride and pour through the Baked Sodium Sulfate. Repeat if remaining volume in the centrifuge tube allows. Rinse Baked Sodium Sulfate with Methylene Chloride if there is enough volume in the centrifuge tube Repeat for the remaining sample elutant(s). Note: If are not followed, then there may be a loss of analytes, especially of the Internal Standards and Surrogates Place the centrifuge tubes in the N-Evap concentrator and concentrate the aliquots under a GENTLE stream of Nitrogen until about 4 ml remains in the tubes. Note: The Nitrogen stream should just dimple the surface of the aliquot, but not penetrate the surface of the aliquot. If the aliquot is concentrated too fast then a loss of analytes will occur At approximately the 4 ml mark, rinse the sides of the centrifuge tube with Ethyl Acetate, and concentrate down to 1 ml mark on the tube. Note: DO NOT allow the concentrate to go below 0.5 ml or loss of analytes will occur With a pipette, transfer sample extract from the centrifuge tube to a 1 ml Class A volumetric flask, and bring to volume with Ethyl Acetate. Place in a vial and cap. Repeat for each sample extraction Complete the prep sheets with finish dates and lot numbers of the reagents and solvents used. Page 28 of 43