Comparison of Cozart | Microplate ELISA and GC-MS Detection of ...

Journal of Analytical Toxicology, Vol. 29, October 2005

Comparison of Cozart| Microplate ELISAand GC-MS Detection of Methadone and Metabolites in Human Hair Gail Cooper 1, Lisa Wilson 1, Claire Reid 1, Dene Baldwin 1, Chris Hand 1, and Vina Spiehler2, *

1CozartBioscience Ltd, Abingdon, Oxfordshire OXI 4 4RY, U.K. and 2Spiehlerand Associates, Newport Beach, California 92663

Abstract The purpose of this study was to determine the performance characteristics of the Cozart Methadone Microplate ELISA assay for the detection of methadone and methadone metabolites in hair specimens. One hundred and ten hair specimens were collected from volunteers (n = 46) with a history of drug use and from drugrelated deaths (n = 64). The hair samples (approximately 20 rag) were extracted by sonication in methanol followed by overnight extraction in methanol at 60~ The methanol extract was evaporated to dryness, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture [methadone-d9 and 2-ethylidene-l,5-dimethyl-3,3diphenylpyrrolidine (EDDP)-d3] and 0.1M HCI were added to approximately 20 mg of specimen or spiked blank hair and sonicated for t h. The pH was adjusted to neutral, and methadone and its primary metabolite, EDDP, were analyzed by GC-MS following solid-phase extraction using Bond Elute Certify columns and pH 7.4 phosphate buffer (0.1M). Forty hair specimens were confirmed positive for methadone by GC-MS. Concentrations ranged from 0.10 to 8.3 n~mg for methadone and 0.1 to 1.2 n~/mg for EDDR The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results with a cutoff of 0.1 ng/mg for both methadone and EDDP as the reference method. The optimum cutoff for the Cozart Methadone Microplate ELISA was determined to he between 200 and 300 pg methadone equivalents/rag hair using a 20 mg hair sample. The Cozart Methadone Microplate EIA for methadone and metabolites in hair using a cut-off of 200 pg/mg hair with a 20 mg hair sample had a sensitivity of 95 _+2% and a specificity of 100 __3.5% (vs GC-MS) and an accuracy of 98.2 _+1.3%.

Introduction

Hair analysis has been recommended for monitoring patients receiving methadone maintenance not only for its analytical and pharmacokinetic advantages, but also because use of * Author to whom correspondenceshould be addressed:Vina Spiehler,Ph.D., DABFT, 422 TustinAve., Newport Beach,CA 92663. E-mail:[email protected].

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hair testing can benefit the nature of the relationship between treatment personnel and patients on methadone maintenance (1-3). Hair testing for drugs has the advantage of a very long window of detection. After an initial lag time of approximately 8 h for drugs entering hair from sweat or between five and ten days for drugs incorporated into hair from blood, the slow growth of hair at about 0.5 in. per month gives a window of detection equal to the length of the hair. The 1.5 in. of hair closest to the scalp is considered to record drug exposure in the past 90 days. Collection of hair is acceptable to both donors and collectors in many situations because the collection of hair is noninvasive and does not require observation by a person of the same sex (4). Hair can be stored and shipped in plain paper envelopes with no problems of spillage or spoilage. Methadone and methadone metabolites are found in hair at concentrations that are within the range of microtiter immunoassays developed for forensic drug screening. After administration of oral methadone, concentrations ranging from 0.2 to 43.9 ng/mg for methadone and from 0.5 to 2.10 ng/mg for 2-ethylidene-l,5-dimethyl-3,3-diphenylpyrrolidine(EDDP) have been reported in hair (5-14). After intravenous administration of methadone at doses of 100 to 200 rag/day, concentrations of methadone found in hair are higher, ranging from 18 to 127.5 ng/mg hair. Several authors, however, have reported that there is no interindividual correlation between dose and methadone or EDDP concentrations in hair (12,13). Hair testing for methadone and methadone metabolites is potentially a valuable part of forensic investigations, including death investigations involving methadone (15,16). For this reason, a validated immunoassay screening method is desirable. ELISA procedures have the advantage in screening of alternative matrices in that the specimen digest and labeled antibody are washed from the plate before development of the enzyme-catalyzed color. A thorough washing of the microtiter plate removes difficult matrices and reduces matrix effects in the detection step. The high sensitivity, low cutoffs, and cross-reactivity with parent drug, that make the Cozart Microplate assays useful for oral fluid and whole blood analysis (17), are also advantageous in analysis of drugs in hair (18). This paper compares ELISA results for methadone and methadone metabolites with GC-MS findings for methadone and EDDP in hair specimens.

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Methods Specimens One hundred and ten hair specimens were obtained from Table I. Cozart Methadone Microplate ELISA and GC-MS Results in Hair Cozart Microplate ELISAMethadone GC-MS Equivalents Methadone EDDP Sample ng/mg* ng/mg ng/mg 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

0,401 > 1,000 > 1.000 > 1.000 > 1.000 0.604 0.424 0.620 0.505 0.831 > 1.000 > 1,000 > 1,000 > 1,000 > 1.000 0.495 0.481 0.406 0.531 0.349 0.403 0.431 0.435 0.518 0.574 0.645 0.404 0.411 0.391 0,182 0.075 > 1.000 > 1.000 0.604 > 1.000 > 1.000 > 1.000 > 1.000 > 1.000 > 1.000 0.058 0.047 0.078 0.103 0.052 0.144 0.094 O.046

0.8 8.3 4.1 1.1 1.7 0.9 0.7 0.8 0.5 0.8 3.1 2.6 2.9 2.8 1.1 1.0 0,5 0.4 0.3 0.1 0.3 0,2 0.3 0.4 0.3 0.4 0.4 0.4 0,4 0.2 0.2 1.0 1,6 0,9 2.8 2.9 0.8 2.5 0.8 0.8 0 0 0 0 0 0 0 0

0.4 1.2 0,4 0,2 0.2 0.1 0.1 0.2 0 0.2 0.5 0.2 0.3 0.5 0,2 0,1 0 0 0,1 0 0 0 0 0 0 0 0 0 0 0 0 0.1 0.2 0,1 0,3 0.5 0.1 0,3 0.1 0 0 0 0 0 0 0 0 0

Total ng/mg

Source

1.2 9.5 4.5 1.0 1.9 1.0 0.8 1.0 0.5 1.0 3.6 2.8 3.2 3,3 1,3 1.1 0.5 0.4 0.4 0.1 0.3 0.2 0.3 0.4 0.3 0.4 0.4 0.4 0,4 0.2 0.2 1.1 1.8 1,0 3,1 3,4 0,9 2,8 0.9 0.8 0 0 0 0 0 0 0 0

D D D D D D D D D D D D D D D D V D V V V V V V V V V V D D D D D D D D D D D D D D D D D D V V

* Expressed as ng methadone equivalents/rag hair; D = drug-related death; and V = volunteer.

volunteers (n = 46) and from drug-involved deaths (n = 64). Hair samples collected from volunteers were cut close to the scalp, wrapped in aluminum foil, sealed, and sent to the laboratory for testing. For immunoassay, 20-30 mg of hair sample was weighed into a labeled vial and cut finely with a pair of clean surgical scissors. The weight was recorded. The weighed hair samples were prepared for analysis by extracting overnight in 2 mL of methanol at 60~ following sonication at room temperature for 60 min. The methanol extract was then taken to dryness under nitrogen and reconstituted in 500 IJL of negative ELISA calibrator matrix buffer. Reconstituted hair extract (25 IJL) was added to duplicate wells of the antibody-coated microtiter plate. Cozart Methadone Microplate ELISA immunoassay The Cozart Methadone Microplate ELISA assay was used to assay the reconstituted hair extracts. Cozart Microplate enzyme immunoassays employ antibody-coated microtiter plates and a drug-derivative, which is labeled with horseradish peroxidase. Twenty-five microliters of sample, calibrator, or control was added to each well of the antibody-coated microtiter plate followed by 100 IJL of working enzyme conjugate. After a 30min incubation at room temperature, the plate was washed four times with 350 IJL wash buffer. Substrate solution (100 IJL, dilute hydrogen peroxide with 3,3',5,5'-tetramethyl benzidine as the chromogen) was added to each well and incubated for a further 30 min. Finally, 100 IJL of stop solution (1M sulfuric acid) was added to each well, and the absorbance was read at 450 nm using a microplate reader within 30 min. All reagents were supplied by the manufacturer in the ELISA kit. Concentrations were determined from the assay calibration curve run on the same plate as the reconstituted hair extract samples. Calibrators were prepared by spiking blank hair samples at concentrations of 0, 50, 100, 200, 300, 500, and 1000 pg/mg of methadone. Concentrations shown in Table I are of total methadone and metabolites expressed as nanograms methadone equivalents per milligram hair after dividing by the weight of the hair sample. The 0, 1, 5, and 20 ng/mL calibrators were run as linearity controls on each plate, which would be equal to 0, 100, 500, and 1000 pg/mg with a 20 mg sample. Precision A calibrator at 200 ng/mg methadone was tested in replicate (n = 16) with one assay and in duplicate for six assays. The precision within assay and between assays was obtained by calculating the mean, standard deviation (SD), and coefficient of variation (CV) for each. To calculate the within-assay and within-day precision, the following formula was used: Mean CV -

N-1

GC-MS procedure for methadone and EDDP For GC-MS analysis, hair samples were washed once in deionized water then in methanol. Each wash step was carried out by sonicating for 5 rain. The hair was then allowed to dry overnight. The dried hair was cut finely with a pair of clean surgical scissors, placed in a silanized vial, and weighed accurately. 679


To prepare standards, approximately20 mg of blank hair was weighed into seven vials and the weight recorded. Then appropriate amounts of standard mixtures of methadone and EDDP (100 ng/mL and 1000 ng/mL) were added to make the following spiked concentrations: 0, 0.2, 0.5, 1.0, 2.5, and 5.0 ng/mg hair. The internal standards mixture contained 1000 ng/mL methadone-d9 and EDDP-d3. Fifty microliters of deuterated internal standards mixture and 1 mL 0.1M hydrochloric acid were added to the specimen or spiked blank hair and sonicated for 1 h at 60~ The samples were allowed to cool to room temperature, and then the supernatant was aliquoted to a clean sample tube. The pH was adjusted to 7 with 1M potassium hydroxide, and 1 mL of pH 6 phosphate buffer (0.1M) was added. Methadone and EDDP were extracted by solid-phase (BondElut Certify 130 rag, 3 mL). The columns were conditioned with I mL methanol followed by I mL phosphate buffer (pH 7.4, 0.1M). The samples were then loadedinto the columns and washed with 1 mL deionized water. The column pH was adjusted with 0.5 mL of 0.01M acetic acid (pH 3.3). The columns were then dried on full vacuum for 10 rain, 50 pL of methanol added, and dried on full vacuum for a further 1 rain. The column was washed with 3 mL acetone/chloroform (1:1) and dried brieflyto remove residual solvent. Methadone and EDDP were eluted with 1 mL 2% ammoniated ethylacetate. The eluates were evaporated to dryness at 40~ under nitrogen, reconstituted in 100 pL ethylacetate, and 2 pL was injected onto the GC-MS.

GC-MS parameters The instrument was an Agilent 5973N mass selective detector with a 6890N GCwith a RestekRTx-5Sil MS column (30 m • 0.25 mm x 0.25 IJm). The temperature program consisted of an initial 2.0 rain at 80~ that was then ramped at 30~ to 230~ then to 300~ (10~ and held for 6 rain. The following ions were monitored: 262, 276, 277 for EDDP; 265, 279, 280 for EDDP-d3;223, 294, 72 for methadone; and 226, 303, 78 for methadone-d9.The abundancesfound for the ions noted in bold were used for quantitation. With a 20-rag hair sample, the limits of detection and quantitation of the GC-MS procedure were equivalent to 0.1 ng/mg for both methadone and EDDP.

+ FN). Specificitywas calculatedas Specificity= true negatives (TN)/[TN + false positives (FP)]. The number of TP, FN, FP, and TN was determined by comparison of the ELISAresults to GC-MS as the reference method. Sensitivity and specificityare probabilities; thus, their uncertainty is expressedas a standard error (SE) using the equation SEp = square root (pq/n) where p is the probability,q is 1 -p, and n is the number of specimens analyzed.

Results and Discussion

Precision The precision of the Cozart Methadone Microplate ELISA using a cut-off of 200 pg/mg equivalents in hair was as follows: intraassay mean absorbance 0.64 + 0.06 SD (9.0% CV), n = 16 and interassay mean absorbance 0.63 + 0.04 SD (6.4% CV),n = 6.

Case results Table I shows the Cozart Methadone Microplate ELISAand GC-MS results for methadone and methadone metabolites in the hair specimens positive by GC-MS and/or ELISA. Forty hair specimenswere confirmed positiveby GC-MS. The TP, FN, FP, and TN rates for the different cutoffs are shown for the Cozart MethadoneMicroplateELISAin Figure 1. From this plot it can be seen that the fewest FP and FN results were found at a cutoff of 200 pg/mg hair. The sensitivity for each cutoff was plotted versus 1 - specificity as a receiver operating curve (ROC) in Figure 2. The optimum cutoff was determined at the inflection point of the smoothed ROC graph to be between 200 and 300 pg methadone equivalents/rag hair. There are currently no approved or proposed guidelines for methadone cutoffs in hair, therefore the performance of the Cozart Methadone Microplate ELISA was assessed at various cutoffs (50, 100, 200, 300, 500, and 750 pg/mg) in comparison with GC-MS as the reference method. Using GC-MS cutoff of 0.1 ng/mg for both methadone and EDDP, the optimum screening cutoff was 200 pg/mg. The Cozart Methadone Microplate ELISA using a cutoff of 200 pg/mg

EIA Methadone in Hair

Matrix controls Hair samples previously analyzed were repeated on subsequent ELISAand GC-MS runs as controls and the results compared to the previous values. From 1 to 10 repeated hair specimens were included in each microtiter plate or GC-MS batch. These control hair samples were used repeatedly until the quantity was no longer sufficient for analysis. Approximately 25% of hair specimens had sufficient quantity for repeated analysis. No single specimen had sufficientquantity for inclusion in all batches. Pooled hair matrix controls were not used.

Sensitivity and specificity Sensitivity, the true positive (TP) rate, was calculated from the tally of TP, and false negatives (FN) as Sensitivity = TP/(TP

680

=~ 1

0

0

~

o]JUUUUU J

50 100 200 300 500 750 Cutoff" concentration (pg/mg hair) Figure1. TP,FN, FP,and TNs for cutoffsof 50-750pg/mgmethadone equivalentsin hair.

Journal of Analytical Toxicology,Vol. 29, October 2005

References

ROC EIA Methadone in Hair 0.8 '~. 0.6

=: 0.4 0.2 0

0.05

i

i

0.1

0.15

0.2

1-specificity Figure 2. ROC curve for Cozart Methadone Microplate ELISA for methadone in hair.

Table U. Sensitivity and Specificity Cozart Methadone Microplate ELISA* Cozart ELISAResultat 200 pg/mg + GC-MS + GC-MS -

38 0

2 70

* Sensitivity = 3 8 / 4 0 = 9 5 . 0 % + 2 % a n d Specificity = 7 0 / 7 0 = 1 0 0 % + 3 . 5 % .

Table III. C ross-Reactiviy of the Cozart | Methadone Microplate ELISA Compound

ng/mL Tested

Methadone LAAM EDDP

100 1000 500 100

EMDP

100,000

100,000

% Cross-Reactivity 100% 1.6% 1.6% 0.69 0.01 < 0.006%

hair and a 20-mg hair sample had a sensitivity for methadone in hair of 95 • 2% and a specificity of 100 • 3.5% versus GC-MS (Table II).

Conclusions The Cozart Methadone Microplate ELISA assay was well suited for screening for methadone and methadone metabolites in hair. The assay has appropriate cross-reactivity for the parent drugs and metabolites found in hair. As shown in Table III, the cross-reactivity for l-(~-acetylmethadol (LAAM) is 1.6%, for EDDP is 0.69%, and for 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) < 0.006%. LAAM and EMDP were not analyzed by GC-MS in this study. There was no matrix effect with hair extracts. The sensitivity and specificity were excellent.

1. C. Brewer. Hair analysis as a tool for monitoring and managing patients on methadone maintenance. A discussion. Forensic Sci. Int. 63(1-3): 277-283 (1993) 2. K. Wolff and J. Strang. Therapeutic drug monitoring for methadone: scanning the horizon. Eur. Addict. Res. 5(1): 36-42 (1999). [Comment Eur. Addict. Res. 6(1): 31-33 (2000).] 3. D.A. Fiellin, P.G. O'Connor, M. Chawarski, J.R Pakes, M.V. Pantalon, and R.S. Schottenfeld. Methadone maintenance in primary care: a randomized controlled trial. J. Am. Med. Assoc. 286(14): 1724-1731 (2001). [Comment J. Am. Med. Assoc. 286(14): 1764-1765 (2001).] 4. M. Fendrich, T. Johnson, S. Sudman, J. Wislar, and V. Spiehler. Validity of drug use reporting in a high-risk community sample: a comparison of cocaine and heroin survey reports with hair tests. Am. J. Epidemiol. 149(10): 955-962 (1999). 5. D.G. Wilkins, P.R. Nagasawa, S.P. Gygi, R.L. Foltz, and D.E. Rollins. Quantitative analysis of methadone and two major metabolites in hair by positive chemical ionization ion trap mass spectrometry. J. Anal. Toxicol. 20(6): 355-361 (1996). 6. P. Kintz, H.P. Eser, A. Tracqui, M. Moeller, V. Crimele, and R Mangin. Enantioselective separation of methadone and its main metabolite in human hair by liquid chromatography/ion spray-mass spectrometry. J. Forensic Sci. 42(2): 291-295 (1997). 7. M. Frost, H. Kohler, and G. Blaske. Enantioselective determination of methadone and its main metabolite 2-ethylidene-l,5-dimethyl3,3-diphenylpyrrolidine (EDDP) in serum, urine and hair by capillalry electrophoresis. Electrophoresis 18(6): 1026-1034 (1997). 8. D.G. Wilkins, A.S. Valdez, G.G. Krueger, and D.E. Rollins. Quantitative analysis of/-c(-acetylmethadol,/-o~-acetyI-N-normethadol and/-u.-acetyI-N,N-dinormethdol in human hair by positive ion chemical ionization mass spectrometry. J. Anal. Toxicol. 21(6): 420-426 (1997). 9. B.A. Goldberger, A.G. Darraj, Y.H. Caplan, and E.J.Cone. Detection of methadone, methadone metabolites and other illicit drugs of abuse in hair of methadone-treatment subjects. J. Anal. Toxicol. 22(6): 526-30 (1998). 10. A.C. Lucas, A.M. Bermejo, M.J. Tabernero, P. Fernadez, and S. Strano-Rossi. Use of solid phase microextraction (SPME)for determination of methadone and EDDP in human hair by GC-MS. Forensic Sci. Int. 107(1-3): 225-232 (2000). 11. F. Sporkert and F. Pragst. Determination of methadone and its metabolites EDDP and EMDP in human hair by headspace solid-phase microextraction and gas chromatography-mass spectrometry. J. Chromatogr. B 746(2): 255-264 (2000). 12. C. Girod and C. Staub. Methadone and EDDP in hair from human subjects following a maintenance program: results of a pilot study. Forensic Sci. Int. 117(3): 175-184 (2001). 13. S. Paterson, R. Cordero, M. McPhillips, and S. Carman. Interindividual dose/concentration relationship for methadone in hair. J. Anal. Toxicol. 27(1): 20-23 (2003). 14. T. Kelly, P. Doble, and M. Dawson. Chiral analysis of methadone and its major metabolites (EDDP and EMDP) by liquid chromatography-mass spectrometry. J. Chromatogr. B 814(2): 315-323 (20O5). 15. G.A.A. Cooper, A. Seymour, M.T. Cassidy, and J.S. Oliver. A study of methadone in fatalities in the Strathclyde Region, 1991-1996. Med. 5ci. Law3:233-242 (1999). 16. A. Seymour, M. Black, J. Jay, G. Cooper, C. Weir, and J.S. Oliver. The role of methadone in drug-related deaths in the west of Scotland. Addiction 8:995-1002 (2003). 17. G. Cooper, L. Wilson, C. Reid, D. Baldwin, C. Hand, and V. Spiehler. Comparison of GC/MS and EIA Resultsfor analysis of methadone in oral fluid. J. Forensic Sci., in press. 18. V. Spiehler. Hair analysis by immunological methods from the beginning to 2000. Forensic Sci. Int. 107:249-59 (2000).

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