Anal. Chem. 1991, 63, 2878-2882
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Analysis of Illicit Drugs in Human Urine by Micellar Electrokinetic Capillary Chromatography with On-Column Fast Scanning Polychrome Absorption Detection Paul Wernly a n d Wolfgang Thormann* Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
Using micellar electrokinetic capillary chromatography (MECC) with a borate/phosphate buffer contalning 75 mM SDS (pH 9.1), common drugs of abuse and/or thelr m e t a b iltes, Including oplokls, benzoylecgonlne, amphetamines, and methaqualone, can easily be analyzed. Afler solid-phase extractbn of 5 mL of urhe, drug concentrations down to about 100 ng/mL can be unambiguously monHored wlth ontoiumn multlwavelength detectlon. Peak assignement Is achleved through comparlson of retentlon t h e s and absorption spectra of elutlng peaks wlth those of computer-stored model runs. The effectiveness of the approach Is demonstrated wlth data obtained from different patlent urlnes which tested posnlvely for one or several drugs using nonlsotoplc Immunoassays. Results suggest that MECC of illlclt drugs Is a hlghly spectflc and sensttive instrumental approach sunable for conflnnatlon testlng foiiowlng a posnlve response of a toxicological screenlng procedure.
INTRODUCTION Screening and confirmation of drugs of abuse in body fluids, including urine, is important for the investigation of intoxications, the detection of potential users of drugs, and the control of drug addicts following withdrawal therapy. Typically, the specimens are first assayed with screening tests, such as thin-layer chromatography and immunological systems, tests which are easy to perform on a mass basis and which detect a broad range of compounds but often lack sensitivity and specificity. Confirmation testing is the necessary second step following the detection of a positive result on a screening procedure. Its purpose is to eliminate any false-positive answer that may have resulted from the initial screening process. For that purpose a highly specific procedure is required. As instrumental approaches for that task, gasliquid chromatography, high-performance liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS) procedures are currently in use (1). These methods are more specific, typically more sensitive, and more time consuming than the screening tests. They also require fairly expensive instrumentation. Recently, high-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC, an interface between electrophoresis and chromatography) were found to be attractive approaches for the analysis of pharmaceuticals in body fluids (2-9). In MECC two distinct phases are used, an aqueous and a micellar phase or pseudostationary phase. These two phases are established by employing buffers containing surfactants (e.g. sodium dodecyl sulfate (SDS)) above their critical micellar concentration. An MECC analysis is performed in equipment designed for HPCE, i.e. in an open-tubular capillary of very small i.d. A high-voltage dc electric field is applied along the column, thus causing both a movement of the entire liquid (the socalled electroosmotic flow) and migration of the charged micelles. Nonionic solutes partition between the two phases 0003-2700/91/0383-2878$02.50/0
and elute with zone velocities between those of the two phases. Elution order is essentially based on the degree of partitioning (10). Fast-scanning, multiwavelength detection in HPCE/ MECC was shown to permit peak confirmation to be assessed via comparison of absorption spectra (9). The effectiveness of this approach was demonstrated with the analysis of barbiturates in human serum and urine. The objectives of the work described in this paper were to investigate the suitability of using MECC with on-column, fast-scanning polychrome UV absorption detection for the analysis of illicit drugs in human urine, including opioids, cocaine metabolites, amphetamines, and hypnotics. EXPERIMENTAL SECTION Chemicals, Origin of Samples, and Drug Screening. All chemicals used were of analytical or research grade. The drugs employed as reference compounds were of European Pharmacopeia quality. Urine samples were collected in our routine drug assay laboratory where they were received for drug screening. Our own urine was employed as blank matrix. The samples were screened for the presence of opiates, cocaine metabolites, methaqualone, amphetamines, barbiturates, and benzodiazepines by automated enzyme immunoassay techniques (EMIT-dau, Syva, Palo Alto, CA) on a Cobas Fara centrifugal analyzer (F. Hoffmann-La Roche, Diagnostica, Basel, Switzerland) and stored at 4 "C until further analysis. The EMIT-dau tests contain calibrators with a cutoff level of 300 ng/mL each. Samples which gave an equal or higher response than the calibrators were interpreted as positive. Electrophoretic Instrumentation and Running Conditions. The instrument with multiwavelength detection employed in this work was described previously (9). Briefly it featured a 75 pm i.d. fused-silica capillary of about 90-cm length (Product TSP/075/375, Polymicro Technologies, Phoenix, AZ) together with a fast-scanning multiwavelength detector Model UVIS 206 PHD with on-column capillary detector cell No. 9550-0155 (both from Linear Instruments, Reno, NV) toward the capillary end. The effective separation distance was 70 cm. A constant voltage of 20 kV was applied using a HCN 14-2oooO power supply (FUG Elektronik, Rosenheim, Germany). The cathode was on the detector side. Sample application occurred manually via gravity through lifting the anodic capillary end, dipped into the sample vial, some 34 cm for a specified time interval (typically 5 8). Multiwavelength data were read, evaluated, and stored by employing a Mandax AT 286 computer system and running the 206 detector software package version 2.0 (Linear Instruments, Reno, NV) with windows 286 version 2.1 (Microsoft, Redmont, WA). Conditioning for each experiment occurred by rinsing the capillary with 0.1 M NaOH for 3 min and with buffer for 5 min. Throughout this work the 206 detector was employed in the high-speed polychrome mode by scanning from 195 to 320 nm at 5-nm intervals (26 wavelengths). With these settings the sampling rate was 3.69 data point/s and wavelength. A buffer composed of 75 mM sodium dodecyl sulfate (SDS),6 mM NazBa407,and 10 mM Na2HP04 (pH about 9.1) was employed. Sample Pretreatment. Standard solutions of drugs of abuse were prepared in methanol at concentrations of 2000 rg/mL. Spiking of urine blank occurred through addition of known aliquots of these standard solutions to the urine prior to extraction. Urine samples were injected as received or, prior to analysis, drugs were extracted using Bond Elut Certify cartridges and a Vac Elut 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
setup (both from Analytichem International, Harbor City, CA). Three extraction procedures were evaluated, the first one being that for cocaine and metabolites recommended by the manufacturer of the solid-phase extraction columns. The Bond Elut Certify cartridges were conditioned immediately prior to use by passing sequentially 2 mL of methanol and an equal volume of 0.1 M phosphate buffer (pH 6)through the columns. The vacuum was turned off to prevent column drying. The columns were loaded by slowly drawing of a mixture of 5 mL of urine and 2 mL of 0.1 M phosphate buffer (pH adjusted to 5). The columns were sequentially rinsed with 3 mL of deionized water, 3 mL of 0.1 M HCl as well as 9 mL of methanol. Elution occurred with 2 mL of methylene chloride/isopropyl alcohol (8020) containing 2% ammonium hydroxide into a test tube. Then the eluate was evaporated to dryness under a gentle stream of nitrogen at room temperature. The residue was dissolved in 100 pL of running buffer. As an alternative for opioids the cartridges were conditioned by passing sequentially 2 mL of methanol and an equal volume of deionized water through the columns. The vacuum was turned off to prevent column drying. For hydrolysis 1.0 mL of concentrated HCl was added to 5 mL of urine, and the solution was vortexed and heated to 120 "C for 30 min. Thereafter about 1.25 mL of 10 M KOH solution was added to reach a pH of 7 . The specimen was then applied and slowly drawn through the cartridge. Without acid hydrolysis, the columns were loaded by slowly drawing 5 mL of urine (adjusted to pH 7 ) . In both cases, the columns were sequentially rinsed with 2 mL of deionized water and 1 mL of 0.1 M acetate buffer (pH 4), as well as 2 mL of methanol. In contrast to the recommendation of the manufacturer of the cartridges, the columns were not dried under full vacuum. Elution occurred with 2 mL of methylene chloride/isopropyl alcohol (8020) containing 2% ammonium hydroxide into a test tube. Evaporation to dryness was achieved under a gentle stream of nitrogen at room temperature. The residue was dissolved in 100 pL of running buffer. For amphetaminesthe cartridges were conditioned immediately prior to use by passing sequentially 2 mL of methanol and an equal volume of 0.1 M phosphate buffer (pH 6) through the columns. The vacuum was turned off to prevent column drying. The columns were loaded by slowly drawing of a mixture of 5 mL of urine and 2 mL of 0.1 M phosphate buffer (pH adjusted to 6). The columns were then rinsed with 1 mL of 1M acetic acid and 6 mL of methanol. The drugs were eluted with 2 mL of 2% ammonium hydroxide in ethyl acetate into a test tube. A drop of 1M HC1 was added to the eluate before evaporation to dryness under a gentle stream of nitrogen at room temperature. The residue was dissolved in 100 pL of running buffer. Recovery. The recovery after sample pretreatment was determined by comparing MECC peak heights after extraction with peak heights obtained by direct injection of equal amounts of the drugs in buffer.
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Figure 1. Single-wavelength (195 nm) electropherograms of three model mixtures of different drugs. The applied voltage was a constant 20 kV in all cases, and the currents were between 76 and 80 HA. Drug concentrations in the samples were 20 pg/mL. Key: (1) benzoylecgonine; (2) morphine; (3) heroin; (4) methamphetamine; (5) codeine: (6)amphetamine; (7) cocaine; (8) methadone; (9)methaqualone, (10) flunitrazepam; (11) oxazepam; (12) diazepam; (X) impurity of benzoylecgonine, benzoic acid. 4
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RESULTS AND DISCUSSION With the experimental conditions used in this work, methanol (neutral marker of electroosmotic flow or tracer of water) and Sudan I11 (tracer for the carrier micelle) were found to elute after 6.3 f 0.2 and 26 f 1.5 min, respectively. In MECC the degree of partitioning between the buffer and the micelles is the major cause of retention and solutes elute between these two tracers (k'values between 0 (water) and (Sudan 111))(IO). Single-wavelength electropherograms of three model mixtures of drugs are depicted in Figure 1. Each compound is characterized by its retention/migration behavior with benzoylecgonine, the major metabolite of cocaine, being the fastest and methadone the slowest of the investigated components. The latter compound was found to be completely absorbed by the micelle and can therefore not be determined with this buffer. It is interesting to note, that the investigated opioids (heroin, codeine, and morphine) separate extremely well with this buffer. The three-dimensional electropherograms depicted in Figure 2 represent the absorbance vs retention time vs wavelength relationships for the three model mixtures of drugs. The absorption spectrum of each com-
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Figure 2. Three-dimensional electropherograms of the model mlxtures
of Figure 1. pound can be extracted from the gathered data points as so-called time slices (see below). Direct injection of urine provides a rather complex electropherogram (panel A of Figure 3) which hinders the analysis
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991 0.070
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Flgure 3. Extraction of cocaine metabolites and opioids: electropherograms of (A) directly injected urine blank, (E) extracted urine blank, and (C) extracted urine blank spiked with benzoylecgonine, morphine, and codeine (10 pg/mL each) and (D) a threedimensional data plot of the extracted spiked blank urine. Other conditions and sample identification are the same as in Figure 1.
of small amounts of drugs which eiuw between 6 and 16 min. Furthermore, for MECC with on-column UV absorption detection, sample concentrations have to be at least on the concentration level (see Figure 1and ref 9). For pg/mL (pM) direct urine injection, this sensitivity limit is not as good compared to that of the commonly used immunological screening methods. Therefore extraction of the drugs or their metabolites remains to be essential for their confirmation by MECC. Panel B of Figure 3 depicts single-wavelength data obtained after Bond Elut Certify extraction of urine blank using the procedure for cocaine and metabolites. A much smaller number of peaks is detected despite that with this approach and 100% recovery a 50-fold concentration of the extracted compounds can be achieved. Panels C and D present data obtained with extracted urine blank which was spiked with benzoylecgonine, morphine, and codeine (10 pg/mL each). All three compounds were found to extract with efficiencies of about 50,60, and 90%, respectively, and spiking on the 200 ng/mL level still provided small peaks which could be unambiguously assigned (data not shown). Methaqualone and amphetamines could also be extracted but with a lower efficiency.
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threedimensional data obtained with the cleaned-up sample. Data of panels B-E all stem from the same experiment. Other conditions and peak identification are the same as in Figure 1.
MECC data of a urine specimen which was found to be markedly positive for cocaine and opiates using enzymemultiplied immunoassay drug screening procedures are depicted in Figure 4. Direct injection (panel A) provided a complex electropherogram. No peak could be assigned to one of the compounds of interest. This, however, was different after extraction with the cocaine procedure using Bond Elut Certify (panels B to E). Having data between 195 and 320 nm, as shown in panels C-E, as well as reference spectra,
ANALYTICAL CHEMISTRY. VOL. 03, NO. 24, DECEMBER 15, 1991
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Flgure 5. Normalized time slices of (A) benzoylecgonine,(B) morphine, (C) codeine, and (D) methaqualone of the data of Figure 4 in comparison to those extracted from the data of Figure 2.
permitted a quick and reliable confirmation of the presence of benzoylecgonine, morphine, and codeine in that sample. The excellent agreement between the time slices at l1.9,16.7, and 22.3 min with those of computer-stored standards is documented with the graphs shown in Figure 5. It is interesting to note that a rather strong peak with a spectrum equal to that of methaqualone was also obtained. EMIT testing for that compound, however, was negative. Heroin is excreted in the urine predominantly as morphine and conjugated morphine, codeine as free and conjugated codeine, conjugated norcodeine, and conjugated morphine, and morphine in its free or glucuronated form (1). Acid hydrolysis prior to sample analysis is often used to increase the content of the free drugs. For that purpose, the MECC behavior of morphine 3-glucuronide was tested. With our experimental configuration, elution of that compound occurred after about 8.5 min (data not shown), i.e. much earlier than morphine. Single-wavelength electropherograms obtained without (panel A) and with (panel B) acid hydrolysis after Bond Elut Certify extraction described for opioids are presented in Figure 6. Blank urine spiked with morphine, morphine 3-glucuronide, codeine, heroine, and methadone (10 pg/mL each) was used. All these compounds except the glucuronide extracted well (panel A). The heroine standard solution was found to be partly decomposed to morphine and
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Flgure 6. Opioide extraction with hydrolysis. MECC data obtained with a urine blank spiked with morphine 3glucuronide, morphine, codeine, heroin, and methadone (10 pgImL each) after extraction without and with acid hydrolysis are shown in panels A and B, respectively. The data depicted in panels C and D were obtained with opiate-positive patient urine without (C) and with (D) acid hydrolysis. Experimental condtions and zone assignements are the same as in Figure 1. Peak X represents a decomposition product of heroin, &acetyl morphine.
6-acetyl morphine (peak X in Figure 6A), a product which eluted between morphine and codeine. With hydrolysis both heroine and its decomposition product vanished and a significantly higher morphine peak was obtained. The peak increase of the latter compound is assumed to originate from the decomposition of both, the glucuronide and heroine. Single-wavelength data obtained with and without hydrolysis of an opiate-positive (benzodiazepine, barbiturate, amphetamine negative) patient sample are depicted in panels C and D of Figure 6. Morphine was positively detected without acid hydrolysis (panel C), its peak, however, being quite small. For complete confirmation this sample was also spiked with morphine (data not shown). After hydrolysis a much increased morphine peak was monitored (panel D). For that case spectral confirmation was easily possible. It was interesting to find, that for all patient urines investigated (urines which were found to be opiate positive with EMIT screening), no hydrolysis was necessary for c o n f i a t i o n of opiates by MECC when the extraction procedure for cocaine and metabolites was used.
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had to be pretreated for analysis of amphetamines. With direct injection of the urine (panel A) no amphetamine or metampetamine peak was found, whereas the opposite was true after extraction (panel B). Amphetamine and metamphetamine were found to elute as close zones and have essentially equal spectra, this making peak confirmation uncertain (panels C and D). Therefore the extract of the sample had to be spiked with metamphetamine and rerun for proper identification of the monitored compound. 15.0
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The feasibility of monitoring illicit drugs and/or their metabolites, including opioids, amphetamines, cocaine metabolites, and hypnotics (methaqualone), in human urine with MECC is demonstrated. The drugs and metabolites have to be extracted but not derivatized or hydrolyzed prior to analysis. Characterization of sample zones by their retention behavior and absorption spectra is a powerful approach for solute identification. HPCE with multiwavelength detection and/or its hyphenation with MS (11, 12) or HPLC (13), represents a new and attractive methodology in instrumental analysis. With this technology, MECC may very well become the most important analysis and confirmation method for illicit drugs and their metabolites in urine and other body fluids.
ACKNOWLEDGMENT
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We acknowledge helpful discussions with T. Zysset as well as valuable technical assistance from P. Gebauer and the technicians of the departmental drug assay laboratory. The generous loan of the 206 UVIS detector by its manufacturer, Linear Instruments, Reno, NV, is gratefully acknowledged. Reference compounds were kindly provided by J. Huber, laboratory of narcotics, Federal Office of Public Health, Bern, Switzerland. Morphine 3-glucuronide was a kind gift of R. Brenneisen and D. Bourquin. Registry No. Benzoylecgonine,519-09-5; morphine, 57-27-2; heroin, 561-27-3;metamphetamine, 537-46-2;codeine, 76-57-3; amphetamine, 300-62-9; cocaine, 50-36-2; methadone, 76-99-3; methaqualone, 72-44-6; flunitrazepam, 1622-62-4; oxazepam, 604-75-1; diazepam,439-14-5; morphine 3-glucuronide, 20290-09-9; 6-acetyl morphine, 2784-73-8.
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(nm) Figure 7. Analysis of amphetamine-positive urine (A) via direct urine injection, (B) after extraction and detection at 195 nm, and (C) after extraction and multiwavelength detection as well as (D) spectral proof. Note that the data of panels E D all stem from the same experiment. Other conditions and peak identification are the same as in Figure 1. WAVELENGTH
The third example presented in this paper (Figure 7) comprises the confirmation of a positively EMIT tested amphetamine patient urine. Using the extraction procedure given for amphetamine (see above), excellent recoveries (>go%) were obtained for both amphetamine and metamphetamine. Concentrations as low as 100 ng/mL in spiked urine could be assessed and positively identified with comparison of time slices. With the same procedure morphine, methaqualone, and methadone could also be extracted. The data presented in panels A and B of Figure 7 reveal that the patient urine
(1) DeCresce, R.; Mazura, A.; Litschitz. M.; Tiison, J. Drug Testing In the Workplace; ASCP Press: Chicago, 1989. (2) Roach, M. C.; Gozel. P.; Zare, R. N. J . Chromtogr. 1988, 426, 129. (3) Tanaka, Y.; Thormann, W. €lecfrophoresls 1990, 1 1 , 760. (4) Nakagawa, T.; Oda, Y.; Shibukawa, A.; Tanaka, H. Chem. pherm. Bull. 1988, 36, 1622. (5) Nakagawa, T.; Oda, Y.; Shibukawa, A.; Fukuda, H.; Tanaka, H. Chem. Phafm. Bull. 1989, 3 7 , 707. (6) Burton, D. E.; Semniak, M. J.: Maskarinec. M. P. J . Chromtwr. Scl. 1988, 2 4 , 347. (7) Nishi, H.; Fukuyama, T.; Matsuo, M. J. Chromtogr. 1990, 575. 245. (8) Nishi. H.; Terabe, S. Electrophoresis 1990, 7 7 , 691. (9) Thormann, W.; Meier, P.; Marcolli, C.; Binder, F. J . Chromtogr. 1991, 545. 445. (10) Terabe, S. Trends Anal. Chem. 1989, 8 , 129. (1 1) Lee, E. D.; Mueck, W.; Henion, J. D.; Covey, T. R. J. Chromtogr. 1988, 458, 313. (12) Edmonds. C. G.; Loo, J. A,; Barinaga, C. J.; Udseth, H. R.; Smith, I?. D. J. Chmmtogr. 1989, 474, 21. (13) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978.
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RECEIVED for review July 8, 1991. Accepted September 19, 1991. This work was sponsored partly by the Swiss National Science Foundation.
