Journal of Analytical Toxicology, VoL 14, NovernberlDecember 1990
Comparison of a TLC Method with EMIT| and GC/MS for Detection of Cannabinoids in Urine* R o d g e r L. Foltz t
Center for Human Toxicology, Univemity of Utah, Sa/t Lake City, Utah 84112 Irving Sunshine
Palo Alto, Ca/ifornia
IA b s t r a c t
I
Urine specimens were analyzed in parallel with a new TLC method, an EMIT| assay, and a reference GC/MS method. At a 9~ cutoff of 20 ng/mL, the TLC method correctly identified 92% of the positive urines and 97% of the negative urines. In contrast, only 63% of the urine specimens shown by GC/MS to contain greater than 20 ng/mL of 9-carboxy-THC were identified as positive by the EMIT d.a.u, assay at the 100-ng/mL cannab]noid cutoff.
throughput than do previ0usly described TLC methods (30, 31). With this TLC procedure the hydrolyzed urine samples are aspirated directly through a biphasic thin-layer chromatogram containing a porous, alkylsilica "extraction layer" located along the lower edge and an upper silica gel/glass fiber phase for the TLC separation. Solid-phase extraction of 9-carboxy-THC within the alkylsilica layer simultaneously removes the metabolite from the matrix and concentrates it on the TLC plate. A rapid TLC development step then sepamtes 9-carboxyTHC from the other components of the extract for subsequent visualization with Fast Blue BB. We present the results of a study in which the new TLC technique was compared with an immunoassay a n d a reference GC/MS method (12).
Introduction
11-nor-Ag-Tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC), the major metabolite of A9-tetrahydrocannabinol, can be detected in urine by various analytical methods. These include enzyme multiplied immunoassay (EMIT| (1-3), radioimmunoassay (RIA) (4-6), fluorescence polarization immunoassay (FPIA) (7,8), gas chromatography (GC) (9,10), gas chromatography/mass spectrometry (GC/MS) (11-18), highperformance liquid chromatography (HPLC) (19-22), and thinlayer chromatography (TLC) (23-28). Recently, the U.S. Department of Health and Human Services (DHHS) issued mandatory guidelines that iisted the types of analytical techniques permissible in federal drug-testing programs (29). For the analysis of "total cannabinoid metabolites" in urine, the guidelines require an initial immunoassay, using a cutoff value of 100 ng/mL. AII presumptive positive immunoassay results must be confirmed by GC/MS at a cutoff concentration of 15 ng/mL of the 9-carboxy-THC. Immunoassays are recommended for the initial test because they are considered to be more sensitive than TLC and more rapid and less expensive than GC or HPLC. Also, immunoassays are more easily automated than tests based on chromatogmphic processes. However, a recently reported TLC technique provides the sensitivity and specificity necessary for ah effective initial test method and permits higher sample *Presenled in part at the meeting of the California Associalion of Toxicologists held in San Diego, California, May 7. 1988. tAuthor to whom correspondence should be addressed.
Materials and Methods
Thin-layer chromatography. The TLC system (TOXI-MS Cannabinoid Test) was obtained from Toxi-Lab, Inc. and was received a s a prototype test package containing all apparatus, reagents, and controls necessary to perform 750 assays for cannabinoids in urine. The tests were performed by a technologist at the Center for Human Toxicology, according to the manufacturer's instructions (31). S imultaneous extraction of 10 specimens and one control (AS-9-carboxy-THC, 20 ng/mL) was achieved by means of a disposable applicator cart¡ that effectively seated the TLC extraction layer to a self-contained vacuum apparatus (31). Before chromatographic separation, a 3-mm diameter standard disc containing 350 ng AS-9-carboxyTHC was inserted into a hole in the chromatogram. The developing rack permitted migration of six chromatograms simultaneously to a distance of 4.5 cm in approximately two minutes. 9-Carboxy-THC in the specimens was visualized asa narrow red band 3.5 cm from the o¡ (Rf0.78) which became purple on exposure to HCI vapor, matching the color and position of both the standard and the control. Only those samples that produced red bands whose size and intensity was greater than or equal to the 20-ng/mL control were classified as positive. Thin-layer chromatograms were preserved by placing transparent tape across both sides of the TLC medium at the
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
375
Journal of AnalyticalToxicology,Vol. 14, NovemberlDecember 1990
Rf of 9-carboxy-THC. The chromatograms were stored protected from light. hnnnmoassay. The EMIT-d.a.u. 100-ng cannabinoid assay (Syva Co.) was performed according to the manufacturer's instructions (32) at Northwest Toxicology, Inc., Salt Lake City, Utah, on an Eppendorf EPOS | Analyzer 5060 made by EM Diagnostics (Darmstadt, West Germany). Gas chromatography/mass spectrometry. All GC/MS assays were performed by personnel of the Center for Human Toxicology using negative ion chemical ionization of the hexafluoroisopropyl/pentafluoropropionyl de¡ of 9-carboxy-THC as previously desc¡ (12). In accordance with the proposed Department of Health and Human Services guidelines under consideration at the time of the study, a 9-carboxy-THC cutoff concentration of 20 ng/mL was used for both the GC/MS and TLC procedures. Sample analysis. A total of 541 samples were aliquoted, coded, and analyzed on a blind basis for both GC/MS and TLC analysis. Of these samples, only 465 were available for analysis by EMIT. The GC/MS results were initially obtained for 187 specimens received at the Center for Human Toxicology between September 4, 1986 and Morch 17, 1987. These samples had been stored frozen until analyzed by TLC (September 15-17, 1987) with an additional 354 specimens acquired from Northwest Toxicology. Initial GC/MS analysis of these 354 samples and reanalysis of 142 of the previously frozen samples were performed between October 27, 1987 and December 2, 1987. AII EMIT assays were also performed during this time.
Results
Comparison of TLC and GC/MS results. Table I compares the qualitative TLC results for 541 samples with the quantitative values obtained with the GC/MS reference method. The GC/MS procedure identified 194 samples (36%) that contained 9-carboxy-THC at concentrations > 20 ng/mL. The TLC method classified 178 of these 194 samples (92%) as true positives under the constraints of the 20-ng/mL cutoff value. The 16 GC/MS positive samples that were judged negative by TLC results were found to contain 9-corboxy-THC concentrations ranging from 20-92 ng/mL (mean = 36.4 ng/mL). The GC/MS analysis of these samples was performed after the TLC analysis, Table I. THC Metabolite Analysis by TLC and GC/MS Category
Numberof Sarnples
so that degradation of the 9-carboxy-THC could not have caused the negative TLC determinations. The GC/MS procedure identified 347 samples (64%) that contained less than 20 ng/mL 9-corboxy-THC. The TLC method correctly identified 336 of these 347 samples (97%) as true negatives (i.e. 9-carboxy-THC < 20 ng/mL). The 11 samples that were incorrectly classified as THC-positive by TLC based on the 20-ng/mL cutoff calibrator actually contained 9-carboxy-THC concentrations ranging from 5-19 ng/mL (mean= 13.7 ng/mL). No falsepositive TLC results (i.e. TLC > 20 ng/mL and GC/MS < 1 ng/mL) were observed. Comparison of EMIT and GC/MS results. The qualitative EMIT results for 465 samples ore compared to the GC/MS quantitations in Table II. The GC/MS method identified 172 (37%) of the 465 samples as having 9-corboxy-THC concentrations greater than 20 ng/mL. Of these 172 GC/MS-positive samples, 109 (63%) gave EMIT responses greater than the 100-ng/mL cutoff calibrator. The 63 samples (37%) negative by EMIT at the 100-ng/mL cutoff contained 9-carboxy-THC concentrations ranging from 20 to 137 ng/mL (mean = 52.4 ng/mL). Surprisingly, eight samples that were EMIT negative were found to contain 9-corboxy-THC concentrations exceeding 100 ng/mL. Five of these samples were available 6 months later and were retested by both methods, with the same results. The GC/MS method indicated that 293 (63.0%) of the 465 samples contained 9-carboxy-THC concentrations below 20 nglmL. The EMIT assay classified 288 (98%) of these as true negatives. Of the five samples that were positive by EMIT but negative by GC/MS, four actually contained 9-corboxy-THC, but at concentrations ranging from 14 to 19 ng/mL (mean = 17.8 ng/mL). GC/MS failed to detect 9-carboxy-THC in one EMIT-positive specimen at the minimum detection limit of 1 ng/mL.
Discussion
The performance of both the EMIT and the TLC were evaluated by the predictive value model (33), and the results are summarized in Table III. For the purpose of these calculations specimens that were positive by EMIT or TLC but which contained less than 20 ng/mL of 9-carboxy-THC were classified as "false positives." Table II. THC Metabolite Analysis by EMIT and GC/MS Category Nurnberof Sarnples
Total number analyzed by both TLC and GC/MS
541
Total number analyzed by both EMIT and GC/MS
465
GC/MS >_20 ng/rnL
194
GC/MS >_20 ng/mL
172
Both TLC and GC/MS _>20 ng/mL
178 (91% of GC/MS positives)
("Unconfirmed positive ") TLC _>20 ng/mL and GC/MS < 20 but > 1 ng/mL
EMIT > 100 ng/mL and GC/MS _>20 ng/mL
(=Unconfirmed positive') 11
EMIT _>100 ng/mL and GC/MS < 20 but > 1 ng/rnL
('False positive ")
('False positive")
TLC 20 ng/mL and GC/MS < 1 ng/mL
EMIT _> 100 ng/rnL and GC/MS < 1 ng/rnL
("False negative") TLC < 20 ng/mL and GC/MS > 20 ng/mL
376
109 (63% of GC/MS positives) 4 1
("False negative ") 17 (9% of GC/MS positives)
EMIT < 100 ng/mL and GC/MS :>20 ng/mL
63 (37% of GC/MS positives)
Journal of AnalyticalToxicology,Vol. 14, November/December1990
Table III. TLC (20 ng/mL) and EMIT d.a.u. (100 ng/mL) Performance vs. GC/MS (20 ng/mL) Prevalence Sensitivity Specificity PredictiveValue(-) PredictiveValue(+) Accuracy
TLC (%)
EMIT (%)
35.9 91.8 96.8 95.5 94.2 95.0
37.0 63.4 98.3 82.0 95.6 85.4
The TLC results correlate better with the GC/MS data than do the EMIT results. This is not surp¡ in view of the fact that the TLC and the GC/MS methods both detect a specific metabolite of THC, whereas other cannabinoids contribute to the total EMIT response. The TLC method did produce a greater incidence of unconfirmed positives (9-carboxy-THC present but less than 20 ng/mL), principally because of the inherent difficulty of visually discriminating between the color intensity in the 20-ng/mL control and sample concentrations of 9-carboxy-THC that were just below the control. Likewise, 11 of 16 samples that produced "false negative" results had 9carboxy-THC spot intensities which were very near the control, but were conservatively called negative. Densitometric measurements would provide more effective discrimination and eliminate the subjective nature of the interpretation. However, even without the use of a scanning densitometer, the technique proved to be an effective screening method, with an overall accuracy of 95%. The throughput of the TLC method was about 60 samples per hour when performed by one technologist. This represents a significant increase over conventional TLC methods (i.e. 10--20 samples/hour). The EMIT-GC/MS comparison indicates that the EMIT d.a.u. 100~ assay may yield an unexpectedly high percentage of "false-negative" tests. Out of 172 specimens found by GC/MS to have 9-carboxy-THC concentrations greater than 20 ng/mL, 63 were negative by EMIT at the 100-ng/mL cutoff. These results are qualitatively consistent with other reports that the EMIT cannabinoid assay gives a high "false negative" rate (34-37). Other immunoassays that employ the 100-ng/mL cutoff value can also give a high frequency of"false negative" results (38,39). These findiqgs suggest a need for reexamination of the current cannabinoid cutoff requirements in the Department of Health and Human Services' Mandatory Guidelines for Federal Workplace Drug Testing Programs.
Conclusion A new TLC system has been shown to be an effective method of screening urine specimens for the major metabolite of A9-tetrahydrocannabinol. The TLC assay permitted detection of 9-carboxy-THC concentrations down to l0 ng/mL. Although the TLC system does not provide the high throughput capability of an automated EMIT system, it does permita substantially greater throughput of samples than is achievable with conventional TLC methods. The agreement between the results obtained with the TLC assay anda reference GC/MS method was
better than that between the EMIT d.a.u. 100-ng assay and the GC/MS method.
Acknowledgment The GC/MS and TLC analyses were performed at the Center for Human Toxicology by Ann M. Caims and the EMIT assays were performed at Northwest Toxicology, Inc. by Claire Richardson.
References 1. M.J. deLaurentis, K. McNeil, A.J. Mann, S. Clark, and H.M. Greenwood. Ah EMIT assay for cannabinoid metabolites in urine. In The Analysis of Cannabinoids in Biological Fluids, NIDA Research Monograph No. 42, R.L. Hawks, Ed., U.S. Government Printing Office, Rockville, MD, 1982, pp. 69-84. 2. R. Rogers, C.P. Crowl, W.M. Eimstad, M.W. Hu, J.K. Kam, R.C. Ronald, G.L. Rowley, and E.F. UIIman. Homogenous enzyme immunoassay for cannabinoids in urine. Clin. Chem. 24. 95-100 (1978). 3. J.E. O'Connor and T.A. Rejent. EMIT cannabinoid assay: confirmation by RIA and GC/MS. J. AnaL ToxicoL 5:168-73 (1981). 4. C.E. Cook, H.H. Seltzman, V.H. Schindier, C.R. Tallent, K.M. Chin, and C.G. Pitt. Radioimmunoassays for cannabinoids. In The Analysis of Cannabinoids in Biological Fluids, NIDA Research Monograph No. 42. R.L. Hawks, Ed., U.S. Government Printing Office, Rockville, MD, 1982, pp. 19-32. 5. A.B. Jones, H.N. EISohly, and M.A. EISohly. Analysis of the major metabolite of delta-9-tetrahydrocannabinol in urine. V. Cross-reactivity of selected compounds in a radioimmunoassay. J. AnaL ToxicoL 8:252-54 (1984). 6. P.S. Childs and H.H. McCurdy. Evaluation of a radioimmunoassay (~251)for cannabinoid metabolites in urine and whole blood. J. AnaL ToxicoL 8- 220-23 (1984). 7. D.E. Moody, L.A. Reinersman, K.M. Monti, D.S. Barberio, A.M. Cairns, and T.A. Jennison. A new TDx procedure for urine cannabinoid detection. In Proceedings of the 24th Intema-
tional Meeting of the Intemational Association of Forensic Toxicologists, Banff, Alberta, G.R. Jones and P.P.Singer, Eds., published by the Alberta Society of Clinical and Forensic Toxicologists, Edmonton, AIberta, 1987, pp. 151-59. 8. L. Karlsson and M. Str5m. Laboratory evaluation of the TDx assay for detection of cannabinoids in urine from prison inmates. J. AnaL ToxicoL 12" 319-21 (1988). 9. M.A. ElSohly, E.S. Arafat, and A.B. Jones. Analysis of the major metabolite of A9-tetrahydrocannabinol in urine. II1. A GC/ECD procedure. J. AnaL ToxicoL 8; 7-9 (1984). 10. J.M. Rosenfeld, Y. Moharir, and S.D. Sandler. Selective sample preparation for determination of 11-nor-9-carboxy-Ag-tetrahydrocannabinol from human urine by gas chromatography with electron capture detection. AnaL Chem. 61- 925-28 (1989). 11. R.L. Foltz. Analysis of cannabinoids in physiological specimens by gas chromatography/mass spectrometry. In Advances in Analytical Toxicology, R.C. Baselt, Ed., Biomedical Publications, Davis, California, 1984, pp. 125-57. 12. K.M. McGinnis, D.S. Barbario, R. Bridges, and R.L. Foltz. A high sensitivity, high through-put assay for the major urinary metabolite of A9-tetrahydrocannabinol, Proceedings of the 33rd Annual Conference on Mass Spectrometry and Allied Topics in San Diego, CA, May 26-31, 1985, pp. 464-5.
377
Journal of Analytical Toxicology, Vol. 14, NovembedDecember 1990
13.T.S. Baker, J.V. Harry, J.W. RusseL[, and R.L Myers_ Rapid method for the GC/MS confirmation of 11-nor-9-carboxy-,~9-THC in urine. J. AnaL ToxicoL 8:255-59 (1984). 14. B.D. Paul, L.D. Mell, J.M. Mitchell, R.M. McKinley, and J. Irving. Detection and quantitation of urinary 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, a metabolite of tetrahydrocannabinol, by capillary gas chromatography and electron impact mass spectrometry. J. AnaL ToxicoL 11: 1-5 (1987). 15. L.J. McBurney, B.A. Bobbie, and L.A. Sepp. GC/MS and EMIT analysis for Ag-tetrahydrocannabinol metabolites in plasma and urine of human subiects. J. AnaL ToxicoL 10:56-64 (1986). 16. S.J. Mul› and G.A. Gasella. Confirmation of marijuana, cocaine, morphine, codeine, amphetamine, methamphetamine, pheneyclidine by GC/MS in urine following immunoassay screening. J. AnaL Toxicol. 12:102-107 (1988). 17. M. Congost, R. De la Torre, and J. Segura. Optimization of the quantitative analysis of the major cannabis metabolite (11-nor-9carboxy-Z~9-tetrahydrocannabinol) in urine by GC/MS. Biorned. Environ. Mass Spectrom. 16:367-72 (1988). 18. W.A. Joern. Detection of past and recurrent marijuana use by a modified GC/MS procedure. J. AnaL ToxicoL 11:49-52 (1987). 19. M.A. EISohly, H.N. EISohly, A.B. Jones, P.A. Dimson, and K.E. Wells. Analysis of the major metabolite of h9-tetrahydrocannabinol in urine. II. A HPLC procedure. J. AnaL ToxicoL 7: 262-84 (1983). 20. D.S. Isenschmid and Y.H. Caplan. A method for the determination of 11-nor-&9-tetrahydrocannabinol-9-earboxylic acid in urine using high performance liquid chromatography with electrochemical detection. J. AnaL ToxicoL 10:170-74 (1986). 21. D. Bourquin and R. Brenneisen. Confirmation of cannabis abuse by the determination of 11-nor-&9-tetrahydrocannabinol-9-ear boxylic acid in urine with high-performance liquid chromatography and electrochemical detection. J. Chromatogr. 414: 187-91 (1987). 22. B.L. Posey and S.N. Kimble. Quantitative determination of 11nor-Ag-tetrahydrocannabinol-9-carboxylic acid in urine by HPLC. J. AnaL ToxicoL 8:234-38 (1984). 23. R.C. Meatheral and J.C. Garriott. A sensitive thin-layer chromatographic procedure for the detection of urinary 11-nor-/t~tetrahydrocannabinol-9-carboxylic acid. J. Anal. ToxicoL 12: 136-40 (1988). 24. C.A. Sutheimer, R. Yarborough, B.R. Hepler, and I. Sunshine. Detection and confirmation of urinary cannabinoids. J. AnaL ToxicoL 9:156-60 (1985). 25. M.J. Kogan, E. Newman, and N.J. Willson. Detection of mari-
378
juana metaboIite 11-nor-A~-tetrahydrocannabSno;-9-carboxylic acid in human urine by bonded-phase adsorption and thin-layer chromatography. J. Chromatogr. 306:441-43 (1984). 26. G.R. Nakamura, W.J. Stall, V.A. Folen, and R.G. Masters. Thinlayer chromatographic-mass spectrometric identification of 11nor-Ag-tetrahydrocannabinol-9-carboxylic acid. J. Chromatogr. 264" 336-38 (1983). 27. J.A. Vinson and D.J. Lopatofsky. A semi-automated extraction and spotting system for drug analysis by TLC. Procedure for analysis of the major metabolite of Ag-tetrahydrocannabinol in urine. Jo Ana/. Toxicol. 9- 6-9 (1985). 28. K.K. Kaistha and R. Tadrus. Semi-quantitative thin-layer massscreening detection of 11-nor-Ag-THC-9-carboxylic acid in human urine. J. Chromatogr. 237:528-33 (1982). 29. Mandatory guide]ines for federal workplace drug testing programs. Federal Register53:11970-89 (1988). 30.D.L King, M.J. Gabor, P.A. Martel, and C.M. O'Donnell. Evaluation of a rapid TLC technique for detection of cannabinoid metabolites in urine. Clin. Chem. 33:972-73 (1987). 31. D.L. King, M.J. Gabor, P.A. Mar'tel, and C.M. O'Donnell. A rapid sample-preparation technique for thin-layer chromatographic analysis for 11-nor-Ag-tetrahydrocannabinol-9-carboxylic acid in human urine. Clin. Chem. 35:163-66 (1989). 32.EMIT 100-ng cannabinoid assay package insert, Syva Co., Palo Alto, California. 33. V.R. Spiehler, C.M. O'Donnell, and D.V. Gokhale. Confirmation and certainty in toxicology screening. Clin. Chem. 34:1535--39 (1988). 34. M.L. Abercrombie and J.S. Jewell. Evaluation of EMIT and R1A high volume test procedures for THC metabolites in urine utilizing GC/MS confirmation. J. AnaL ToxicoL 10:178-80 (1986). 35. D.R. Berkabile and A. Meyers. False negative rate for EMIT cannabinoids. J. AnaL ToxicoL 13:63 (1989). 36. W.A. Joem. Further observations: False negative EMIT cannabinoids. J. AnaL ToxicoL 13:126 (1989). 37. M. Lehrer and G.M. Meenan. More on the false negative rate for EMIT cannabinoids. J. AnaL ToxicoL 14:62 (1990). 38. J. Standefer. EMIT and TDx tests for THC: Same cutoff, different results. Forensic Urine Drug Testing Newsletter, Sept,: 4-5 (1988). 39. D.J. Wells and M.T. Barnhill, Jr. Comparative results with five cannabTnoTdimmunoassay systems at t~.e screening threshoId of 100 pg/L Clin. Chem. 35:2241-43 (1989). Manuscript received January 2, 1990; revision received March 30, 1990.
Comparison of a TLC Method with EMIT| and GC/MS for Detection of Cannabinoids in Urine* R o d g e r L. Foltz t
Center for Human Toxicology, Univemity of Utah, Sa/t Lake City, Utah 84112 Irving Sunshine
Palo Alto, Ca/ifornia
IA b s t r a c t
I
Urine specimens were analyzed in parallel with a new TLC method, an EMIT| assay, and a reference GC/MS method. At a 9~ cutoff of 20 ng/mL, the TLC method correctly identified 92% of the positive urines and 97% of the negative urines. In contrast, only 63% of the urine specimens shown by GC/MS to contain greater than 20 ng/mL of 9-carboxy-THC were identified as positive by the EMIT d.a.u, assay at the 100-ng/mL cannab]noid cutoff.
throughput than do previ0usly described TLC methods (30, 31). With this TLC procedure the hydrolyzed urine samples are aspirated directly through a biphasic thin-layer chromatogram containing a porous, alkylsilica "extraction layer" located along the lower edge and an upper silica gel/glass fiber phase for the TLC separation. Solid-phase extraction of 9-carboxy-THC within the alkylsilica layer simultaneously removes the metabolite from the matrix and concentrates it on the TLC plate. A rapid TLC development step then sepamtes 9-carboxyTHC from the other components of the extract for subsequent visualization with Fast Blue BB. We present the results of a study in which the new TLC technique was compared with an immunoassay a n d a reference GC/MS method (12).
Introduction
11-nor-Ag-Tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC), the major metabolite of A9-tetrahydrocannabinol, can be detected in urine by various analytical methods. These include enzyme multiplied immunoassay (EMIT| (1-3), radioimmunoassay (RIA) (4-6), fluorescence polarization immunoassay (FPIA) (7,8), gas chromatography (GC) (9,10), gas chromatography/mass spectrometry (GC/MS) (11-18), highperformance liquid chromatography (HPLC) (19-22), and thinlayer chromatography (TLC) (23-28). Recently, the U.S. Department of Health and Human Services (DHHS) issued mandatory guidelines that iisted the types of analytical techniques permissible in federal drug-testing programs (29). For the analysis of "total cannabinoid metabolites" in urine, the guidelines require an initial immunoassay, using a cutoff value of 100 ng/mL. AII presumptive positive immunoassay results must be confirmed by GC/MS at a cutoff concentration of 15 ng/mL of the 9-carboxy-THC. Immunoassays are recommended for the initial test because they are considered to be more sensitive than TLC and more rapid and less expensive than GC or HPLC. Also, immunoassays are more easily automated than tests based on chromatogmphic processes. However, a recently reported TLC technique provides the sensitivity and specificity necessary for ah effective initial test method and permits higher sample *Presenled in part at the meeting of the California Associalion of Toxicologists held in San Diego, California, May 7. 1988. tAuthor to whom correspondence should be addressed.
Materials and Methods
Thin-layer chromatography. The TLC system (TOXI-MS Cannabinoid Test) was obtained from Toxi-Lab, Inc. and was received a s a prototype test package containing all apparatus, reagents, and controls necessary to perform 750 assays for cannabinoids in urine. The tests were performed by a technologist at the Center for Human Toxicology, according to the manufacturer's instructions (31). S imultaneous extraction of 10 specimens and one control (AS-9-carboxy-THC, 20 ng/mL) was achieved by means of a disposable applicator cart¡ that effectively seated the TLC extraction layer to a self-contained vacuum apparatus (31). Before chromatographic separation, a 3-mm diameter standard disc containing 350 ng AS-9-carboxyTHC was inserted into a hole in the chromatogram. The developing rack permitted migration of six chromatograms simultaneously to a distance of 4.5 cm in approximately two minutes. 9-Carboxy-THC in the specimens was visualized asa narrow red band 3.5 cm from the o¡ (Rf0.78) which became purple on exposure to HCI vapor, matching the color and position of both the standard and the control. Only those samples that produced red bands whose size and intensity was greater than or equal to the 20-ng/mL control were classified as positive. Thin-layer chromatograms were preserved by placing transparent tape across both sides of the TLC medium at the
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
375
Journal of AnalyticalToxicology,Vol. 14, NovemberlDecember 1990
Rf of 9-carboxy-THC. The chromatograms were stored protected from light. hnnnmoassay. The EMIT-d.a.u. 100-ng cannabinoid assay (Syva Co.) was performed according to the manufacturer's instructions (32) at Northwest Toxicology, Inc., Salt Lake City, Utah, on an Eppendorf EPOS | Analyzer 5060 made by EM Diagnostics (Darmstadt, West Germany). Gas chromatography/mass spectrometry. All GC/MS assays were performed by personnel of the Center for Human Toxicology using negative ion chemical ionization of the hexafluoroisopropyl/pentafluoropropionyl de¡ of 9-carboxy-THC as previously desc¡ (12). In accordance with the proposed Department of Health and Human Services guidelines under consideration at the time of the study, a 9-carboxy-THC cutoff concentration of 20 ng/mL was used for both the GC/MS and TLC procedures. Sample analysis. A total of 541 samples were aliquoted, coded, and analyzed on a blind basis for both GC/MS and TLC analysis. Of these samples, only 465 were available for analysis by EMIT. The GC/MS results were initially obtained for 187 specimens received at the Center for Human Toxicology between September 4, 1986 and Morch 17, 1987. These samples had been stored frozen until analyzed by TLC (September 15-17, 1987) with an additional 354 specimens acquired from Northwest Toxicology. Initial GC/MS analysis of these 354 samples and reanalysis of 142 of the previously frozen samples were performed between October 27, 1987 and December 2, 1987. AII EMIT assays were also performed during this time.
Results
Comparison of TLC and GC/MS results. Table I compares the qualitative TLC results for 541 samples with the quantitative values obtained with the GC/MS reference method. The GC/MS procedure identified 194 samples (36%) that contained 9-carboxy-THC at concentrations > 20 ng/mL. The TLC method classified 178 of these 194 samples (92%) as true positives under the constraints of the 20-ng/mL cutoff value. The 16 GC/MS positive samples that were judged negative by TLC results were found to contain 9-corboxy-THC concentrations ranging from 20-92 ng/mL (mean = 36.4 ng/mL). The GC/MS analysis of these samples was performed after the TLC analysis, Table I. THC Metabolite Analysis by TLC and GC/MS Category
Numberof Sarnples
so that degradation of the 9-carboxy-THC could not have caused the negative TLC determinations. The GC/MS procedure identified 347 samples (64%) that contained less than 20 ng/mL 9-corboxy-THC. The TLC method correctly identified 336 of these 347 samples (97%) as true negatives (i.e. 9-carboxy-THC < 20 ng/mL). The 11 samples that were incorrectly classified as THC-positive by TLC based on the 20-ng/mL cutoff calibrator actually contained 9-carboxy-THC concentrations ranging from 5-19 ng/mL (mean= 13.7 ng/mL). No falsepositive TLC results (i.e. TLC > 20 ng/mL and GC/MS < 1 ng/mL) were observed. Comparison of EMIT and GC/MS results. The qualitative EMIT results for 465 samples ore compared to the GC/MS quantitations in Table II. The GC/MS method identified 172 (37%) of the 465 samples as having 9-corboxy-THC concentrations greater than 20 ng/mL. Of these 172 GC/MS-positive samples, 109 (63%) gave EMIT responses greater than the 100-ng/mL cutoff calibrator. The 63 samples (37%) negative by EMIT at the 100-ng/mL cutoff contained 9-carboxy-THC concentrations ranging from 20 to 137 ng/mL (mean = 52.4 ng/mL). Surprisingly, eight samples that were EMIT negative were found to contain 9-corboxy-THC concentrations exceeding 100 ng/mL. Five of these samples were available 6 months later and were retested by both methods, with the same results. The GC/MS method indicated that 293 (63.0%) of the 465 samples contained 9-carboxy-THC concentrations below 20 nglmL. The EMIT assay classified 288 (98%) of these as true negatives. Of the five samples that were positive by EMIT but negative by GC/MS, four actually contained 9-corboxy-THC, but at concentrations ranging from 14 to 19 ng/mL (mean = 17.8 ng/mL). GC/MS failed to detect 9-carboxy-THC in one EMIT-positive specimen at the minimum detection limit of 1 ng/mL.
Discussion
The performance of both the EMIT and the TLC were evaluated by the predictive value model (33), and the results are summarized in Table III. For the purpose of these calculations specimens that were positive by EMIT or TLC but which contained less than 20 ng/mL of 9-carboxy-THC were classified as "false positives." Table II. THC Metabolite Analysis by EMIT and GC/MS Category Nurnberof Sarnples
Total number analyzed by both TLC and GC/MS
541
Total number analyzed by both EMIT and GC/MS
465
GC/MS >_20 ng/rnL
194
GC/MS >_20 ng/mL
172
Both TLC and GC/MS _>20 ng/mL
178 (91% of GC/MS positives)
("Unconfirmed positive ") TLC _>20 ng/mL and GC/MS < 20 but > 1 ng/mL
EMIT > 100 ng/mL and GC/MS _>20 ng/mL
(=Unconfirmed positive') 11
EMIT _>100 ng/mL and GC/MS < 20 but > 1 ng/rnL
('False positive ")
('False positive")
TLC 20 ng/mL and GC/MS < 1 ng/mL
EMIT _> 100 ng/rnL and GC/MS < 1 ng/rnL
("False negative") TLC < 20 ng/mL and GC/MS > 20 ng/mL
376
109 (63% of GC/MS positives) 4 1
("False negative ") 17 (9% of GC/MS positives)
EMIT < 100 ng/mL and GC/MS :>20 ng/mL
63 (37% of GC/MS positives)
Journal of AnalyticalToxicology,Vol. 14, November/December1990
Table III. TLC (20 ng/mL) and EMIT d.a.u. (100 ng/mL) Performance vs. GC/MS (20 ng/mL) Prevalence Sensitivity Specificity PredictiveValue(-) PredictiveValue(+) Accuracy
TLC (%)
EMIT (%)
35.9 91.8 96.8 95.5 94.2 95.0
37.0 63.4 98.3 82.0 95.6 85.4
The TLC results correlate better with the GC/MS data than do the EMIT results. This is not surp¡ in view of the fact that the TLC and the GC/MS methods both detect a specific metabolite of THC, whereas other cannabinoids contribute to the total EMIT response. The TLC method did produce a greater incidence of unconfirmed positives (9-carboxy-THC present but less than 20 ng/mL), principally because of the inherent difficulty of visually discriminating between the color intensity in the 20-ng/mL control and sample concentrations of 9-carboxy-THC that were just below the control. Likewise, 11 of 16 samples that produced "false negative" results had 9carboxy-THC spot intensities which were very near the control, but were conservatively called negative. Densitometric measurements would provide more effective discrimination and eliminate the subjective nature of the interpretation. However, even without the use of a scanning densitometer, the technique proved to be an effective screening method, with an overall accuracy of 95%. The throughput of the TLC method was about 60 samples per hour when performed by one technologist. This represents a significant increase over conventional TLC methods (i.e. 10--20 samples/hour). The EMIT-GC/MS comparison indicates that the EMIT d.a.u. 100~ assay may yield an unexpectedly high percentage of "false-negative" tests. Out of 172 specimens found by GC/MS to have 9-carboxy-THC concentrations greater than 20 ng/mL, 63 were negative by EMIT at the 100-ng/mL cutoff. These results are qualitatively consistent with other reports that the EMIT cannabinoid assay gives a high "false negative" rate (34-37). Other immunoassays that employ the 100-ng/mL cutoff value can also give a high frequency of"false negative" results (38,39). These findiqgs suggest a need for reexamination of the current cannabinoid cutoff requirements in the Department of Health and Human Services' Mandatory Guidelines for Federal Workplace Drug Testing Programs.
Conclusion A new TLC system has been shown to be an effective method of screening urine specimens for the major metabolite of A9-tetrahydrocannabinol. The TLC assay permitted detection of 9-carboxy-THC concentrations down to l0 ng/mL. Although the TLC system does not provide the high throughput capability of an automated EMIT system, it does permita substantially greater throughput of samples than is achievable with conventional TLC methods. The agreement between the results obtained with the TLC assay anda reference GC/MS method was
better than that between the EMIT d.a.u. 100-ng assay and the GC/MS method.
Acknowledgment The GC/MS and TLC analyses were performed at the Center for Human Toxicology by Ann M. Caims and the EMIT assays were performed at Northwest Toxicology, Inc. by Claire Richardson.
References 1. M.J. deLaurentis, K. McNeil, A.J. Mann, S. Clark, and H.M. Greenwood. Ah EMIT assay for cannabinoid metabolites in urine. In The Analysis of Cannabinoids in Biological Fluids, NIDA Research Monograph No. 42, R.L. Hawks, Ed., U.S. Government Printing Office, Rockville, MD, 1982, pp. 69-84. 2. R. Rogers, C.P. Crowl, W.M. Eimstad, M.W. Hu, J.K. Kam, R.C. Ronald, G.L. Rowley, and E.F. UIIman. Homogenous enzyme immunoassay for cannabinoids in urine. Clin. Chem. 24. 95-100 (1978). 3. J.E. O'Connor and T.A. Rejent. EMIT cannabinoid assay: confirmation by RIA and GC/MS. J. AnaL ToxicoL 5:168-73 (1981). 4. C.E. Cook, H.H. Seltzman, V.H. Schindier, C.R. Tallent, K.M. Chin, and C.G. Pitt. Radioimmunoassays for cannabinoids. In The Analysis of Cannabinoids in Biological Fluids, NIDA Research Monograph No. 42. R.L. Hawks, Ed., U.S. Government Printing Office, Rockville, MD, 1982, pp. 19-32. 5. A.B. Jones, H.N. EISohly, and M.A. EISohly. Analysis of the major metabolite of delta-9-tetrahydrocannabinol in urine. V. Cross-reactivity of selected compounds in a radioimmunoassay. J. AnaL ToxicoL 8:252-54 (1984). 6. P.S. Childs and H.H. McCurdy. Evaluation of a radioimmunoassay (~251)for cannabinoid metabolites in urine and whole blood. J. AnaL ToxicoL 8- 220-23 (1984). 7. D.E. Moody, L.A. Reinersman, K.M. Monti, D.S. Barberio, A.M. Cairns, and T.A. Jennison. A new TDx procedure for urine cannabinoid detection. In Proceedings of the 24th Intema-
tional Meeting of the Intemational Association of Forensic Toxicologists, Banff, Alberta, G.R. Jones and P.P.Singer, Eds., published by the Alberta Society of Clinical and Forensic Toxicologists, Edmonton, AIberta, 1987, pp. 151-59. 8. L. Karlsson and M. Str5m. Laboratory evaluation of the TDx assay for detection of cannabinoids in urine from prison inmates. J. AnaL ToxicoL 12" 319-21 (1988). 9. M.A. ElSohly, E.S. Arafat, and A.B. Jones. Analysis of the major metabolite of A9-tetrahydrocannabinol in urine. II1. A GC/ECD procedure. J. AnaL ToxicoL 8; 7-9 (1984). 10. J.M. Rosenfeld, Y. Moharir, and S.D. Sandler. Selective sample preparation for determination of 11-nor-9-carboxy-Ag-tetrahydrocannabinol from human urine by gas chromatography with electron capture detection. AnaL Chem. 61- 925-28 (1989). 11. R.L. Foltz. Analysis of cannabinoids in physiological specimens by gas chromatography/mass spectrometry. In Advances in Analytical Toxicology, R.C. Baselt, Ed., Biomedical Publications, Davis, California, 1984, pp. 125-57. 12. K.M. McGinnis, D.S. Barbario, R. Bridges, and R.L. Foltz. A high sensitivity, high through-put assay for the major urinary metabolite of A9-tetrahydrocannabinol, Proceedings of the 33rd Annual Conference on Mass Spectrometry and Allied Topics in San Diego, CA, May 26-31, 1985, pp. 464-5.
377
Journal of Analytical Toxicology, Vol. 14, NovembedDecember 1990
13.T.S. Baker, J.V. Harry, J.W. RusseL[, and R.L Myers_ Rapid method for the GC/MS confirmation of 11-nor-9-carboxy-,~9-THC in urine. J. AnaL ToxicoL 8:255-59 (1984). 14. B.D. Paul, L.D. Mell, J.M. Mitchell, R.M. McKinley, and J. Irving. Detection and quantitation of urinary 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, a metabolite of tetrahydrocannabinol, by capillary gas chromatography and electron impact mass spectrometry. J. AnaL ToxicoL 11: 1-5 (1987). 15. L.J. McBurney, B.A. Bobbie, and L.A. Sepp. GC/MS and EMIT analysis for Ag-tetrahydrocannabinol metabolites in plasma and urine of human subiects. J. AnaL ToxicoL 10:56-64 (1986). 16. S.J. Mul› and G.A. Gasella. Confirmation of marijuana, cocaine, morphine, codeine, amphetamine, methamphetamine, pheneyclidine by GC/MS in urine following immunoassay screening. J. AnaL Toxicol. 12:102-107 (1988). 17. M. Congost, R. De la Torre, and J. Segura. Optimization of the quantitative analysis of the major cannabis metabolite (11-nor-9carboxy-Z~9-tetrahydrocannabinol) in urine by GC/MS. Biorned. Environ. Mass Spectrom. 16:367-72 (1988). 18. W.A. Joern. Detection of past and recurrent marijuana use by a modified GC/MS procedure. J. AnaL ToxicoL 11:49-52 (1987). 19. M.A. EISohly, H.N. EISohly, A.B. Jones, P.A. Dimson, and K.E. Wells. Analysis of the major metabolite of h9-tetrahydrocannabinol in urine. II. A HPLC procedure. J. AnaL ToxicoL 7: 262-84 (1983). 20. D.S. Isenschmid and Y.H. Caplan. A method for the determination of 11-nor-&9-tetrahydrocannabinol-9-earboxylic acid in urine using high performance liquid chromatography with electrochemical detection. J. AnaL ToxicoL 10:170-74 (1986). 21. D. Bourquin and R. Brenneisen. Confirmation of cannabis abuse by the determination of 11-nor-&9-tetrahydrocannabinol-9-ear boxylic acid in urine with high-performance liquid chromatography and electrochemical detection. J. Chromatogr. 414: 187-91 (1987). 22. B.L. Posey and S.N. Kimble. Quantitative determination of 11nor-Ag-tetrahydrocannabinol-9-carboxylic acid in urine by HPLC. J. AnaL ToxicoL 8:234-38 (1984). 23. R.C. Meatheral and J.C. Garriott. A sensitive thin-layer chromatographic procedure for the detection of urinary 11-nor-/t~tetrahydrocannabinol-9-carboxylic acid. J. Anal. ToxicoL 12: 136-40 (1988). 24. C.A. Sutheimer, R. Yarborough, B.R. Hepler, and I. Sunshine. Detection and confirmation of urinary cannabinoids. J. AnaL ToxicoL 9:156-60 (1985). 25. M.J. Kogan, E. Newman, and N.J. Willson. Detection of mari-
378
juana metaboIite 11-nor-A~-tetrahydrocannabSno;-9-carboxylic acid in human urine by bonded-phase adsorption and thin-layer chromatography. J. Chromatogr. 306:441-43 (1984). 26. G.R. Nakamura, W.J. Stall, V.A. Folen, and R.G. Masters. Thinlayer chromatographic-mass spectrometric identification of 11nor-Ag-tetrahydrocannabinol-9-carboxylic acid. J. Chromatogr. 264" 336-38 (1983). 27. J.A. Vinson and D.J. Lopatofsky. A semi-automated extraction and spotting system for drug analysis by TLC. Procedure for analysis of the major metabolite of Ag-tetrahydrocannabinol in urine. Jo Ana/. Toxicol. 9- 6-9 (1985). 28. K.K. Kaistha and R. Tadrus. Semi-quantitative thin-layer massscreening detection of 11-nor-Ag-THC-9-carboxylic acid in human urine. J. Chromatogr. 237:528-33 (1982). 29. Mandatory guide]ines for federal workplace drug testing programs. Federal Register53:11970-89 (1988). 30.D.L King, M.J. Gabor, P.A. Martel, and C.M. O'Donnell. Evaluation of a rapid TLC technique for detection of cannabinoid metabolites in urine. Clin. Chem. 33:972-73 (1987). 31. D.L. King, M.J. Gabor, P.A. Mar'tel, and C.M. O'Donnell. A rapid sample-preparation technique for thin-layer chromatographic analysis for 11-nor-Ag-tetrahydrocannabinol-9-carboxylic acid in human urine. Clin. Chem. 35:163-66 (1989). 32.EMIT 100-ng cannabinoid assay package insert, Syva Co., Palo Alto, California. 33. V.R. Spiehler, C.M. O'Donnell, and D.V. Gokhale. Confirmation and certainty in toxicology screening. Clin. Chem. 34:1535--39 (1988). 34. M.L. Abercrombie and J.S. Jewell. Evaluation of EMIT and R1A high volume test procedures for THC metabolites in urine utilizing GC/MS confirmation. J. AnaL ToxicoL 10:178-80 (1986). 35. D.R. Berkabile and A. Meyers. False negative rate for EMIT cannabinoids. J. AnaL ToxicoL 13:63 (1989). 36. W.A. Joem. Further observations: False negative EMIT cannabinoids. J. AnaL ToxicoL 13:126 (1989). 37. M. Lehrer and G.M. Meenan. More on the false negative rate for EMIT cannabinoids. J. AnaL ToxicoL 14:62 (1990). 38. J. Standefer. EMIT and TDx tests for THC: Same cutoff, different results. Forensic Urine Drug Testing Newsletter, Sept,: 4-5 (1988). 39. D.J. Wells and M.T. Barnhill, Jr. Comparative results with five cannabTnoTdimmunoassay systems at t~.e screening threshoId of 100 pg/L Clin. Chem. 35:2241-43 (1989). Manuscript received January 2, 1990; revision received March 30, 1990.
