Analytical Letters, 39: 1393–1399, 2006 Copyright # Taylor & Francis Group, LLC ISSN 0003-2719 print/1532-236X online DOI: 10.1080/00032710600668699
BIOANALYTICAL
Simultaneous Determination of Methadone, Heroin, Cocaine and their Metabolites in Urine by GC-MS ´ lvarez, F. Palos, A. M. Bermejo, P. Ferna´ndez, and M. J. Tabernero I. A Institute of Legal Medicine, Forensic Toxicology Service, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
Abstract: A gas chromatographic-mass spectrometric (GC-MS) method for the determination of methadone, heroin, cocaine, and their metabolites in urine using Selected Ion Monitoring (SIM) was developed. Following a liquid-liquid extraction with Toxitubes Aw and using their deuterated analogs as internal standards, the analytes were derivatized with 99:1 (v/v) N,O-bis-trimethylsilyl-trifluoroacetamide/ trimethylchlorosilane and injected by hand, in the splitless mode, at 2408C and a purging time of 0.75 min. The mass selective detector was kept at 3008C and molecules were ionized in the electron impact mode, using an energy of 70 eV. The detector response was linear for all drugs studied over the range 50 – 1000 ng/mL. Keywords: Drugs of abuse, GC-MS, urine, liquid-liquid extraction, derivatization
INTRODUCTION The fast social and economic evolution in recent years, together with the increased availability of drugs have contributed to worsen the problem of their unsuitable use, especially among young people (Klaassen and Watkins 2003; OED 2002; Navarro and Sa´nchez 2002; Barrio et al. 1998). Received 22 September 2005; accepted 20 January 2006 Financial support of CICYT (Project BFI2002-03385) of the Spanish Government is gratefully acknowledged. Address correspondence to Purificacio´n Ferna´ndez, Institute of Legal Medicine, Forensic Toxicology Service, Faculty of Medicine, University of Santiago de Compostela, C/ San Francisco, s/n. 15782, Santiago de Compostela, Spain. E-mail:
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From an analytical and toxicological point of view, the high consumption of abuse drugs justifies a continuous research in this area (Buja´n et al. 2001; Bermejo et al. 2000). To save time and costs and maintain sensitivity and reproducibility, the techniques for preparing biological samples and determining drugs of abuse must be optimized (Baselt 2000; Dart 2004; Moffat et al. 2004; Mills and Roberson 1987; Wang et al. 1994). Urine is the most used sample to detect abuse drugs because it can be obtained non-invasively and in high amounts. The purpose of this study was to develop an analytical method for the determination of methadone, 2-ethylen-1,5-dimethyl-3,3-diphenylpirrolidine (EDDP), heroin, morphine, 6-acethylmorphine (6AM), codeine, cocaine, benzoylecgonine (BEG), and ecgonine methyl ester (EME) in urine by gas chromatography-mass spectrometry (CG-MS). This method is mainly focused towards the analysis of samples from heroin and/or cocaine addicts under detoxification treatment with methadone.
EXPERIMENTAL Standards Drugs stock solutions (1 mg/mL) were purchased from Cerilliantw (Round Rock, TX, USA). Calibration curves were prepared at standard concentrations of 0.05, 0.1, 0.4, 0.6, 0.8, and 1 mg/mL.
Extraction Procedure Liquid-liquid extraction was applied in urine by using Toxitubes Aw (Dipesa, Madrid, Spain) that contain a mixture of organic solvents and a buffer salt of alkaline pH. Ten microlitres of each deuterated analog (0.1 mg/mL) was added to a 1 mL sample spiked with the drugs; samples were homogenized by shaking and poured into a Toxitube A, mechanically shaken for 10 min, and centrifuged at 5000 r.p.m. for 8 min. The organic layer was then transferred into a conical-bottom flask and evaporated to dryness in a bath at 608C under a nitrogen stream. The dry residue was derivatized with 40 mL of bistrimethylsilyltrifluoroacetamide (BSTFA) –trimethylchlorosilane (TMCS) (99:1, v/v) by heating at 708C for 20 minutes and 2 mL were injected into the chromatograph.
Chromatographic Conditions Analysis was carried out on a Hewlett-Packard 6890 Gas Chromatograph interfaced to a model HP 5973 Mass Selective Detector, with the electron
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impact of 70 eV for the ionization of the compounds. Chromatographic elution was done with a 12 m 0.2 mm ID capillary column internally coated with a 0.33 mm thick film of 5% phenylmethylsiloxane. The injector temperature used was 2408C. A volume of 2 mL was injected by hand in the splitless mode, and a purging time of 0.75 min was used. The carrier gas (helium) flow rate was 1 mL/min. The temperature program was started at 908C (1 min), followed by a 308C/min ramp to 1808C (held for 1 min) and a second 48C/min ramp to 2508C (held for 5 min). The mass selective detector was kept at 3008C, the ion source at 2308C and the quadrupole at 1508C. The m/z range spanned was 50 –550, the scan rate was 1.5 scan/s, and the dwell time for each mass was 100 ms. In order to obtain a better precision in the quantitative analysis, the deuterated analytes of each drug were used as internal standards.
RESULTS AND DISCUSSION The identification of the nine compounds was carried out using their retention times and their mass spectra (Pfleger and Weber 1992), which were obtained by injecting the solutions of the drugs and their deuterated forms in methanol (Fig. 1) in scan mode. The retention times for drugs and their deuterated homologue were identical, because substitution of hydrogen for deuterium atoms changes molecular weight and mass spectrum but does not affect the physicochemical properties of the compounds. For each drug, ions showing higher abundances were selected whenever there was no coincidence with its deuterated analog. The ion with highest abundance was chosen as quantifier ion for each drug and its deuterated
Figure 1. Scan mode chromatogram of the nine drugs
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analog, keeping the rest of the selected ions as qualifiers. Chromatographic determination was done in the single ion monitoring (SIM) mode, which resulted in substancially improved sensitivity and specificity for each compound (Table 1). Due to the chemical properties of some analytes, a derivatization was necessary in order to convert them to sylilated derivates and increase their volatilities. So, a clear differenciation with the possible interferences was achieved, choosing a reagent that only derivatizes one functional group of the molecules. The linearity of the developed method was studied through the calibration curves for each analite in methanol and urine (Table 2). They were obtained by plotting the drug/internal standard area ratios against the concentration in the range 0.05– 1.00 mg/mL. The results obtained were good, showing that the slopes of the calibration graphs were statistically different from 0 and the correlation coefficients were not statistically different from 1 (Bressolle et al. 1996). Limits of detection (LOD) and quantitation (LOQ) were obtained from 10 blank samples spiked with deuterated internal standards. The LOD is defined as the mean value of the blank samples plus 3 standard deviations (SD) of the mean. The LOQ is defined as the mean value of the blank samples plus 10 SD of the mean (Armbruster et al. 1994). The results oscillated between 9.61 and 15.02 ng/mL (average: 13.57 ng/mL) for LOD, and between 32 and 50 ng/mL (average: 45 ng/mL) for LOQ. Table 1. Retention times and ions selected for monitorization Compound EME EME-D3 EDDP EDDP-D3 Methadone Methadone-D3 Cocaı´ne Cocaı´ne-D3 BEG BEG-D3 Codeı´ne Codeı´ne-D3 Morphine Morphine-D3 6AM 6AM-D3 Heroı´n Heroı´n-D9
Quantifier ion (m/z)
Qualifier ions (m/z)
Retention time (minutes)
96 99 277 280 294 297 182 185 240 243 371 374 429 432 399 402 369 378
198 171 262 265 295 298 303, 198 306, 201 82, 361 85, 364 178, 234 237 236, 414 239 340, 287 343 310, 204 334
3.8 3.8 7.3 7.3 8.7 8.7 9.6 9.6 10.6 10.6 13.3 13.3 14.4 14.4 15.3 15.3 17.8 17.8
Determination of Drugs in Urine by GC-MS Table 2.
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Parameters of the calibration curves in urine
Compound EME EDDP METHADONE COCAI´NE BEG CODEI´NE MORPHINE 6AM HEROI´N
Slope (a)
Intercept (b)
Coefficient of correlation
2.1366 2.1532 1.3590 2.0945 1.3245 2.9148 2.0762 4.1307 3.4876
0.0507 0.0163 1.8816 0.4305 0.1250 0.3596 0.1020 0.2338 3.4291
0.9925 0.9964 0.9848 0.9835 0.9930 0.9857 0.9935 0.9920 0.9948
Toxitubes Aw, used for extraction of drugs from urine, do not require additional solvents or special equipment. They need a shorter time than the solid-phase extraction and make it possible to obtain clean chromatograms, as confirmed by the analyses of 10 drug-free specimens. To calculate the recoveries of the urine extraction procedure, the mean value of the areas ratio drug/internal standard from each concentration level of calibration curve in methanol and urine were compared. The average recoveries were found to be 88.87% for methadone, 89.94% for EDDP, 94.11% for heroin, 92.64% for morphine, 89.53% for codeine, 92.91% for 6AM, 89.36% for cocaine, 89.41% for BEG and 90.24% for EME. These values were good, irrespective of the concentration levels (no significant differences were encountered). The precision and accuracy of the proposed method were evaluated by analazing 10 urine solutions at two different concentrations (0.1 and 0.8 mg/mL) of each drug. The precision, expressed as the coefficient of variation, was less than 9% in all drugs. The accuracy, expressed as the Table 3.
Precision and accuracy at two different concentrations CONC.: 0.1 mg/mL
CONC.: 0.8 mg/mL
Compound
CV(%)
REL. E.(%)
CV(%)
REL. E.(%)
EME EDDP Methadone Cocaine BEG Codeine Morphine 6AM Heroı´n
6.14 7.80 5.08 4.73 4.17 5.12 6.04 5.19 2.93
9.95 8.01 2.51 3.14 4.33 23.76 24.21 5.18 0.83
2.07 2.32 1.65 1.66 0.84 1.13 0.93 0.77 1.79
0.31 0.22 0.66 0.83 0.25 0.10 20.15 20.03 0.97
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Urinary levels (mg/mL) in nine real cases of intoxication
Compound
1
2
3
4
5
6
7
8
9
EME EDDP Methadone Cocaı´ne BEG Codeı´ne Morphine 6AM Heroı´n
1.82 2.09 0.42 0.08 1.05 — — — —
0.21 1.94 0.78 0.28 1.40 0.01 0.19 0.03 —
3.58 — — 0.51 1.61 — — — —
— — — — 0.64 — — — —
0.81 — 1.56 0.05 1.63 2.66 0.10 — —
— 9.08 4.40 — — 0.11 0.54 — —
0.09 — — 0.02 0.39 — — — —
— — — — — 0.99 — — —
— 8.70 4.23 — — — — — —
relative error obtained from the theoretical concentration of the samples tested, was in the range +12% (Table 3). These results were acceptable according to Bressolle et al. (1996). Finally, the method proposed was applied to nine real urine samples coming from individuals intoxicated with one or more of the drugs studied. The results obtained are shown in Table 4. As was to be expected, the concentrations of metabolites are bigger than those of the original durgs. The mean EME/COC and BEG/COC ratios were 6.8 and 5.9 respectively. EDDP was 2.4 times bigger than methadone, and morphine was 9.3 times bigger than 6AM, both metabolites of heroin.
CONCLUSIONS The proposed analytical method for simultaneous determination of the nine drugs in urine presents a good linearity, precision, accuracy, and sensitivity to be applied to cases of intoxication. Toxitubes A eliminate or separate all compounds that could interfere in the analysis, providing high recoveries of the drugs extracted from urine.
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Bermejo, A.M., Lucas, A.C.S., Tabernero, M.J., and Ferna´ndez, P. 2000. Simultaneous determination of methadone, heroin and their metabolites in hair by GC-MS. Anal. Lett., 33: 739– 752. Bressolle, F., Bromet-Petit, M., and Audran, M.M. 1996. Validation of liquid chromatographic and gas chromatographic methods. Applications to pharmacokinetics. J. Chromatogr. B, 686: 3 – 10. Buja´n, L., Ferna´ndez, P., Lafuente, N., Aldonza, M., and Bermejo, A.M. 2001. Comparison of two chromatographic methods for the determination of cocaine and its metabolites in blood and urine. Anal. Lett., 34 (13): 2263– 2275. Dart, R.C. 2004. Medical Toxicology; Lippincott Williams & Wilkins: Philadelphia, USA. Klaassen, C.D. and Watkins, J.B., III Eds.; 2003. Casarett & Doull’s Essentials of Toxicology. McGraw-Hill: New York, USA. Mills, T., III and Roberson, J.C. 1987. Instrumental Data for Drug Analysis. Elsevier: New York, USA. Moffat, A.C., Osselton, M.D., and Widdop, B. Eds.; 2004. Clarke’s Analysis of Drugs and Poisons in Pharmaceuticals, Body Fluids and Postmortem Material. Pharmaceutical Press: London, UK. Navarro, J. and Sa´nchez, L. 2002. Observatorio de Galicia sobre Drogas. Informe General 2002. Xunta de Galicia: Santiago de Compostela, Spain. Observatorio Espan˜ol sobre Drogas (OED). Informe n8 5. Ministerio del Interior: Madrid, Spain, 2002. Pfleger, K. and Weber, M.A. 1992. Mass Spectral and GC Data of Drugs, Poisons, Pesticides, Pollutants and Their Metabolites. VCH: Weinheim, Germany. Wang, W.-L., Darwin, W.D., and Cone, E.J. 1994. Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatographymass spectrometry. J. Chromatogr. B, 660: 279– 290.
