Journal of Analytical Toxicology, Vol. 27, October 2003

Sensitivity, Specificity, and Efficiency in Detecting in Oral Fluid with the Cozart| Microplate EIA and GC-MS Following Controlled Administration Downloaded from https://academic.oup.com/jat/article/27/7/402/784027 by guest on 23 September 2021

Allan J. Barnes 1, Insook Kim 1, Raf Schepers 1, Eric T. Moolchan 1, Lisa Wilson 2, Gail Cooper 2, Claire Reid 2, Chris Hand 2, and Marilyn A. Huestis 1,* 1Chemistry and Metabolism, IRP, NIDA, NIH, Baltimore, Maryland21124, and 2Cozart Bioscience Ltd, Oxfordshire, OX144RU, United Kingdom

[Abstract [ Introduction

Oral fluid specimens (N = 1406) were collected from 19 subjects Currently, there is strong interest in monitoring drug use prior to and up to 72 h following controlled administration of oral through oral fluid testing in driving under the influence, drug codeine. Volunteers provided informed consent to participate in treatment, criminal justice, and workplace drug-testing pro- this National Institute on Drug Abuse Institutional Review Board- grams. Oral fluid, as a matrix for biological testing of opiates, approved protocol. A modification of Cozart Microplate Opiate EIA Oral Fluid Kit (Opiate ELISA), employing codeine calibrators, was has advantages over urine testing including the ease and non- used for semiquantitative analysis of opiates, followed by gas invasiveness of specimen collection and a reduction in the po- chromatography-massspectrometry (GC-MS) for the confirmation tential for specimen adulteration and substitution (1-4). Oral and quanlitation of codeine, norcodeine, , and fluid testing is a greater analytical challenge because drug con- normorphine in oral fluid. GC-MS limits of detection and centrations are generally lower than in urine, leading to shorter quantitation were 2.5 pg/L for all analytes. The Substance Abuse detection time windows (2,5). and Mental Health Services Administration (SAMHSA) has Codeine is commonly used as an and antitussive and proposed a 40-pg/L opiate screening and a 40-pg/L morphine or can be taken alone or in combination with other substances codeine confirmation cutoff for the detection of opiate use. Oral such as acetaminophen, caffeine, and acetylsalicylic acid, with fluid opiate screening and confirmation cutoffs of 30 pg/L are in use in the U.K. Utilizing 2.5-, 20-, 30-, and 40-pg/L GC-MS cutoffs, total daily codeine doses ranging from 60 to 240 mg (6). Al- 26%, 20%, 19%, and 18% of the oral fluid specimens were though used for pain relief and as a suppressant, codeine positive for codeine or one of its metabolites. Six Opiate also is abused for its euphoric and depressant effects (7-9). The ELISA/confirmation cutoff criteria (2.5/2.5, 10/2.5, 20/20, 30/20, detection of codeine is included in military, workplace, and 30/30, and 40/40 pg/L) were evaluated. Calculations for Opiate drug-testing programs because of its abuse and performance- ELISA sensitivity, specificity, and efficiency were determined from impairing effects. the number of true-positive, true-negative, false-positive, and false- Microtiter plate enzyme immunoassays have been used to negative results at each screening/confirmation cutoff. Sensitivity, screen blood, urine, serum, oral fluid, sweat, and hair for a va- specificity, and efficiency for the lowest cutoff were 91.5%, 88.6%, riety of of abuse (10-14). Improved performance charac- and 89.3%. Application of the cutoff currently used in the U.K. yielded sensitivity, specificity, and efficiency results of 79.7%, teristics, increased throughput, smaller sample volumes, and 99.0%, and 95.4% and similar results of 76.7%, 99.1%, and decreased costs have increased their utility for qualitative and 95.1% when applying the SAMHSA criteria. These data indicate semiquantitative analysis (14,15). Microplate immunoassays that the Opiate ELISA efficiently detects oral codeine use. In are the primary replacement for radioimmunoassays of drugs in addition, the data, collected following controlled oral codeine whole blood, utilized in many medical examiner and driving administration, may aid in the interpretation of opiate oral fluid test under the influence laboratories. Neogen Opiates Group (Lex- results and in the selection of appropriate oral fluid screening and ington, KY) microtiter plate assay results for 127 postmortem confirmation cutoffs. blood specimens were compared with gas chromatography- mass spectrometry (GC-MS) results at two cutoffs (20 and 50 9Author to whom correspondenceshould be addressed:Marilyn A. Hueslis,Ph.D., Acting Chief, CDM, IRP, NIDA, 5500 Na[han Shock Drive, Baltimore, MD 21224. E-mail: ]Jg/L) (16). Sensitivity, specificity, and efficiency results uti- [email protected],gov. lizing the 20- and 50-1Jg/L cutoffs were 95.2%, 92.2%, and

402 Reproduction (photocopying} of editorial content of this journal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 27, October 2003

93.7% and 88.8%, 96.8%, and 92.9%, respectively.Comparable to 72 h after codeine administration using three different performance was documented using microtiter plates from STC methods. Specimens were collected following citric acid candy Technologies (Bethlehem, PA) for over 200 postmortem blood stimulation and expectoration (N = 1077), and smaller subsets specimens from selected coroner's cases (15). Microtiter plate were collected with Salivette| neutral cotton swabs (N = 147) or enzyme immunoassays have also proven useful in oral fluid for Salivette citric acid-treated cotton swabs (N = 182). Oral fluid drug monitoring. Moore et al. (17) evaluated performance char- specimens collected with Salivette were extracted from the acteristics of the Cozart Microplate Opiate EtA oral fluid kit swabs by centrifugation and stored in polypropylene tubes at (Opiate ELISA)when testing 71 oral fluid specimens from vol- -20~ until Opiate ELISAand GC-MS analysis. unteers entering a substance abuse clinic. Sensitivity, speci- ficity, and efficiency were 89%, 92%, and 92%, respectively, Cozart Microplate Opiate EIA Oral Fluid Kit when utilizing a 30-lag/L Opiate ELISA cutoff and a GC-MS The Opiate ELISAis a competitive enzyme immunoassay for cutoff of 30 lag/L for morphine, codeine, , 6- the detection of opiates in human oral fluid, developedby Cozart acetylmorphine, and/or . Bioscience Ltd. (Oxfordshire, U.K.) (18). The assay was per-

Limited, controlled drug administration data in oral fluid are formed according to the manufacturer's protocol, with the ex- Downloaded from https://academic.oup.com/jat/article/27/7/402/784027 by guest on 23 September 2021 available for the interpretation of oral fluid test results. One of ception that the commercially available opiate kit was modified the undetermined issues for oral fluid testing is the selection of to optimize detection of codeine by substituting five codeine cal- appropriate screening and confirmation cutoffs. Recently, the ibrators (0, 1, 5, 10, and 50 lag/L) for the morphine calibrators. Substance Abuse and Mental Health Services Administration Codeine calibrators were employed because codeine has a (SAMHSA) proposed an oral fluid opiate screening cutoff of 40 greater cross-reactivity with these opiate antibodies than mor- lag/L and confirmation cutoffs of 40 pg/L for codeine and/or phine, and semiquantitative results were required to evaluate morphine. Oral fluid testing in the U.K. has utilized a 30-lag/L performance at different opiate cutoffs. Quality control sam- opiate screening cutoff and a confirmation cutoff of 30 lag/L ples were prepared by adding codeine to drug-free human oral for codeine and/or morphine. Pairing semiquantitative oral fluid fluid diluted 1:3 with 2 mol/L sodium acetate, the buffer from results from the Opiate ELISAand quantitative GC-MS data al- the Cozart fluid collection system. lowed us to investigate immunoassay performance at these and Each microtiter plate was calibrated in duplicate, yielding other relevant cutoffs. semiquantitative results between I and 50 pg/L. Results greater The goals of this controlled drug administration study were to than the upper limit of linearity were not extrapolated beyond evaluate the sensitivity, specificity, and efficiency of the Opiate the curve limits and were reported as greater than the highest ELISA, as compared with GC-MS for detecting codeine use and calibrator. Concentrations of oral fluid specimens were deter- to evaluate potential opiate oral fluid cutoffs for screening and mined in singlate, and duplicate quality control samples were in- confirmation assays. cluded to monitor assay performance. Between-run precision for the low and high quality control samples was calculated from ab- sorbances across the 19 Opiate ELISAplates (N = 37). Interassay Materials and Methods precision for the low control was 21.3% with a mean absorbance of 0.179 • 0.038 and 27.2% for the high control with a mean of Participants 0.125 • 0.034. Twelve men and seven women (14 African American, 3 Cau- casian, and 2 Hispanic) participated in a controlled codeine ad- ministration protocol approved by the National Institute on Experimental Drug Abuse Institutional Review Board. Volunteers provided informed consent, remained on a closed clinical research ward under continuous medical surveillance for 10 weeks and re- Chemicals and reagents ported a history of opiate use that was verified by a positive Chemicals were obtained from the following sources: urine opiate test. codeine.P04, [2Ha]-codeine hydrochloride.2H20, norcodeine hydrochloride.3H20, morphine sulfate, [2Ha]-morphine Drug administration hydrochoride.3H20, normorphine hydrochloride.H20 from Codeine sulfate, obtained from Roxane Laboratories Sigma Chemicals (St Louis, MO) and N,O-bis(trimethyi)triflu- (Columbus, OH), was administered orally in capsules. After an oroacetamide (BSTFA)with 1% trimethylchlorosilane (TMCS) initial washout period of three weeks to permit elimination of and N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide previously self-administered opiates, subjects received three (MTBSTFA)with 1% tert-butyldimethylchlorosilane (TBDMCS) low doses of oral codeine sulfate (60 mg/70 kg) within seven days from Pierce Chemical (Rockford, IL). Deuterated codeine and, following a three-week interval, three high doses (120 was used as the internal standard for codeine and norcodeine, mg/70 kg) within the same time frame. The dosage regimen and and deuterated morphine was used as the internal standard route of administration were selected to minimize adverse drug for morphine and normorphine. Solid-phase extraction (SPE) effects. columns (Clean Screen ZSDAU020) and filtration columns (RFV02F4P) were obtained from United Chemical Technologies Oral fluid collection (Bristol, PA). Solvents were HPLC grade and purchased from Oral fluid specimens (AT= 1406) were collected prior to and up the following sources: methylene chloride, 2-propanol, and

403 Journal of Analytical Toxicology,Vol. 27, October 2003 acetonitrile from Mallinckroft Baker Inc. (Phillipsburg, NJ) Screening and confirmation cutoff concentrations and methanol from Fisher Scientific (Fair Lawn, NJ). Sodium Six Opiate ELISA/GC-MScutoffs were evaluated. The lowest acetate, acetic acid, ammonium hydroxide, and hydrochloric cutoffs, 2.5/2.5 and 10/2.5, were at the LOQ of the GC-MS assay. acid were ACS-reagent grade and obtained from Mallinckroft In addition to the SAMHSA (40/40) and U.K. (30/30) cutoffs, Baker Inc. thresholds at 20/20 and 30/20 were assessed. Adhering to the criteria for the U.K. and SAMHSA cutoffs, only codeine and Extraction morphine were used in calculations to determine TP, TN, FP, Samples were analyzed for codeine and three metabolites and FN tests. However, for the other ELISA/confirmation cut- using SPE and GC-MS according to a previously published offs, codeine, norcodeine, morphine, and normorphine con- procedure (19-22). Drug-free oral fluid was spiked with codeine, centrations were evaluated. norcodeine, morphine, and normorphine at concentrations ranging from 2.5 to 500 lag/L. For each analytical run, two calibration curves were generated, 2.5-50 lag/L and 50-

500 pg/L. Splitting the curves improved sensitivity and Results Downloaded from https://academic.oup.com/jat/article/27/7/402/784027 by guest on 23 September 2021 linearity for all analytes of interest (r > 0.98) and allowed us to achieve limits of detection and quantitation (LOQ) of Cross-reactivity 2.5 pg/L for all analytes. Quality control samples utilized We evaluated the cross-reactivity of the Opiate ELISA to de- stock solutions from a different source and were spiked to termine which prescription or illicit could produce pos- contain 0, 12.5, 25, and 250 pg/L of codeine, norcodeine, itive immunoassay results. Table I lists the cross-reactivity of morphine, and normorphine. Quantitation of control samples and closely related compounds as compared with codeine was required to be within + 20% of theoretical values. Ion ratios activity. Even at elevated concentrations, norcodeine exhibited of quality control samples and participant specimens were poor cross-reactivity. In contrast to norcodeine, there was good required to be within • 20% of those observed for the 10 and cross-reactivity and, hence, high sensitivity for , di- 100-ng calibrators' ion ratios for the low and high calibration hydrocodone, and pholcodine. curves, respectively. Briefly, 1 mL of oral fluid and 100 pL of (1 Iag/mL) internal standard solution (COD-d3, MOR-d3) were Codeine and metabolites in oral fluid extracted by SPE (Clean Screen ZSDAU020) and derivatized Following controlled administration of three 60-mg/70-kg with MTBSTFAcontaining 1% TBDMCSand BSTFAcontaining and three 120-mg/70-kg oral codeine sulfate doses, codeine 1% TMCS. was the primary analyte and norcodeine the only metabolite de- GC-MS analysis of the derivatized extract was performed on tected in oral fluid with our GC-MS LOQ of 2.5 lag/L. Mor- a Hewlett-Packard (Palo Alto, CA) 5890A GC interfaced with a Hewlett-Packard 5972 mass selective detector or a Hewlett- Packard 6890 GC interfaced with a Hewlett-Packard 5973 mass Table I. Cross-Reactivity of the Cozart Microplate Opiate selective detector utilizing electron impact operated in the EIA Oral Fluid Kit splitless mode. Separation of analytes was achieved using an Apparent HP-1 fused-silicacapillary column (12 m x 0.2-ram i.d., 0.33 lam Codeine film thickness). Three ions for each analyte were monitored Concentration Concentration % Cross- (quantitative ion in parenthesis): [2H3]-codeine,m/z (374), 237, Compound (pg/L) (pg/L) Reactivity* 181; codeine, m/z (371), 234, 178; norcodeine, m/z (429), 254, 292; [2H3]-morphine,m/z (417), 474, 281; morphine, m/z (414), 100,000 12.6 0.00126 471,278; and normorphine, m/z (472), 529, 350. 10,000 2.3 0.0226 Norcodeine 10,000 19.8 0.198 Performance evaluation Norcodeine 1000 2.8 0.28 True-positive (TP), true-negative (TN), false-positive (FP), 1000 4.4 0.44 and false-negative (FN) results were determined by comparing Morphine-3-glucoronide 1000 8.1 0.81 immunoassay with GC-MS results. A sample was considered Heroin 100 2.7 2.7 positive if the analytical result was greater than or equal to the 100 4.4 4.4 specified cutoff. Therefore, a sample was considered TP if there 6-Monoacetylrnorphine 100 4.8 4.8 was a positive immunoassay result and codeine, norcodeine, Morphine 100 8.5 8.5 Morphine 10 1.4 14.0 morphine, and/or normorphine were positive by GC-MS. If Hydrocodone 100 16.5 16.5 both results were negative, the sample was a TN. A positive Hydrocodone 10 3.4 34.2 immunoassay result and a GC-MS result negative for all ana- Dihydrocodeine 10 5.1 51,0 lytes was considered an FP. A negative immunoassay result and Pholcodine 10 5.6 56.2 a GC-MS result positive for at least one of the opiate analytes Pholcodine 5 3.5 69.9 was considered an FN. Sensitivity of the immunoassay at a spe- Codeinet 100 100 100 cific cutoff was calculated as TP/(TP + FN) x 100 and specificity ' Cross-reactivity= (ApparentConcentration/Target Concentration) x 100. as TN/(TN + FP) x 100. Efficiency was calculated as (TP + Codeinecalibrators were used as the reference. TN)/total number x 100.

404 Journal of Analytical Toxicology, Vol. 27, October 2003 phine and normorphine were not detected in any oral fluid but decreased the number of FP results by 109. Specificity and specimens. Norcodeine was never present without concurrent efficiency were high, 99.1% and 95.1%, respectively, but there codeine and, on only one occasion, did the concentration of was a 14.8% decrease in sensitivity to 76.7%. We anticipated norcodeine exceed that of codeine. At the GC-MS LOQ, 26% that lowering the SAMHSAthreshold cutoffs to those currently (N = 365) of the specimens were positive for codeine and 13% utilized in the U.K. (30/30) would improve performance char- (N = 183) for norcodeine. Codeine concentrations ranged from acteristics. Although efficiencyand specificitydiffered only frac- 2.5 to 3961 lag/Land norcodeine from 2.6 to 191 lag/L. In addi- tionally, 17 additional TP specimens were identified with the tion, 1041 specimens (74%) had codeine and norcodeine con- 30/30 U.K. cutoff. centrations below the GC-MS LOQ and were defined as negative We evaluated two additional cutoffs with concentrations specimens. below the U.K. and SAMHSAthresholds. When comparing assay performance at 20/20 and 30/20, as expected, we found that the Performance characteristics at different cutoffs higher Opiate ELISAscreening cutoff increased specificity and The designation of individual specimens as TP, TN, FP, and FN decreased sensitivity. In contrast, sensitivity increased when and the performance characteristics of the Opiate ELISAat dif- the screening cutoff was maintained and the confirmation Downloaded from https://academic.oup.com/jat/article/27/7/402/784027 by guest on 23 September 2021 ferent immunoassay and GC-MS cutoffs are summarized in threshold increased, as illustrated with the 30/20 and 30/30 Table II. As expected, the lowest cutoff (2.5/2.5) exhibited the cutoffs. The best assay efficiency(95.6%) of the six evaluated was greatest overall sensitivity (91.5%), yet produced the greatest obtained with the 20/20 cutoff. Sensitivity was greater than number (119) of FP results. Specificityand efficiencywere both 80% and specificity greater than 98%. Assay performance char- greater than 88%, but this large number of FP tests would put acteristics for the four higher cutoffs were comparable with an unnecessary burden on the confirmation section of a forensic total differences of 36 TP results and 5 FP results between the laboratory or, if employed in a screening only treatment pro- 20/20 and 40/40 cutoffs. All four threshold concentrations are gram, would produce too many unsupportable results. Raising within the analytical capabilities of the Opiate ELISAand rou- the Opiate ELISA cutoff from 2.5 to 10 lag/L and maintaining tine GC-MS analysis, however the 20/20 cutoff showed im- the GC-MS cutoff of 2.5 lag/Lyielded an 18% loss of assay sen- proved performance characteristics over both SAMHSA and sitivity because of the large number (97) of FN specimens. U.K. cutoffs. However, this higher Opiate ELISA screening cutoff greatly improved specificity and decreased the number of FP results. A similar efficiencyof 9196 was found. The use of a 2.5-lag/L cutoff may not be practical for forensic applications because of ana- Discussion lytical limitations of the confirmation assay. Performance char- acteristics at higher confirmation cutoffs of 20 lag/Lor greater The testing for opiate use and abuse historically has included had efficienciesof better than 94%. detection and confirmation of codeine and morphine. The in- SAMHSAhas proposed a 40-lag/L opiate screening cutoffwith clusion of norcodeine in the GC-MS confirmation analysis had a confirmation cutoff of 40 ]ag/Lmorphine and/or codeine to de- no effect on assay performance characteristics because nor- tect opiate use in oral fluid for federally mandated drug testing codeine was never present without concurrent codeine. In ad- programs. Comparing assay performance at these cutoffs with dition, norcodeine has low cross-reactivity with Opiate ELISA our lowest cutoff (2.5/2.5) identified 143 fewer TP specimens, antibodies. Following our controlled codeine administration, norcodeine was detected at a concentration range of 2.6-191 lag/L, concentrations well Table II. Sensitivity, Specificity, and Efficieny of the Cozart Microplate EIA below those needed to generate a positive Opiate Oral Fluid Kit for Detection of Codeine and Metabolites in Oral Opiate ELISA result. Compounds with higher Fluid at Different Immunoassay/GC-MS Cutoffs (N = 1406) cross-reactivity (hydrocodone, dihydrocodeine, and pholcodine) were not present in our con- Cutoff(pg/L) 2.5/2.5' 10/2.5' 20/20* 30/20' U.K.~ SAMHSAt trolled study and did not contribute to semi-

TP 334 268 227 210 208 191 tivity, specificity, and efficiency results. How- I=P 119 21 15 9 11 10 ever, if present in routine oral fluid specimens, TN 922 1020 1107 1113 1124 1137 these compounds most likely would produce FN 31 97 47 64 53 58 positive Opiate ELISA tests that would not be Sensitivityw (%) 91.5 73.4 82.9 76.6 79.7 76.7 confirmed if only codeine and morphine were Specificity~ (%) 88.6 98.0 98.7 99.2 99.0 99.1 targeted analytes. Efficiencyw (%) 89.3 91.6 95.6 94.8 95.4 95.1 These oral fluid screening and confirmation data following controlled codeine drug admin- * Cozart Micro#ate Opiate EIA Oral Fluid Kit screening cutoff/morphine, normorphine, codeine, and/or norcodeineconfirmation cutoff by GC-MS. istration will aid in the interpretation of oral t Cutoffs currently utilized in the U.K. for the analysisof opiates in oral fluid: 30-pg/L screening cutoff and 30- fluid drug tests. In addition, they permit quan- pg/L confirmation cutoff for morphine and/or codeine. * ProposedSAMHSA cutoffs for opiates in oral fluid: 40-1Jg/Lscreening cutoff and 40-pg/I. confirmation cutoff titative evaluation of potential immunoassay for morphine and/or codeine. and confirmation cutoffs. The selection of ap- s Sensitivity,specificity, and efficiency were calculated using the following formula: sensitivity = TP/(TP + FN) x 100, specificity = TN/(TN + FP) x 1000, and efficiency = (TP + TN)/(TP + TN + FP + FN) x 100. propriate screening and confirmation cutoffs will be important in establishing oral fluid as an

405 Journal of Analytical Toxicology,Vol. 27, October 2003 alternative matrix for drug testing. This controlled codeine ad- lected oral fluid specimens for opiates by immunoassay. J. Anal. ministration study demonstrates that the Cozart Opiate ELISA Toxicol. 25:310-315 (2001). 12. M.A. Huestis, E.J. Cone, C.J. Wong, A. Umbricht, and K.L. Preston. provides a suitable an adequate screening procedure for de- Monitoring opiate use in substance abuse treatment patients with tecting codeine exposure, with supporting evidence that a 20/20 sweat and urine drug testing. J. Anal. Toxicol. 24:509-521 (2000). cutoff improves performance characteristics. 13. C. Moore, D. Deitermann, D. Lewis, B. Feeley, and R.S. Niedbala. The detection of cocaine in hair specimens using micro-plate en- zyme immunoassay. J. Forensic Sci. 44:609-612 (1999). 14. B.J. Perrigo and B.P. Joynt. Use of ELISA for the detection of References common drugs of abuse in forensic whole blood samples. Can. Soc. Forensic Sci. 28:261-269 (1995). 15. V.R. Spiehler, I.B. Collison, P.R. Sedgwick, S.L. Perez, S.D. Le, 1. H.W. Peel, B.J. Perrigo, and N.Z. Mikhael. Detection of drugs in and D.A. Farnin. Validation of an automated microplate enzyme saliva of impaired drivers. J. Forensic ScL 29:185-189 (1984). immunoassay for screening of postmortem blood for drugs of 2. W. Schramm, R.H. Smith, flA. Craig, and D.A. Kidwell. Drugs of abuse. J. Anal. Toxicol. 22:573-579 (1998). abuse in saliva: a review. J. Anal. ToxicoL 16:1-9 (1992). 16. P. Kemp, G. Sneed, T. Kupiec, and V. Spiehler. Validation of a mi- 3. E.J. Cone. Saliva testing for drugs of abuse. Ann. N.Y. Acad. 5ci. crotiter plate ELISA for screening of postmortem blood for opiates Downloaded from https://academic.oup.com/jat/article/27/7/402/784027 by guest on 23 September 2021 694:91-127 (1993). and benzodiazepines. J. Anal. Toxicol. 26:504-512 (2002). 4. A. Jehanli, S. Brannan, L. Moore, and V.R. Spiehler. Blind trials of 17. L. Moore, J. Wicks, V. Spiehler, and R. Holgate. Gas chromatog- an onsite saliva drug test for marijuana and opiates. J. Forensic Sci. raphy-mass spectrometry confirmation of Cozart RapiScan saliva 46" 121 4-1220 (2001). and opiates tests. J. Anal. Toxicol. 25:520-524 (2001). 5. D.A. Kidwell. Discussion: caveats in testing for drugs of abuse. 18. Cozart Bioscience, Ltd. Cozart Microplate EIA Opiate Oral Fluid Kit NIDA Res. Monogr. 117:98-120 (1992). Technical Sheet (Version 05/01). 6. R.C. Baselt. Disposition of Toxic Drugs and Chemicals in Man, 2nd 19. M.A. Huestis, J.M. Oyler, E.J. Cone, A.T. Wstadik, D. Schoen- ed. Biomedical Publications, Davis, CA, 1989, pp 214-218. doffer, and R.E. Joseph, Jr. Sweat testing for cocaine, codeine and 7. B.A. Sproule, U.E. Busto, G. Somer, M.K. Romach, and E.M. metabolites by gas chromatography-mass spectrometry. J. Chro- Sellers. Characteristics of dependent and nondependent regular matogr. B 733:247-264 (1999). users of codeine. J. Clin. Psychopharmacol. 19:367-372 (1999). 20. J.M. Oyler, E.J. Cone, R.E. Joseph, Jr., E.T. Moolchan, and M.A. 8. M.K. Romach, B.A. Sproule, E.M. Sellers, G. Somer, and U.E. Huestis. Duration of detectable methamphetamine and am- Busto. Long-term codeine use is associated with depressive symp- phetamine in urine after controlled oral administration of toms. J. Clin. Psychopharmacol. 19:373-376 (1999). methamphetamine to humans. Clin. Chem. 48:1703-1714 (2002). 9. P. Isaac, W. Seto, K.L. Lanctot, and U.E. Busto. Use and abuse of 21. I. Kim, A.J. Barnes, J.M. Oyler, R. Schepers, R.E. Joseph, Jr., E.J. prescription opioids in Canada 1978 to 1989. Can. J. Clin. Phar- Cone, D. Lafko, E.T. Moolchan, and M.A. Huestis. Plasma and oral macol. 2:81-86 (1995). fluid and pharmacodynamics after oral codeine 10. S. Kerrigan and W.H. Phillips, Jr. Comparison of ELISAs for opiates, administration. Clin. Chem. 48:1486-1496 (2002). methamphetamine, cocaine metabolite, benzodiazepines, phen- 22. R.J. Schepers, J.M. Oyter, R.E. Joseph, Jr., E.J.Cone, E.T. Moolchan, cyclidine, and cannabinoids in whole blood and urine. Clin. and M.A. Huestis. Methamphetamine and amphetamine pharma- Chem. 47:540-547 (2001). cokinetics in oral fluid and plasma after controlled oral metham- 11. R.S. Niedbala, K. Kardos, J. Waga, D. Fritch, L. Yeager, S. Dod- phetamine administration to human volunteers. Clin. Chem. 49: damane, and E. Schoener. Laboratory analysis of remotely col- 121-132 (2003).

406