RTI International, Center for Forensic Sciences, 3040 Cornwallis Rd., P.O. Box 12194, Research Triangle Park, North Carolina

A Comparison of the Validity of Gas Chromatography Mass Spectrometry and Liquid Chromatography Tandem Mass Spectrometry Analysis of Urine Samples II: Amphetamine, Methamphetamine, (±)-3,4-Methylenedioxyamphetamine, (±)-3,4-Methylenedioxymethamphetamine, (±)-3,4-Methylenedioxyethylamphetamine, Phencyclidine, and (±)-11-nor-9-Carboxy- 9 -tetrahydrocannabinol * Peter R. Stout, Nichole D. Bynum, Cynthia M. Lewallen, John M. Mitchell, Michael R. Baylor, and Jeri D. Ropero-Miller RTI International, Center for Forensic Sciences, 3040 Cornwallis Rd., P.O. Box 12194, Research Triangle Park, North Carolina 27709 Abstract On November 25, 2008, the U.S. Department of Health and Human Services posted a final notice in the Federal Register authorizing the use of liquid chromatography tandem mass spectrometry (LC MS MS) and other technologies in federally regulated workplace drug testing (WPDT) programs. To support this change, it is essential to explicitly demonstrate that LC MS MS, as a technology, can produce results at least as valid as gas chromatography (GC) MS, the long-accepted standard in confirmatory analytical technologies for drugs of abuse. A series of manufactured control urine samples (n = 10 for each analyte) containing amphetamine, methamphetamine, (±)-3,4-methylenedioxyamphetamine, (±)-3,4- methylenedioxymethamphetamine, (±)-3,4- methylenedioxyethylamphetamine, phencyclidine, and (±)-11-nor- 9-carboxy- 9 -tetrahydrocannabinol at concentrations ranging from 10% to 2000% of federal cutoffs were analyzed with replication by five federally regulated laboratories using GC MS and at RTI International using LC MS MS. Interference samples as described in the National Laboratory Certification Program 2009 Manual were analyzed by GC MS and LC MS MS as well as previously * This paper was developed (in part) under contract 277-2003-00044 from the Substance Abuse and Mental Health Services Administration (SAMHSA), U.S. Department of Health and Human Services (HHS). The views, policies, and opinions are those of the authors and do not reflect those of SAMHSA or HHS. Author to whom correspondence should be addressed: Peter R. Stout Ph.D., Senior Research Forensic Scientist, RTI International, Center for Forensic Sciences, 3040 Cornwallis Rd., P.O. Box 12194, Research Triangle Park, NC 27709. Email: pstout@rti.org. confirmed urine specimens of WPDT origin. Matrix effects were assessed for LC MS MS. Results indicated that LC MS MS analysis produced results at least as precise, accurate, and specific as GC MS for the analytes investigated in this study. Matrix effects, while evident, could be controlled by the use of matrix-matched controls and calibrators with deuterated internal standards. Introduction On November 25, 2008, the U.S. Department of Health and Human Services (HHS) published a final notice in the Federal Register allowing the use of liquid chromatography tandem mass spectrometry (LC MS MS) and other technologies in federally regulated workplace drug testing (WPDT) programs (1). This federal guideline is anticipated to become effective in October 2010. To support this change, it is essential to explicitly demonstrate that LC MS MS as a technology can produce results at least as valid as gas chromatography (GC) MS, the accepted standard in confirmatory testing for drugs of abuse in urine. GC MS has been the accepted standard in confirmatory analytical technologies for drugs of abuse in urine and is currently the only confirmatory method allowed for use in support of federally regulated WPDT programs. A very large body of literature is available with validation studies for GC MS methods (2,3). 430 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission.

Numerous validation studies have been published utilizing LC MS MS methods for a wide variety of target analytes in urine, oral fluid, and blood (4 12). Most of these studies provide data documenting accuracy, precision, linearity, and interferences (4,13,14). These criteria are required to demonstrate that a method is valid and capable of producing accurate, precise, and reliable results. Additional data are needed to support the minimal technical criteria that define acceptable instrument and batch performance for the application of LC MS MS in WPDT. In a previous study by Stout et al. (4), direct comparisons of results obtained from GC MS and LC MS MS using concurrent analyses of the same sample materials were reported for codeine (COD), morphine (MOR), 6-acetylmorphine (6-AM), and benzoylecgonine (BZE). This study attempted to evaluate comparisons of the analysis of phencyclidine (PCP), amphetamine (AMP), methamphetamine (MAMP), (±)-3,4- methylenedioxyamphetamine (MDA), (±)-3,4-methylenedioxymethamphetamine (MDMA), (±)-3,4-methylenedioxyethylamphetamine (MDEA), and (±)-11-nor-9-carboxy- 9 - tetrahydrocannabinol (THCA) in urine by LC MS MS with results from GC MS. LC MS MS has some notable differences from GC MS. It is generally more sensitive than GC MS; however, there is the potential for greater variability in product ion formation in analyses using multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) (3). Additionally, LC methods are more susceptible to matrix effects the potential for components of the sample matrix to either suppress or enhance the ionization of target analytes (15 18). Therefore, for LC MS MS method validation, it is critical to understand the potential matrix effect, as well as interference(s). Interference studies for GC MS are required to investigate structurally similar drug analytes that may interfere with testing, while interference studies for LC MS MS should investigate interference from both structural analogues and matrices. Direct comparisons of results obtained from GC MS and LC MS MS using concurrent analyses of the same sample materials have not been apparent in the scientific literature. This study was a continuation of prior work to include such analyses to assess the ability of LC MS MS to produce results at least as valid as GC MS by directly comparing results from both analytical technologies for the same sets of manufactured urine samples and the same sets of previously confirmed urine specimens. Methods Table I. Details of the Distribution of Sample Concentrations in the Linearity, Precision, and Accuracy (LPA) Samples Reagents and analytes All solvents used at RTI were analytical-grade solvents and reagents purchased from Fisher Scientific (Fair Lawn, NJ), Burdick and Jackson (Muskegon, MI), Sigma-Aldrich (Deisenhofen, Germany), EMD (Gibbstown, NJ), Aqua Solutions (Deer Park, TX), Strem Chemicals (Newburyport, MA), BDH (VWR West Chester, PA), and ACROS (Morris Plains, NJ). Solid-phase extraction columns were purchased from SPEware (Baldwin Park, CA). Drug-free human urine was collected from volunteers under Institutional Review Board-approved protocols with consent. Validation samples were manufactured at RTI International (Research Triangle Park, NC) using manufactured stock materials separate from the stock materials used for controls and calibrators. The AMP, MDA, MDMA, MDEA, THCA, and PCP used in preparing the validation samples were all obtained through the National Institute on Drug Abuse (NIDA) from the NIDA drug supply repository housed at RTI, and MAMP was purchased from Sigma-Aldrich. Calibrators for LC MS MS analyses were prepared by RTI using drug-free human urine. All stock drug materials used to prepare calibrators were obtained from Cerilliant (Round Rock, TX). Controls for GC MS and LC MS MS analyses were obtained from National Laboratory Certification Program (NLCP) Performance Testing (PT) program. The drug concentrations in the controls were 625 ng/ml for AMP; 350 ng/ml for MDA; 625 ng/ml for MAMP; 2500 ng/ml for MDMA; 350 ng/ml for MDEA; 20 ng/ml for PCP; and 10 and 175 ng/ml for THCA. Drug Concentration (ng/ml) Amps series Sample % of Number cutoff AMP MAMP MDA MDMA MDEA PCP THCA 1 10 50 50 50 50 50 2.5 1.5 2 20 100 100 100 100 100 5 3 3 40 200 200 200 200 200 10 6 4 75 375 375 375 375 375 18.7 11.25 5 100 500 500 500 500 500 25 15 6 125 625 625 625 625 625 31.2 18.75 7 200 1000 1000 1000 1000 1000 50 30 8 500 2500 2500 2500 2500 2500 125 75 9 1000 5000 5000 5000 5000 5000 250 150 10 2000 10000 10000 10000 10000 10000 500 300 Manufacture of validation samples In accordance with the NLCP guidance for method validation, the expanded series of samples was manufactured in drug-free human urine. Three series of fortified urine samples were manufactured: one contained PCP, one contained AMP/MDA/MAMP/MDMA/MDEA (AMPS series), and one contained THCA. Each series contained the analytes at concentrations across three orders of magnitude. The validation samples for PCP, the AMPS series, and THCA for linearity, precision, and accuracy (LPA) determinations are described in Table I. 431

Linearity, precision, and accuracy (LPA) determination by LC MS MS and GC MS analyses The LPA sample series was analyzed by five HHS-certified drug testing laboratories using GC MS and was also analyzed by RTI using LC MS MS. The samples, along with control materials derived from past NLCP PT materials, were shipped frozen overnight from RTI to the laboratories. These control materials provided known reference points to allow deviations from expected performance to either be attributed to issues within the laboratory or to the manufactured sample materials. Each laboratory was provided with sufficient sample volume for all requested analyses and was compensated for their participation. The PCP, THCA, and AMPS series of manufactured samples were analyzed at each laboratory using its standard validated GC MS methods. Each laboratory extracted one set of samples and analyzed each extract five times using five separate calibrations within a five-day period. Because the purpose of the study was to compare the differences in analytical method performance, this protocol was designed to limit variations arising from extraction procedures. RTI analyzed the PCP, THCA, and AMPS series samples using LC MS MS analyses of 10 extractions for each series. Interference samples Amphetamine interference samples (Table II) were also shipped to the five separate reference laboratories for GC MS analysis. These samples contained 50,000 ng/ml of phentermine and 1,000,000 ng/ml of ephedrine, pseudoephedrine, and phenypropanolamine (PPA) in addition to the other drugs in the AMPS LPA samples (40% cutoff). The samples were extracted both with and without periodate treatment to evaluate the effects on potential interferences. Laboratories were instructed to analyze the materials once using their standard amphetamines extraction and GC MS procedures. Five replicate LC MS MS analyses of these materials were performed at RTI. Matrix effect samples Matrix effect samples were analyzed by LC MS MS at RTI. Target analyte concentrations were prepared at cutoff concentrations for each drug series: 500 ng/ml of AMP/MAMP/MDMA/ MDA/MDEA, 15 ng/ml of THCA and 25 ng/ml of PCP. For the purposes of this study, 10 lots of urine each from a different donor were collected and 432 Table III. Agilent LC Method Parameters samples were analyzed once. Matrix effects were evaluated using the methods described by Matuszewski et al. (18). Three sets of samples were created for each target analyte. Type A Table II. Detail of Drugs and Sample Concentrations (ng/ml) Included in AMPS Interference Study Drug Sample 1 Sample 2 Amphetamine 200 Methamphetamine 200 MDA 200 MDMA 200 MDEA 200 Phentermine 50000 50000 Ephedrine 1000000 1000000 Pseudoephedrine 1000000 1000000 PPA 1000000 1000000 Flow Stop Post Injection Analyte Temp. Rate Time Time Volume Series Column ( C) (ml/min) Gradient (min) (min) (µl) PCP Zorbax XDB-C18 50 0.8 5% B at 0 min 7 2 5 (1.8 µm, 4.6 x 50 mm) 40% B at 6 min 95% B at 7 min AMPS Zorbax XDB-C18 50 0.5 0% B at 0 min (1.8 µm, 4.6 x 50 mm) 10% B at 3 min 8 3 2 75% B at 8 min THCA Zorbax XDB-C18 50 0.6 5% B at 0 min 6 3 5 (3.5 µm, 2.1 x 50 mm) 95% B at 3 min Table IV. Agilent MS MS Method Parameters and Ions Used* Collision Collision Precursor Fragmentation Energy Product Energy Product Ion Voltage Voltage Ion 1 Voltage Ion 2 Drug (m/z) (V) (V) (m/z) (V) (m/z) PCP 244 90 10 159 40 91 PCP-d 5 249 90 10 164 35 96 AMP 136 70 5 119 20 91 AMP-d 6 142 70 7 125 7 93 MAMP 150 90 5 119 15 91 MAMP-d 9 159 90 5 125 20 93 MDA 180 70 5 163 10 134 MDA-d 5 185 70 10 168 20 110 MDMA 194 90 10 163 25 105 MDMA-d 5 199 90 10 165 5 107 MDEA 208 90 10 163 25 105 MDEA-d 5 213 90 10 163 15 135 THCA 345 110 10 327 15 299 THCA-d 3 348 110 18 330 18 302 * Dry gas flow for all analytes was 13 L/min, temperature 350 C, and nebulizer gas at 45 psi.

samples (neat in mobile phase) were made by preparing an amount of target analyte and internal standard in mobile phase equivalent as in sample types B and C. Type B samples (post-extraction spike) were made by fortifying the eluent from the solid-phase extraction of negative urine matrix with target analytes and internal standard. Type C samples (pre-extraction spike) were made by fortifying negative urine matrix with target analytes and internal standard prior to solid-phase extraction. Comparative calculations were used to evaluate the data: ME (%) = B/A 100 RE (%) = C/B 100 PE (%) = C/A 100 where A, B, and C = the mean responses as represented by the area under the peaks for target and internal standard quantitative transitions, ME = the matrix effect, RE = the recovery effect ( recovery of the extraction procedure ), and PE = the process effect ( process efficiency ). The mean responses for A, B, and C were determined across the 10 lots of urine. Additionally, a comparison of the relative matrix effect was accomplished by comparing the % CV in the response across the 10 matrix lots for sample types B and A. This provided a comparison of the variation due to analysis as represented by the % CV of sample type A (neat in mobile phase) with the variation due to matrix represented by the % CV of sample type B (post-extraction spike). Previously confirmed specimens Previously confirmed urine specimens were obtained from Laboratory Corporation of America Holdings (LabCorp, Research Triangle Park, NC). All specimens had been slated for destruction, and all identifiers were removed prior to arrival at RTI. Urine specimens from LabCorp were previously confirmed positive for AMP, MAMP, PCP, or THCA. LabCorp analyzed the specimens using GC MS, and RTI analyzed the specimens using LC MS MS. Thirty-six AMP/MAMP, 6 PCP, and 140 THCA previously confirmed positive samples were utilized. No samples with MDA/MDMA/MDEA were available for use in this study. LC MS MS analysis conducted at RTI Samples for PCP were extracted using the same procedure as detailed for BZE in Stout et al. (4). AMPS extraction. Fifty microliters of methanolic deuterated internal standard (ISTD) (final concentration: 500 ng/ml AMP-d 6, MAMP-d 9, MDA-d 5, MDMA-d 5, and MDEA-d 5 ) was added to each 1 ml sample of urine. For calibrators, 1 ml of negative urine was used and drug standards were added to produce concentrations of 40, 500, 2000, 5000, and 12000 ng/ml. Then 1 ml of 1 M NaOH was added to all tubes, followed by 1.5 ml of 1 M sodium periodate. Interference samples were run with and without periodate addition, but results of these samples indicated the benefit of using periodate and all samples were processed using periodate addition. The samples were capped, vortex mixed, and incubated at 50 60 C for 15 min. The samples were allowed to cool to room temperature and then centrifuged for 5 min at 3000 rpm. Each sample was transferred to a solid-phase extraction column (SPEware Cerex Polychrom Clin II, 35 mg) and allowed to flow by gravity before the column was rinsed with 1 ml of deionized (DI) water followed by 1 ml of 0.1 M hydrochloric acid (HCl) and then dried under nitrogen (N 2 ) at 25 psi for 2 min. The columns were washed with 1 ml each of methanol then ethyl acetate and dried for 2 min at 25 psi N 2. The analytes were eluted by gravity followed by the addition of 1 ml of ethyl acetate/methanol/ammonium hydroxide (80:20:2) and 50 µl of 1 N HCl to each tube. The eluent was evaporated to dryness under a stream of N 2 at 30 C and reconstituted in 100 µl of 5 Table V. Summary of Five-Point Calibration, Internal Standard (ISTD), QC Sample Concentrations (ng/ml), and Calibration Types Used for LC MS MS Analyses Calculation Calibration Points ISTD Conc. QC Conc. Average Maximum Minimum Drug Type (ng/ml) (ng/ml) (ng/ml) r 2 r 2 r 2 AMP Quadratic 40, 500, 2000, 5000, 12000 500 2000 0.9993 0.9999 0.9980 (y = ax 2 + bx + c) MAMP Quadratic 40, 500, 2000, 5000, 12000 500 625 0.9984 0.9996 0.9962 (y = ax 2 + bx + c) MDA Quadratic 40, 500, 2000, 5000, 12000 500 350 0.9999 1.0000 0.9996 (y = ax 2 + bx + c) MDMA Quadratic 40, 500, 2000, 5000, 12000 500 2500 0.9996 1.0000 0.9989 (y = ax 2 + bx + c) MDEA Quadratic 40, 500, 2000, 5000, 12000 500 350 0.9998 1.0000 0.9995 (y = ax 2 + bx + c) PCP Linear 2, 25, 50, 100, 600 50 20 0.9975 0.9995 0.9892 (y = mx + b) THCA Quadratic 2, 15, 100, 200, 350 30 10 and 175 0.9940 0.9998 0.9877 (y = ax 2 + bx + c) 433

mm ammonium formate/acetonitrile (95:5) with 0.1% formic acid. THCA extraction. Thirty microliters of methanolic deuterated ISTD (final concentration: 30 ng/ml THCA-d 3 ) was added to each 1-mL sample of urine. For calibrators, 1 ml of negative urine was used and drug standards were added to produce concentrations of 2, 15, 100, 200, and 350 ng/ml. Then, 70 µl of 45% KOH was added and each tube was capped, vortex mixed, and heated at 60 C for 15 min. After the samples cooled to room temperature, 350 µl of glacial acetic acid was added, and each tube was vortex mixed. Solid-phase extraction columns (SPEware Trace-N, 15 mg, 10 microns) were conditioned with 150 µl of methanol containing 0.1M acetic acid, which was allowed to flow through the column by gravity for 2 min. The samples were added to the columns and allowed to flow through at a rate of approximately 1 ml/min before the columns were washed with 1 ml of 80:20 DI H 2 O/acetic acid, and then 750 µl of 40:60 DI H 2 O/methanol. The columns were dried for 10 min at 30 psi N 2. The analytes were eluted with 800 µl of 98:2 hexane/acetic acid. The eluent was evaporated to dryness under a stream of N 2 at 30 C. The samples were reconstituted with 100 µl of isopropanol. LC MS MS method. The LC MS MS equipment consisted of an Agilent 1200 series LC coupled to an Agilent 6410 triplequadrupole MS with an electrospray source operating in positive mode (Santa Clara, CA). The mobile phases consisted of (A) 5 mm ammonium formate with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The Agilent LC method parameters are given in Table III. All analyses were conducted in MRM mode using the MS MS conditions listed in Table IV. Collision energies and fragmentation voltages were optimized for each transition. The capillary voltage used was 3500 for all analytes. Calibration. Details about the calibration curves used for the quantification of each drug analyte are given in Table V. Data were collected; calibration and data reduction were performed Table VI. Evaluation of Precision and Accuracy of GC MS Results Precision Evaluation Regression Comparison to Target Concentrations and ANOVA Analysis of Line Fit Average Average 95% 95% between within Accuracy Evaluation Confidence Confidence laboratory laboratory Average Accuracy interval of interval of p Drug n %CV %CV accuracy %CV r 2 Slope* slope Intercept intercept value AMP 220 13.08 3.00 101.2 17.54 0.961 0.94 0.91 0.96 41.4 37.69 120.59 0.3031 MAMP 224 9.56 1.60 106.1 9.80 0.976 0.97 0.95 0.99 65.8 2.65 134.33 0.0595 MDA 169 8.00 4.25 101.4 15.56 0.991 0.86 0.84 0.87 80.5 37.05 124.04 0.0003 MDMA 182 9.51 2.66 100.4 12.32 0.965 0.84 0.82 0.86 86.8 11.46 162.09 0.0242 MDEA 147 9.53 3.10 99.8 12.63 0.952 0.86 0.82 0.89 76.5 30.27 183.35 0.1588 PCP 233 4.24 1.99 98.5 5.94 0.999 0.95 0.94 0.95 0.8 0.14 1.48 0.0187 THCA 220 17.09 3.92 80.4 16.41 0.982 0.87 0.86 0.89 1.4 3.03 0.23 0.0928 * Note: all slopes were significantly different from zero at the p < 0.00001 level. The intercepts are significantly different (p < 0.05) than zero for MDA, MDMA, and PCP. Table VII. Evaluation of Precision and Accuracy of LC MS MS Results Precision Accuracy Regression Comparison to Target Concentrations and Evaluation Evaluation ANOVA Analysis of Line Fit Average Average Average Average 95% Confidence 95% Confidence Overall Overall Overall Accuracy Overall Accuracy interval of interval of p Drug n %CV* %CV Accuracy* %CV Accuracy %CV r 2 Slope slope Intercept intercept value AMP 100 3.76 3.23 99.9 5.73 97.3 5.76 0.999 1.03 1.02 1.03 21.8 47.40 3.76 0.094 MAMP 100 3.54 3.61 102.4 6.57 97.7 8.17 0.995 0.969 0.95 0.98 9.9 39.91 59.68 0.694 MDA 100 2.08 2.73 101.6 3.01 104.6 8.07 0.999 1.02 1.01 1.02 24.3 51.38 2.75 0.078 MDMA 100 3.54 4.10 105.5 4.18 112.1 6.43 0.996 0.970 0.95 0.98 9.75 58.41 38.91 0.692 MDEA 100 2.37 3.06 105.8 2.88 111.0 6.00 0.998 0.962 0.95 0.96 12.1 41.56 17.32 0.416 PCP 100 5.93 6.67 93.4 7.92 101.1 7.88 0.995 0.966 0.95 0.97 4.6 2.01 7.22 0.001 THCA 100 8.56 8.54 77.9 12.59 74.9 10.61 0.988 1.06 1.04 1.08 5.1 2.75 7.44 3.76 E 05 * Five-point calibration One-point calibration. Note: all slopes were significantly different from zero at the p < 0.00001 level. The intercepts for PCP and THCA were significantly different from zero (p < 0.05). 434

using Mass Hunter software (version B.01.04, San Jose, CA). For all analytes, LC MS MS analyses were conducted using both a five-point calibration and a one-point calibration at the federally mandated cutoff concentrations (1). The type of curve best fitting the data was chosen, and used for the entire validation study. For the data to be acceptable, control quantitation for each batch had to be within 20% of the target concentration. The ratio of the product ions had to be within 20% of the average of the calibrator ion ratios, and the retention time had to be within 2% of the average of the calibrators. Calibrators were back-calculated against the model curve, and the back calculated value had to be within 20% of the theoretical concentrations. Statistical analysis. Statistical tests conducted using Microsoft Excel 2007 (Seattle, WA) included linear regression analyses with subsequent analysis of variance (ANOVA) of the regression fit, calculation of average, standard deviation, and % CV. Student t-tests were used for the comparison of analyses of the previously confirmed specimens. For these tests, significance was assigned at the p < 0.05 level. For the comparison of GC MS and LC MS MS analyses of the LPA samples, a two-way imbalanced ANOVA was conducted (SAS PROC GLM using SAS, Raleigh, NC). If the crossed term of analytical method and concentration was significant, the two methods were compared after adjusting for the effect of the expected concentration. This allowed for the determination of significant differences between the two analytical methods at each concentration in the analyzed series. Again, significance was assigned at the p < 0.05 level. Results and Discussion Materials analyzed by both GC MS and LC MS MS The LPA sample series was analyzed by both GC MS and LC MS MS. The precision and accuracy of results from the five HHS-certified laboratories conducting GC MS analyses are shown in Table VI. Results are from five HHS-certified laboratories, each analyzing five replicates of Figure 1. Typical chromatography and monitored MRM for each of the target compounds and their respective internal standards from the LC MS MS analysis. Figure 1 is continued on next page. 435

Figure 1. Typical chromatography and monitored MRM for each of the target compounds and their respective internal standards from the LC MS MS analysis. Figure 1 is continued on next page. each sample type. The corresponding LC MS MS results are shown in Table VII. For GC MS results, within-laboratory precision represented by % CV was low (< 5% CV). Between laboratory % CV was less than 14% for all compounds except THCA, which had greater variability as shown by a 17% CV. The number of samples for analysis (n) varied for each compound because samples not meeting acceptance criteria for GC MS (e.g., ion ratios out of range, or IR confirmation unacceptable) were excluded from calculations. Several samples were also excluded because the concentrations were not within the calibration range of some laboratories, and some were excluded because not all laboratories test for all drugs present in the samples. All five HHS-certified laboratories tested for amphetamine, methamphetamine, PCP, and THCA. One of the five laboratories did not test for MDA or MDMA and two of the labs did not test for MDEA. Control materials consisting of prior NLCP PT materials were inserted into sample sets (a positive and negative control were included with each sample set sent to laboratories). The results of the control materials are not included in the results shown in Tables VI and VII. In Table VI, GC MS accuracy was assessed by determining % accuracy and by regression analysis comparing target concentration with measured concentration. First, % accuracy (measured concentration/target concentration 100) was determined. This allowed the calculation of the average % accuracy for each analyte and the associated % CV. All drugs except THCA were 98.5 to 106.1% accurate for GC MS with accuracy % CVs less than 18%. The accuracy for THCA was 80.4%. Based upon RTI s experience in manufacturing proficiency testing samples for THCA this decreased response relative to idea concentration is typical. Concentration points that were either below a laboratory s limit of detection (LOD) or above a laboratory s upper limit of linearity (ULOL) were not used in the regression and accuracy analyses of the GC MS results. The second method of demonstrating accuracy and precision was a regression analysis comparing target concentration with measured concentration. If the results were completely accurate and pre- 436

cise over the entire range tested, the regression results would theoretically be an r 2 = 1.000, a slope of 1.000, and an intercept of 0.000. A theoretically perfect analytical precision would yield an ANOVA analysis indicating a model-fit with a slope significantly different than 0.000 and an intercept not significantly different than 0.000. As indicated in Table VI, GC MS results were very close to ideal, indicating the results were highly precise and accurate. MDA, MDMA, and PCP deviate from ideal in that the intercepts are significantly different than 0.000; however, these differences are small. Qualitatively, LC MS MS results demonstrated good chromatographic performance, exhibiting symmetrical peaks and at least 90% resolution of adjacent peaks. An example of typical chromatography is shown in Figure 1. Samples analyzed by LC MS MS were quantitated using both a five-point calibration, as described in the methods for each drug, and a one-point calibration using only the calibrator at the cutoff for that analyte (i.e., the calibration method most commonly used in WPDT). LC MS MS results for precision and accuracy are presented in Table VII. The average % CV was highly comparable for the five-point and one-point LC MS MS calibrations, with the values from the fivepoint calibrations ranging from 2.08 to 8.56% and 2.73 to 8.54% for the onepoint calibrations. The average overall % CV was higher for most of the analytes using the one-point LC MS MS calibration than the average within-laboratory % CV for GC MS results. This is due to the exclusion of points below the LOD or above the reported ULOL for GC MS analyses but not for LC MS MS analysis. The two exceptions are for MDA and MDEA, which had lower % CVs for the LC MS MS analysis regardless of the number of calibration points used. Accuracy was also assessed by calculating the average accuracy and performing the same regression analysis as for GC MS results. The LC MS MS average accuracy for all analytes except THCA ranged from 93.4 to 105.8% using a five-point calibration and 97.3 to 112.1% using a one-point calibration. The average accuracy for THCA was 77.9% using a five-point calibration and 74.9 % using a one-point calibration. This is similar to the GC MS accuracy of 80% and again based upon our experience in manufacturing proficiency testing samples for THCA this decreased response relative to theoretical concentration is typical. For the purposes of this study, the LC MS MS results obtained with a five-point calibration and best fit to a quadratic equation were used for comparison with GC MS results. Intercepts for PCP and THCA were significantly different than 0.000 indicating a small but real deviation from ideal results. When GC MS and LC MS MS results at each concentration were compared using a two-way ANOVA, significant difference (p < 0.05) was observed for the upper concentrations for all compounds (Table VIII). These differences were likely due to Figure 1. Typical chromatography and monitored MRM for each of the target compounds and their respective internal standards from the LC MS MS analysis. Table VIII. Significant Two-Way ANOVA Interactions from the Comparison of GC MS and LC MS MS Analyses GC MS LC MS MS Target Least Squares Least Squares % Concentration Mean Mean Drug (ng/ml) (ng/ml) (ng/ml) p* Difference AMP 5000 4659 5001 0.0383 6.8% AMP 10000 9386 10374 <.0001 9.5% MAMP 10000 9669 10358 <.0001 6.7% MDA 5000 4509 4947 <.0001 8.9% MDA 10000 8524 9803 <.0001 13.0% MDMA 2500 2295 2636 0.0284 12.9% MDMA 5000 4512 5201 <.0001 13.2% MDMA 10000 8327 10245 <.0001 18.7% MDEA 5000 4533 5210 0.0002 13.0% MDEA 10000 8508 10383 <.0001 18.1% PCP 125 121 115 0.0241 5.0% PCP 250 234 245 <.0001 4.7% PCP 500 475 517 <.0001 8.1% THCA 300 261 282 <.0001 7.6% * Significance assigned for values of p < 0.05. 437

Table IX. Summary of the Deviation of Quantitation of Samples by Each Method (GC MS and LC MS MS)* 10% 20% 40% 75% 100% 125% 200% 500% 1000% 2000% Drug LC GC LC GC LC GC LC GC LC GC LC GC LC GC LC GC LC GC LC GC AMP 1 (20) 5 (20) 5 (20) 5 (20) 5 (20) 1 (20) 1 (50) 5 (20) 5 (50) MAMP 9 (20) 4(20) 5 (20) 5 (20) 5 (20) 1 (20) 5 (20) MDA 9 (20) 8(20) 1 (20) 4 (20) 2 (50) MDMA 5 (20) 3 (20) 1 (20) 1 (20) 9 (20) MDEA 6 (20) 4 (20) 7 (20) PCP 1 (20) THCA 3 (20) 4(20) 1 (20) 10 (20) 2(20) 5 (20) 1 (20) 4 (20) 5 (20) 2 (20) 1 (20) 5 (20) * The deviation of quantitation of samples are the number of sample values occurring either outside of a 20% or 50% window away from the target by concentration % of the cutoff (NLCP grading criteria for urine PT samples). Number in parentheses indicates either a 20% error or a 50% error. For THCA, as there was an expected loss of THCA in the manufacture of the samples, the comparison was against the mean of the five GC MS laboratory results. the difference in calibration between GC MS (one point) and LC MS MS (multipoint) lending to differences at higher concentrations. In the evaluation of performance testing samples in the NLCP, results are evaluated for errors of quantitation deviating more than 20% or 50% from the target for any given sample (19). Table IX summarizes the comparison of GC MS and LC MS MS results evaluated with this 20% and 50% criteria. For concentrations between 40% and 1000% of the cutoff for each drug, LC MS MS had no quantitation errors while there were several GC MS errors. This indicates that LC MS MS is at least as capable as GC MS at producing acceptable results as would be required on performance testing samples in the NLCP. Table X compares the results of the interference samples detailed in Table II when analyzed by both GC MS and LC MS MS. These samples contained the interferants ephedrine, pseudoephedrine, PPA (all at 1,000,000 ng/ml) and phentermine (50,000 ng/ml) in the presence of analytes of interest at 40% of cutoff concentrations and in the absence of the analytes altogether. The mean quantitative result from GC MS and LC MS MS analyses were compared using the student t-test with significance assigned at p < 0.05. Only MAMP results from LC MS MS with interferants present were significantly different than GC MS results (p < 0.05). The GC MS value was within 10% and the LC MS MS value was within 5% of the theoretical 200 ng/ml concentration. The two values were approximately 12% different, and so, although statistically significant, the difference was small. Figure 2 presents typical chromatography observed for the 438 Table X. Summary of Comparison of Interference Samples using Mean Quantitative Results of GC MS and LC MS MS Analyses of 40% Cutoff Control Material Containing Potential Interferants* GC MS Analysis LC MS MS Analysis Drug n Mean %CV n Mean %CV AMP 5 203.2 10.8 5 198.9 3.7 MAMP 5 218.8 7.1 5 191.6 3.0 MDA # 3 210.0 10.0 5 204.9 2.1 MDMA # 3 205.3 7.3 5 200.9 2.6 MDEA # 2 207.0 14.3 5 203.4 2.9 * A comparison of the results was performed by student t-test with significance assigned at p < 0.05. Phentermine (50000 ng/ml) and Ephedrine/Pseudoephedrine/PPA (1000000 ng/ml). Phentermine (50000 ng/ml) and Ephedrine/Pseudoephedrine/PPA (1000000 ng/ml). All values were within 10% of the theoretical concentration of 200 ng/ml. # Not all GC MS laboratories analyzed for MDA, MDMA, or MDEA. Limited sample size precluded statistical analysis. LC MS MS analysis of samples containing structural analogue compounds as potential interferants in the presence of target analytes at 40% of cutoff concentrations. The left and right columns show samples extracted without and with periodate pretreatment, respectively. The top row of chromatograms is the target compounds; the lower row is the ISTD. There was a slight shoulder on the MDMA peak; however, it was eliminated during the periodate pre-treatment. The qualitative improvement in the chromatography with the use of periodate led to the continued use of periodate treatment in the method for all analyses of the AMPS series. LC methods do not pose the same potential for the formation of MAMP, but the periodate pretreatment can still improve chromatographic performance in the method. None of the target analytes were detected in the interference samples without the addition of the target. Figure 3 presents a comparison of the average accuracy of

Figure 2. Example chromatography for amphetamine, methamphetamine, MDA, MDMA, and MDEA with the presence of the interfering structural analogues phentermine (at 50,000 ng/ml), epedrine, pseudoephedrine, and PPA (at 1,000,000 ng/ml each). The top row of chromatograms is the target compound; the bottom row is the ISTD. 439

GC MS and LC MS MS results across the concentration range tested for each target compound. The error bars represent one standard deviation (SD) for each average. From this, it is apparent that the average accuracies across the concentration range are very similar for GC MS and LC MS MS. The notable exceptions are for AMP GC MS results at 125% of the cutoff concentration and for THCA at 10% and 20% of the cutoff concentration. The divergent result at 125% of cutoff for AMP is due to one GC MS laboratory reported values divergent from the other four laboratories. Evaluation of LC MS MS data components and matrix effects For LC MS MS results, Table XI summarizes the average and distributions of other qualifier data for the results, such as retention time (RT), ion ratios, and internal standard (ISTD) responses. The distribution of RTs for both target analyte and ISTD were very small, with %CVs less than 1.3% for all compounds. The average and %CV of RTs for PCP were analyzed separately for validations 1 7 and 8 10. The LC column was changed during this validation and resulted in a slight retention time shift between runs. Within-run RTs were consistent. The ratio of quantitation product ion to qualifier product ion for both target and ISTD demonstrated little variation with %CVs less than 4.5 for all target analytes with the exception of THCA. As would be expected, the internal standard responses were more variable. The NLCP requires that internal standard ion Figure 3. A comparison of the average accuracy of GC MS and LC MS MS results across the concentration range tested for each target compound. The error bars represent one standard deviation (SD) for each average. 440

responses for the quantifying ion of a specimen be within 0.5 and 2 times of that obtained with the calibrator, the average of calibrator and controls, or the average of all calibrators, controls, and specimens in the analytical batch (19). Because CV = SD/mean, if the CV is less than 1, the interval of two times the mean will represent at least 2SD, or 95.4% of the population. Therefore, the majority of points in the population will be within 2SD or 2 times the mean. Although the %CVs are large, few samples fell outside of current NLCP criteria. The number of samples falling outside these criteria for AMP, MAMP, MDMA, MDA, and MDEA, were 5, 0, 0, 4, and 1, respectively. All exhibited low internal standard response outside of the acceptable range for the 10,000 ng/ml sample and this is consistent with ion suppression of the internal standard by the elevated concentration of the analytes. The number of samples outside of the acceptable range for THCA was 20. All unacceptable responses were for the samples at concentration levels 150 and 300 ng/ml and one sample at 75 ng/ml. The 22 unacceptable samples for PCP however, did not correlate with the high concentrations. Fourteen of the 22 unacceptable PCP results were less than 0.5 the mean response. No clear reason for this was determined other than PCP had more variable performance in this analysis than other compounds. Matrix effect was assessed only for LC MS MS results. Table XII summarizes the results of the matrix effect analysis. All ME values were 100 ± 10% indicating negligible matrix effect. The same calculations, although slightly lower with the exception of PCP are presented for the internal standard. The difference in variability (%CV) of the responses of target compounds spiked into 10 different lots of urine (sample type B) compared to those of target compounds spiked into mobile phase (sample type A) indicates a more obvious relative matrix effect. This suggests that the presence of matrix results in more variable ion response. In all cases except PCP and MDEA, the variability associated with multiple lots of matrix is less than that associated with a single lot of matrix suggesting that there is a minimal relative matrix effect. For PCP and MDEA the difference in variability is not large. These single lot samples were comprised of 10 separate cutoff calibrators analyzed over 5 days. THCA has a notably higher variability than the other compounds, but a similar pattern of no indication that the variability is due to different lots of urine. More likely, it is due to solubility issues with THCA in the manufacture of samples. The material used to prepare these samples came from the same lot of urine that was used for all analyses and provides an indication of the variability due to a single lot of matrix. This suggests that the effect is common to all individuals and not a result of variation between individuals. Though the matrix effect was small, there was a significant difference in the quantitation values calculated for AMP, MAMP, Table XI. Comparison of LC MS MS Data Components and Variability in these Components Average ISTD Average Average Target Qualifier Qualifier Target ISTD ISTD Drug n Ratio %CV Ratio %CV RT* %CV RT* %CV Response %CV AMP 100 58.84 0.87 86.51 1.44 4.80 1.27 4.74 1.29 133100.06 28.21 MAMP 100 34.21 1.47 45.15 1.50 5.52 1.07 5.45 1.04 240200.94 23.75 MDA 100 17.87 1.50 39.54 1.30 5.20 0.99 5.15 0.97 178478.05 27.88 MDMA 100 37.30 1.44 2.31 2.05 5.84 0.92 5.79 0.97 331079.22 22.19 MDEA 100 38.49 1.06 27.70 1.13 7.18 0.43 7.15 0.50 387028.55 25.00 PCP 100 70.98 3.14 76.72 4.06 5.72 0.12 5.70 0.12 53778.31 80.29 THCA 100 58.88 8.12 80.74 10.56 3.16 0.17 3.15 0.16 5647.62 44.95 *PCP average RT in table is for validation 1 7. The RT for validation 8 10 are 5.82 and 5.79 for analyte and IS, respectively. Table XII. Evaluation of Matrix Effect Based on Matuszewski et al. (18). Responses Based on the Quantitative Transition Ion Areas Relative Matrix Effect Target Ion Response ISTD Ion Response from 10 Lots Variability in Single Lot of Matrix ME RE) PE ME RE PE %CV for %CV for Average % CV of cutoff Drug (%) (%) (%) (%) (%) (%) sample B sample A calibrator analyzed over 5 days AMP 102.84 91.21 93.80 92.96 89.35 83.05 2.43 0.52 6.74 MAMP 98.54 89.30 87.99 82.89 84.81 70.30 3.51 0.59 13.35 MDA 98.29 93.73 92.13 92.66 93.04 86.22 7.98 1.24 8.20 MDMA 91.63 91.33 83.69 88.78 88.73 78.78 12.57 0.65 12.87 MDEA 91.52 92.04 84.24 91.22 91.93 83.86 11.38 0.38 7.68 PCP 98.13 71.79 70.45 109.43 70.90 77.58 13.46 6.91 11.18 THCA 102.51 90.33 92.60 97.19 92.25 89.66 11.79 22.36 25.19 441

MDA, and MDMA, in sample type A (neat in mobile phase) from samples type B (post-extraction) and C (pre-extraction) as seen in Table XIII. There is no significant difference between B and C. For all analytes, with the exception of MDEA and PCP, the neat in mobile phase samples quantitation are slightly below the target concentrations and the matrix containing samples are slightly above the target concentrations. This result, coupled with the apparent slight difference in matrix effect for the target compounds and ISTD evident in Table XII, highlights the importance of the use of matrixmatched calibrators and controls that are subjected to the same treatment as samples and the use of stable isotope-labeled ISTDs. Of particular note even though THCA had a larger variability in responses, the quantitated values were Table XIII. Summary of Quantitative Results Associated with Each Matrix Effect Evaluation Sample Type. Differences Between Each Group Evaluated by One-Way ANOVA Sample Type A* B C AMP Mean (ng/ml) 441.0 491.7 503.3 % CV 0.2 2.0 2.8 % Accuracy 88.2 98.3 100.7 MAMP Mean (ng/ml) 412.8 498.2 526.8 % CV 0.9 2.7 2.2 % Accuracy 94.0 100.1 100.8 MDA Mean (ng/ml) 470.1 500.5 504.2 % CV 1.0 2.4 1.3 % Accuracy 94.0 97.9 101.0 MDMA Mean (ng/ml) 474.2 489.5 505.0 % CV 0.8 2.9 2.0 % Accuracy 94.8 97.9 101.0 MDEA Mean (ng/ml) 502.2 503.8 504.6 % CV 0.5 2.9 1.4 % Accuracy 100.4 100.8 100.9 PCP Mean (ng/ml) 23.7 21.1 21.3 % CV 1.4 6.4 7.4 % Accuracy 95.0 84.7 85.5 THCA Mean (ng/ml) 14.9 15.8 15.4 % CV 4.7 4.0 6.0 % Accuracy 99.2 105.3 102.8 * Neat in mobile phase. Significantly different than samples B and C for AMP, MAMP, MDA, MDMA, and PCP (p < 0.001). Post extraction fortification. Pre extraction fortification. not significantly different again highlighting the importance of matrix-matched calibrators and stable isotope-labeled ISTDs. As there was no apparent relative matrix effect or differing matrix effect between urine lots, the matrix effect observed could be controlled for by the use of matrixmatched controls and calibrators. Further work is needed to determine whether synthetic urine has a comparable matrix effect to human urine used in this study and would be appropriate to use in general. Validation of any method using a synthetic matrix should include evaluation of the potential matrix effect. Previously confirmed specimens Urine specimens obtained from LabCorp that were previously confirmed positive for AMP, MAMP, PCP, or THCA were analyzed by both GC MS and LC MS MS. A comparison of the results for these specimens presented in Table XIV summarizes the regression analysis for the results of both analyses. Previously confirmed samples were compared by a paired t-test and regression analysis. ISTD responses from GC MS and LC MS MS analyses are presented for comparison. The ion responses for THCA analyses by GC MS were unavailable, but the variability in LC MS MS ISTD responses for THCA were comparable to the other results from both GC MS and LC MS MS analyses. Also, average RTs and product ion ratios are presented for the LC MS MS data. The ion ratios and RTs were tightly distributed. There were no significant differences (p < 0.05) between GC MS and LC MS MS results for any of the drugs with the exception of PCP (p = 0.0280). Table XIV. Comparison Summary of GC MS and LC MS MS Analysis of Previously Confirmed Samples Drug AMP MAMP PCP* THCA n 36 4 6 140 r 2 0.993 1.000 0.999 0.847 Regression slope 0.96 0.98 1.14 0.69 t-test result (p) 0.2589 0.6081 0.0280 0.1645 Average GC MS 46382 76356 98486 ISTD response % CV 21.73 35.23 29.35 Average LC MS MS 65791 93975 29698 3194 ISTD response % CV 15.89 21.99 31.68 30.79 Average LC MS MS 58.79 34.07 71.95 67.29 ion ratio % CV 1.00 1.26 0.85 5.62 Average LC MS MS 4.78 5.51 5.84 3.17 retention time % CV 0.47 0.53 0.23 0.08 * The GC MS and LC MS MS results are significantly different (p < 0.05) for PCP. 442

Conclusions Based upon the data collected, GC MS and LC MS MS were comparable for the analyses of AMP, MAMP, MDMA, MDA, MDEA, PCP, and THCA. Both technologies provided accurate, precise, and specific results without interference from structural analogues. In previously confirmed specimens, results from both technologies were directly comparable. In manufactured materials in human urine, results were highly reproducible both within and between the two technologies. Evaluation of specific parameters of the LC MS MS data demonstrated that LC MS MS retention times were highly reproducible over several months and across multiple different samples. Product ion ratios were also very reproducible, and internal standard responses were consistently within 0.5 to 2 times the mean ISTD response from the controls, calibrators and specimens with the exception of THCA and PCP. Minimal matrix effect was observed for all analytes with the exception and AMP and MDA; however, it did not impact the overall results. These results did emphasize the need for matrix-matched controls and calibrators that are subjected to the same treatment as samples, as well as the use of stable isotope-labeled ISTD as a means of controlling for matrix effects. This study demonstrated that LC MS MS technology can produce results at least as valid as GC MS for confirmatory testing of AMP, MAMP, MDMA, MDA, MDEA, PCP, and THCA at urine concentrations and conditions appropriate for federally regulated WPDT. It was demonstrated that LC MS MS can meet the NLCP s chromatographic criteria for retention time stability, peak shape, ion ratio, and calibration in both manufactured and archived samples. References 1. 73 CFR 71858. November 25, 2008. Mandatory guidelines for Federal Workplace Drug Testing Programs. http://edocket.access. gpo.gov/2008/e8-26726.htm (accessed April 2009). 2. M.R. Moeller and T. Kraemer. Drug abuse monitoring in blood for control of driving under the influence of drugs. Ther. Drug Monit. 24(2): 210 221 (2002). 3. S.P. Vorce and D.J. Kuntz. Confirmation: traditional and newer analytical techniques. In Handbook of Workplace Drug Testing, 2nd ed., J.D. Ropero-Miller and B.A. Goldberger, Eds. AACC Press, Washington D.C., 2009, pp 61 98. 4. P.R. Stout, N.D. Bynum, J.M. Mitchell, M.R. Baylor, and J.D. Ropero-Miller. A comparison of the validity of gas chromatography mass spectrometry and liquid chromatographytandem mass spectrometry analysis of urine samples for morphine, codeine, 6-acetylmorphine, and benzoylecgonine. J. Anal. Toxicol. 33: 398 408 (2009). 5. X. Xu, M.M. Iba, and C.P. Weisel. Simultaneous and sensitive measurement of anabasine, nicotine and nicotine metabolites in human urine by LC tandem mass spectrometry. Clin. Chem. 50: 2323 2330 (2004). 6. S.S. Johansen and H.M. Bhatia. Quantitative analysis of cocaine and its metabolites in whole blood and urine by high performance liquid chromatography coupled with tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 852: 338 344 (2007). 7. S.V. Kala, S.E. Harris, T.D. Freijo, and S. Gerlich. Validation of analysis of amphetamines, opiates, PCP, cocaine and benzoylecgonine in oral fluid by liquid chromatography tandem mass spectrometry. J. Anal. Toxicol. 32: 605 611 (2008). 8. M. Wood, M. Laloup, F.M.M. Ramirez, K.M. Jenkins, M.S. Young, J.G. Ramaekers, G. DeBoeck, and N. Samyn Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid phase extraction and liquid chromatography tandem mass spectrometry. Forensic Sci. Int. 150: 227 238 (2005). 9. C.J. Briscoe, M.R. Stiles, and D.S. Hage. System suitability in bioanalytical LC/MS/MS. J. Pharm. Biomed. Anal. 44: 484 491 (2007). 10. S. Ma and M. Zhu. Recent advances in applications of liquid chromatography tandem mass spectrometry to the analysis of reactive drug metabolites. Chem. Biol. Interact. 179: 25 37 (2009). 11. M. Careri and A. Mangia. Validation and qualification: the fitness for purpose of mass spectrometry-based analytical methods and analytical systems. Anal. Bioanal. Chem. 386: 38 45(2006). 12. E.J. Fox, S. Twigger, and K.R. Allen. Criteria for opiate identification using liquid chromatography linked to tandem mass spectrometry: problems in routine practice. Ann. Clin. Biochem. 46: 50 57 (2009). 13. B. Maralikova and W. Weinmann. Confirmatory analysis for drugs of abuse in plasma and urine by high-performance liquid chromatography tandem mass spectrometry with respect to criteria for compound identification. J.Chromatogr. B Analyt. Technol. Biomed. Life Sci. 811: 21 30 (2004). 14. M. Jemal, Z. Ouyang, and M.L. Powell. A strategy for a post method validation use of incurred biological samples for establishing the acceptability of a liquid chromatography/tandem mass spectrometric method for quantitation of drugs in biological samples. Rapid Commun. Mass Spectrom. 16: 1538 1547 (2002). 15. P.J. Taylor. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography electrospray-tandem mass spectrometry. Clin. Biochem. 38: 328 334 (2005). 16. A. Van Eeckhaut, K. Lanckmans, S. Sarre, I. Smolders, and Y. Michotte. Validation of bioanalytical LC MS/MS assays: evaluation of matrix effects. J. Chromatogr. B Analyt. Technol.Biomed. Life Sci. 877(23): 2198 2207 (2009). 17. E. Chambers, D.M. Wagrowski-Diehl, Z. Lu, and J.R. Mazzeo. Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. J. Chromatogr. B852: 22 34 (2007). 18. B.K. Matuszewski, M.L. Constanzer, and C.M. Chavez-Eng. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC MS/MS. Anal. Chem. 75: 3019 3030 (2003). 19. National Laboratory Certification Program, Manual for Urine Laboratories. Effective date: 1 January 2009. RTI International, Research Triangle Park, NC. 443