Technical Notes Anal. Chem. 1996, 68, 3021-3028
Analysis of Cocaine, Benzoylecgonine, and Cocaethylene in Urine by HPLC with Diode Array Detection Karine M. Clauwaert, Jan F. Van Bocxlaer, Willy E. Lambert, and Andre´ P. De Leenheer*
Laboratorium voor Toxicologie, Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium
A solid phase extraction method was developed for the isolation of cocaine, benzoylecgonine, and cocaethylene from urine followed by high-performance liquid chromatography/diode array detection. The application of a new solid hybrid phase extraction technology produced much cleaner extracts than conventional extraction procedures and made the selective extraction of substances with different polarities possible. Two internal standards with great structural resemblance to benzoylecgonine (a carboxylic acid) and to the two esters, cocaine and cocaethylene, respectively, were synthesized. A linear response over a broad concentration range was obtained. The sensitivity, specificity, and accuracy were satisfactory for each analyte. Hydrolysis of cocaine and cocaethylene to benzoylecgonine during extraction and analysis was less than 0.5%. The method described can be used to corroborate cocaine use, to establish cocaine overdoses, and to study pharmacological effects of cocaine and its metabolites. Cocaine, the major alkaloid of Erythroxylum coca, has a long history of human use and abuse. More than 4000 years ago, people chewed the coca leaves. In the beginning of this century, cocaine was an ingredient in tonics and sodas. Now, cocaine is almost exclusively associated with abuse, where it is snorted, injected, or smoked as “freebase” or “crack”.1 The instantaneous and overwhelming effects of such applications explain the popularity of cocaine among drug users. The concomitant extreme danger and high mortality rate readily attract the attention of the physicians and analysts. The effects of cocaine on the central nervous system are well known. It blocks the re-uptake of norepinephrine, dopamine, and serotonin, monoamines implicated in memory function. The high synaptic concentrations of monoamines, and especially of dopamine, result in an increased sense of alertness, well-being, and euphoria. Subsequent monoamine depletion in the presynaps results in a “crash” syndrome, with depression and physical discomfort. This cycle is responsible for the reinforcing properties of cocaine abuse: drug users prevent the crash by taking new and higher doses.1 (1) Jatlow, P. Clin. Chem. 1987, 33, 66B-71B. S0003-2700(96)00060-1 CCC: $12.00
© 1996 American Chemical Society
Cocaine is rapidly and almost completely metabolized and deactivated. Benzoylecgonine is one of the main degradation products, formed by either an hepatic carboxylesterase or spontaneous hydrolysis.2 Although benzoylecgonine has no pharmacological activity, it is of great toxicological and analytical interest: due to its long half-life (6 times longer than that of cocaine), it remains detectable longer than the parent compound, cocaine. Unfortunately, its hydrophilic nature renders it difficult to extract. Cocaine is hydrolyzed by hepatic and plasma esterases to ecgonine methyl ester,1,2 another inactive metabolite, and both benzoylecgonine and ecgonine methyl ester are further hydrolyzed to ecgonine.1 Cocaethylene, an active homologue that arises through transesterification of cocaine following co-consumption of cocaine and alcohol, shares many neurochemical and behavioral properties with cocaine and can reach significant blood concentrations.3 N-Demethylation of cocaine to norcocaine has been identified as a minor metabolic pathway.1 Recently, a pyrolysis product, anhydroecgonine methyl ester, resulting from the elimination of benzoic acid from cocaine, was found in urine and hair of crack smokers.4 Because of the high hydrophilic nature of benzoylecgonine, liquid-liquid extraction solvents require an alcohol to improve extraction efficiency. Although alcohols increase the recovery of benzoylecgonine, they also increase the recovery of other endogenous, water-soluble metabolites typically present in urine. This increases the chemical background noise, generates interfering signals, and reduces column lifetime.5 We found that some authors have had difficulties in achieving clean chromatograms as well as good extraction efficiency for benzoylecgonine (60%6 and 66%5). In addition, reproducibility sometimes is low (>10%).7 Solid phase extractions (SPEs) have become popular, as they give high recoveries and clean extracts. The SPE of cocaine and metabolites in previously reported methods can be divided into three groups, according to the nature of the sorbents. A first (2) Bailey, D. N. J. Anal. Toxicol. 1994, 18, 13-15. (3) Jatlow, P. Ther. Drug. Monit. 1993, 15, 533-536. (4) Kintz, P.; Cirimele, V.; Sengler, C.; Mangin, P. J. Anal. Toxicol. 1995, 19, 479-482. (5) Balı´kova´, M.; Vecerkova´, J. J. Chromatogr. 1994, 656, 267-273. (6) Nakashima, K.; Okamoto, M.; Yoshida, K.; Kuroda, N.; Akiyama, S.; Yamaghuci, M. J. Chromatogr. 1992, 584, 275-279. (7) Rop, P. P.; Grimaldi, F.; Bresson, M.; Fornaris, M.; Vial, A. J. Liq. Chromatogr. 1993, 16, 2797-2811.
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group is based on nonpolar sorbents such as ethylsilica8 or octadecylsilica.9 Svensson9 used a C18 sorbent for the determination of benzoylecgonine but needed two consecutive C18 SPE steps to acquire the necessary cleanup for his samples. This resulted in an extraction efficiency of only 41% for benzoylecgonine. Moreover, this approach entails the use of an identical chemical analytical principle for extraction and chromatographic separation, thus limiting the method’s intrinsic selectivity. A second group is based on ion exchange interactions, where strong cation exchange columns (SCX) are used that retain basic compounds, positively charged at a very low pH.10-12 Extraction efficiencies of 75% and 86%, comparable to ours, were reported12 when the cation exchange approach was used. Unfortunately, this method also requires an extra, consecutive C18 extraction step, in fact doubling the analysis cost and complexity. Recently, a third group of solid phase sorbents, the mixedmode sorbents, became popular for the analysis of drugs of abuse.13-16 These extraction cartridges contain a nonpolar C8 sorbent and a strong cation exchanger. Analytes are strongly retained on the sorbent by a combination of Coulombic and nonpolar interactions, allowing a series of drastic aqueous and organic wash steps. This results in a superior sample cleanup prior to screening or confirmation analyses such as TLC, HPLC, or GC/MS. Mills et al.17 previously compared chromatographic results after extraction with C8 sorbents versus mixed-mode extractions and concluded that the latter were clearly superior, resulting in chromatograms with much fewer interferences. As an alternative, they also used a combination of two different extraction columns, C18 and SCX, in sequence. An additional advantage of the mixed-mode sorbents is the moderate pH condition used as compared with, e.g., the classical strong cation exchangers, reducing procedural hydrolysis of cocaine to a minimum. Batch-to-batch irreproducibility for SPE preparations is, however, generally recognized as a possible problem. A recent report nullifies this concern, but it uses GC/MS and deuterated internal standards.18 SPE can, indeed, give reliable quantitative results if an internal standard is used that has close structural similarity to the compounds analyzed. In the literature, some solid phase methods8-10 do not use an internal standard at all. In our opinion, this should be avoided, since control of the extraction is an absolute necessity in order to anticipate unexpected losses. Other internal standards that are used differ completely in structure, pKa and lipophilicity from cocaine and metabolites, e.g., bupivacaine,13 tetracaine,14 methadone,15 and benzoctamine.5 The use (8) Levine, B. S.; Tebbett, I. R. Drug Metab. and Dispos. 1994, 22, 498-500. (9) Svensson, J.-O. J. Anal. Toxicol. 1986, 10, 122-124. (10) Browne, S. P.; Tebbett, I. R.; Moore, C. M.; Dusick, A.; Covert, R.; Yee, G. T. J. Chromatogr. 1992, 575, 158-161. (11) Logan B. K.; Stafford D. T.; Tebbett I. R.; Moore C. M. J. Anal. Toxicol. 1990, 14, 154-159. (12) Lampert, B. M.; Stewart, J. T. J. Chromatogr. 1989, 495, 153-165. (13) Moore, C.; Browne, S.; Tebbett, I.; Negrusz, A. Forensic Sci. Int. 1992, 53, 215-219. (14) Ferna´ndez, P.; Rodrı´guez, P.; Bermejo, A. M.; Lo´pez-Rivadulla, M.; Cruz, A. J. Liq. Chromatogr. 1994, 17, 883-890. (15) Roy, I. M.; Jefferies, T. M.; Threadgill, M. D.; Dewar, G. H. J. Pharm. Biomed. Anal. 1993, 10, 943-948. (16) Moore, C. M.; Browne, S. P.; Negrusz, A.; Tebbett, I.; Meyer, W.; Jain, L. J. Anal. Toxicol. 1993, 17, 388. (17) Mills, M. S.; Thurman, E. M.; Pedersen, M. J. J. Chromatogr. 1993, 629, 11-21. (18) Jennison,, T. A.; Jones, C. W.; Wozniak, W.; Urry, F. M. J. Chromatogr. Sci. 1994, 32, 126-131.
3022 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 1. Chemical structures of benzoylecgonine and its internal standard, 2′-methylbenzoylecgonine, and cocaine and cocaethylene and their internal standard, 2′-methylcocaine.
of local anesthetic agents and other drugs should be avoided, since they are often used to adulterate street drugs;19 moreover polydrug abuse is common among cocaine users. Hexylbenzoylecgonine15 is more closely related to cocaine, but it is markedly less polar. None of the previously reported internal standards can counterbalance eventual losses of benzoylecgonine during the extraction. Part of the retention mechanism on the solid phase cartridge is based on ionic interactions with the carboxylic acid function of benzoylecgonine. None of the proposed internal standards possess such a carboxylic acid function. An internal standard that perfectly compensates for analytical errors and eventual degradation phenomena cannot be the same for benzoylecgonine, a carboxylic acid, and cocaine or cocaethylene, two ester compounds. Such internal standards are not commercially available, and for that reason, an internal standard for benzoylecgonine and one for cocaine and cocaethylene were synthesized (see Figure 1). HPLC/UV has been used to identify cocaine and especially its more polar metabolite, benzoylecgonine, in urine.20 In situations where cocaine use is the major concern, the focus is, appropriately, on the detection of cocaine’s more slowly eliminated but inactive metabolite, benzoylecgonine. On the other hand, measurement of cocaine and cocaethylene in urine and blood can be required for correlation with acute clinical effects whether for clinical, forensic, or research purposes. HPLC procedures described for measuring cocaine and its metabolites are considerably less sensitive than desired, are often tested in a narrow concentration range, which is a severe limitation in view of the strongly different metabolite concentrations, are generally based on UV detection, resulting in lower specificity, and use internal standards not structurally related to benzoylecgonine nor cocaine and cocaethylene. Our aim was to develop a method that circumvents all these limitations. This paper describes its results and practical application in the fields of both forensic and clinical toxicology. EXPERIMENTAL SECTION Apparatus. The HPLC unit was composed of a ternary lowpressure gradient system (Model 325, Kontron Instruments, (19) Analytical profiles of cocaine, local anesthetics, and common diluents found with cocaine; CND Analytical Inc.: Auburn, 1990. (20) Clauwaert, K.; Lambert, W.; De Leenheer, A. J. Liq. Chromatogr. 1995, 18, 2097-2114.
Milano, Italy) and an injector Model 7725i with a 50-µL loop (Rheodyne). A diode array detector (DAD 440, Kontron Instruments) linked to a data system (450-MT2/DAD, Kontron Instruments) was used for data acquisition and storage. The Fourier transform infrared spectra (KBr) were obtained on a Fourier transform infrared spectrometer (System 2000, Perkin Elmer, Buckinghamshire, U.K.). The proton nuclear magnetic resonance (1H-NMR) spectra were obtained on a Bru¨cker WH 360 instrument (Karlsruhe, Germany). A saturated solution of the powder samples in deuterated methanol was used to record chemical shifts relative to tetramethylsilane (in ppm). For gas chromatography/mass spectrometry (GC/MS), a 3400 Series Varian gas chromatograph (Varian, Sunnyvale, CA) was used in combination with a Finnigan Mat Magnum mass-selective ion trap detector (Finnigan, San Jose´, CA). For gas chromatography/Fourier transform infrared spectrometry (GC/FT-IR), a Perkin Elmer Autosystem gas chromatograph was used (Perkin Elmer, Buckinghamshire, U.K.) in combination with a Perkin Elmer GC/IR System 2000 interface and a FT-IR System 2000 detector. Reagents. All the reagents and products were of analytical grade and were from E. Merck (Darmstadt, Germany). Methanol and acetonitrile were of HPLC grade from Romil Chemicals Ltd. (Loughborough, U.K.), and water was of HPLC grade from Prosan (Ghent, Belgium). Ammonium hydroxyde, pentafluoropropanol, and heptafluorobutyric anhydride were obtained from Aldrich (Milwaukee, WI). The benzoylecgonine tetrahydrate, cocaethylene, and cocaine hydrochloride standards were from Makor Chemicals (Jerusalem, Israel). Bond Elut Certify columns (300 mg/3 mL) were obtained from Varian Sample Preparation Products (Harbor City, CA). Samples. Urine samples were immediately analyzed for drugs of abuse using an enzyme immunologic technique. The commercial enzyme immunoassay used for the detection of benzoylecgonine was the EMIT d.a.u. Cocaine Metabolite Assay (Syva Co., San Jose´, CA). The samples that tested positive by EMIT were stored at -20 °C until time of HPLC analysis. Synthesis of Internal Standards. To enhance the precision of the analysis, appropriate internal standards were synthesized in our laboratory. Starting from pure cocaine hydrochloride, methylecgonine and ecgonine were prepared by an acid hydrolysis and a subsequent methylation.21 Cocaine hydrochloride (1 g) was refluxed in 1.2 N HCl (70 mL) for 24 h. After evaporation to 10 mL on a Bu¨chi rotary evaporator (Bu¨chi, Switzerland), the solution was cooled and the resulting benzoic acid precipitate filtered off. Further evaporation to 2 µg/mL), was unreasonably low, resulting in an inacceptable limited linearity interval. To improve this deflection of linearity, the capacity of the cartridge was increased to 300 mg.
Figure 3. Blank urine (f) eluting position of benzoylecgonine (1), 2′-methylbenzoylecgonine (2), cocaine (3), cocaethylene (4) and 2′methylcocaine (5).
This immediately raised the recovery for benzoylecgonine to an acceptable level, up to a concentration of 5 µg/mL. The increased cartridge size also retained additional endogenous components from the urine. In an effort to counteract this and to intensify the cleanup, the volume of wash solvents was increased. Initially, methanol alone was used as the wash solvent, but it soon became clear that a combination of methanol and acetonitrile gave cleaner extracts: 9 mL of both seemed to be the maximum, as using more wash solvent produced breakthrough in this step, resulting in a lower recovery and a much worse reproducibility. The use of more elution solvent (in excess of 2 mL) or of a higher percentage of ammonia in the elution mixture increased the procedural recovery of the compounds, unfortunately at the expense of the purity. Large peaks of unknown origin appeared and confused the evaluation of the compounds of interest. The flow speed through the sorbent is an important analytical variable, since reducing the contact time by using a higher flow speed results in a marked reduction of extraction efficiency, especially for the important metabolite benzoylecgonine. Chromatography. Using the HPLC conditions described, benzoylecgonine, cocaine, and cocaethylene were all well resolved. Attempts to use isocratic conditions were unsatisfactory. Optimal resolution for benzoylecgonine resulted in unacceptably long retention times for cocaine and cocaethylene. When a higher percentage of organic solvent was used, benzoylecgonine was inadequately resolved from early-eluting endogenous compounds. The retention times of benzoylecgonine, 2′-methylbenzoylecgonine, cocaine, cocaethylene, and 2′-methylcocaine were 6.3, 9.7, 13.7, 16.5, and 17.7 min, respectively, yielding capacity factors (k′) of 2.5, 4.4, 6.6, 8.2, and 8.8 (Figure 2). As described in the Experimental Section, our chromatographic analysis accomplishes optimum resolution in a more than acceptable run time. A major advantage of our approach is also the possibility of multiple sequential injections, without the risk of interfering, late-eluting peaks. Method Evaluation. The calibration curves were linear over the specified ranges (0.1-5.0 µg/mL for cocaine and benzoylecgonine and 0.05-2.5 µg/mL for cocaethylene). A correlation Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
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Table 2. HPLC Retention Data for Compounds Evaluated as Possible Interferents compound
capacity factor, k′
acetaminophen morphine caffeine hydromorphone dihydrocodeine N-methylephedrine amphetamine metamphetamine benzoylecgonine 3,4-methylenedioxy-N-methylamphetamine oxycodone codeine 6-monoacetylmorphine 3,4-methylenedioxy-N-ethylamphetamine pholcodine hydrocodone pindolol 2′-methylbenzoylecgonine viloxazine phenobarbital apomorphine thebacon
0.54 0.92 1.13 1.26 1.64 1.69 1.69 2.23 2.47 2.73 2.80 2.99 3.19 3.31 3.74 3.96 4.12 4.56 5.38 5.42 5.68 6.40
compound cocaine naloxone acetylcodeine bromazepam cocaethylene alprenolol 2′-methylcocaine propranolol citalopram nitrazepam alprazolam, amitryptiline, brotizolam, butryptiline, camazepam, chlordiazepoxide, clobazam, clomipramine, clonazepam, clotiazepam, cloxazolam, desipramine, diazepam, dothiepine, doxepine, ethylloflazepate, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, halazepam, imipramine, lofepramine, loprazolam, lorazepam, lormetazepam, maprotiline, medazepam, melitracen, mianserin, midazolam, nordazepam, nortryptiline, opipramol, oxazepam, paroxetine, sertraline, temazepam, trazodone, triazolam, trimipramine
coefficient of 0.9995 or higher was obtained for the relationship between the peak height ratio (compound/IS) and the corresponding calibration concentrations. The method shows good linearity over a broad concentration range. Some of the previously reported methods tested their linearity in a narrow concentration range: 36-1445,6 50-1000,7 or 200-1000 ng/mL.15 This should be avoided since cocaine and its metabolites appear in vastly different concentrations in urine. The various regression line slopes were 0.001 29 (CV% ) 3.9 for n ) 8 determinations on separate days) for cocaethylene, 0.001 28 (CV% ) 4.7, n ) 8) for benzoylecgonine, and 0.001 48 (CV% ) 5.0, n ) 8) for cocaine, and the intercepts were all close to zero. These linearity data indicate a good day-to-day match for all calibration curves. For concentrations exceeding the calibration ranges, the response deviated from linearity, in which cases we diluted and reassayed the sample. Table 1 presents the within-day and total (between-day) reproducibility data obtained (n ) 8) for the different concentrations tested. The coefficients of variation ranged from 1.6% to a maximum of 7.0%. The precision varied slightly, although it was not concentration-dependent, but met with our objective of a routinely applicable analysis for cocaine and two of its diagnostically important metabolites. These results clearly indicate that reproducibility is acceptable over the studied concentration range, a criterion which is not discussed5,8,13 or met12 by previously published SPE extraction methods. When our internal standard for cocaine is used for calculation of the benzoylecgonine reproducibility data and vice versa, the resulting coefficients of variation are much higher (up to 15%). This clearly proves that the use of an internal standard with close structural similarity to the analyte of interest, a criterion often neglected, is vital in order to get sound analytical results. We found that the recovery was high, reproducible, and fully concentration independent in the studied interval, as mentioned above. Mean recoveries of benzoylecgonine, cocaine, and cocaethylene were 95%, 89%, and 88%, respectively. These recoveries 3026
Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
capacity factor, k′ 6.44 6.91 7.16 8.02 8.11 8.44 8.72 8.79 9.32 9.51 >9.55
are higher than the ones obtained with most of the other procedures. For validation purposes, we also determined accuracy for benzoylecgonine, cocaine, and cocaethylene at two separate concentration levels. We found 102.7% and 101.7% for benzoylecgonine at the respective concentrations of 0.159 and 1.592 µg/mL; 101.7% and 103.1% at 0.153 and 1.530 µg/mL for cocaine, and 98.6% and 103.9% at 0.089 and 0.893 µg/mL for cocaethylene. The limit of detection, as defined in the Experimental Section, was 20 ng/mL for the three compounds. The quantitation limit, the lowest point of the calibration graph, was 0.1 µg/mL for benzoylecgonine and cocaine and 0.05 µg/mL for cocaethylene, quantitatively measurable with acceptable reproducibility [total reproducibilities (CV%) were 7.0% for benzoylecgonine, 4.5% for cocaine, and 5.5% for cocaethylene]. All UV spectra were recognized by the system with a spectral matching factor greater than 950 at these concentrations. It is obvious that LOD and LOQ can be much lower if the spectral demands are not taken into consideration and merely the signal-to-noise definition is used. We confined ourselves to the more stringent definitions, including spectral criteria, in order to preserve, even in the lowest range, the double identification potential (retention time and UV spectrum), vital in toxicological analysis. Figure 2 shows the chromatogram of a urine sample enriched with 0.5 µg/mL of cocaine and benzoylecgonine as well as 0.25 µg/mL cocaethylene and 0.65 µg/mL of the two internal standards. Comparison of this chromatogram with that of a blank urine (Figure 3) shows that there are no interfering peaks from endogenous compounds. We obtained clean chromatograms, even for postmortem urine extracts, with little chemical background noise and no interfering peaks after a relatively simple extraction method. This is in contrast to previously published chromatograms of urine extracts monitored with UV detection.5,6,9 As our method is very suitable for use in a toxicological laboratory, selectivity considerations, especially pertaining to prescription drug and drugs of abuse, are of prime importance.
Table 3. Urinary Concentrations of Cocaine and Metabolites of Cases Found Positive by the EMIT Screening Procedurea concn found, µg/mL patient no.
case
other drugs found
benzoylecgonine
cocaine
cocaethylene
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
JC PM JC JC CC CC CC CC CC CC JC PM PM JC PM JC CC JC JC JC CC CC CC CC JC JC CC JC JC CC PM
opiates, cannabinoids opiates, benzodiazepines, tricyclic antidepressants opiates, MDMA, MDEA, cannabinoids opiates, MDMA, MDEA, cannabinoids opiates opiates nd nd opiates nd opiates, cannabinoids opiates, ethanol opiates, ethanol, benzodiazepines opiates, cannabinoids cannabinoids, ethanol opiates, cannabinoids nd cannabinoids opiates, benzodiazepines opiates, benzodiazepines cannabinoids opiates, cannabinoids opiates opiates opiates cannabinoids opiates opiates, cannabinoids, benzodiazepines benzodiazepines, ethanol, MDMA cannabinoids opiates, benzodiazepines, methadone
0.29 48.58 10.47 3.87 3.38 25.82 1.03 93.6 33.1 136.9 604.9 7.94 7.04 5.89 147.66 7.23 1.72 0.59 232.62 243.40 1.23 0.15 5.06 0.39 4.42 5.83 6.51 6.27 18.95 1.13 40.82
nd 41.02 nd nd nd nd nd 20.6 nd 6.99 9.72 8.82 17.30 nd 6.82 0.91 nd nd 2.98 3.25 nd nd 0.10 0.17 0.54 nd nd 0.82 0.93 nd nd
nd nd nd nd nd nd nd nd nd nd nd 0.74 1.42 nd 0.410 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd
a Abbreviations used: nd, not detected; PM, post mortem case; CC, clinical case; JC, judicial case; MDMA, 3,4-methylenedioxy-Nmethylamphetamine; MDEA, 3,4-methylenedioxy-N-ethylamphetamine.
In that respect Table 2 shows the k′ of drugs considered to be possible interferences, in order of ascending k′ value. As can be seen, thebacon partially overlapped with cocaine, bromazepam was insufficiently resolved from cocaethylene, and propranolol interfered with 2′-methylcocaine. Fortunately, the UV spectra of the interfering substances were distinctly different from those of either analyte under investigation, allowing a reliable differential diagnosis. Furthermore, only a slight percentage of the dose that is taken from either bromazepam or propranolol (2% and
