Anal. Chem. 1998, 70, 163-172
Matrix and Modifier Effects in the Supercritical Fluid Extraction of Cocaine and Benzoylecgonine from Human Hair Janet F. Morrison,*,† Stephen N. Chesler,† Wesley J. Yoo,†,‡ and Carl M. Selavka§,|
Analytical Chemistry Division, National Institute of Standards and Technology, 222/A213, Gaithersburg, Maryland 20899, and National Medical Services, Inc., 2300 Stratford Avenue, Willow Grove, Pennsylvania 19090
The analysis of human hair for the presence of drugs of abuse was first reported in 1979, when Baumgartner and co-workers1 used radioimmunoassay to detect opiates in extracts of hair from suspected heroin abusers. In the decade and a half since that
first published report, the literature has flourished with applications of hair testing for both illicit and, more recently, therapeutic drugs. Despite legal challenges regarding constitutionality and admissibility, the results of hair drug testing have been submitted to American courts as scientific evidence in criminal cases, military courts-martial, child custody and adoption cases, probation revocation proceedings, unemployment compensation cases, and cases involving workplace drug testing programs.2 Hair provides a unique toxicological specimen because it greatly expands the time window for drug detection compared with more common biological samples, such as urine and serum, which generally provide information only on recent drug use (e.g., hours to days). Following their deposition in hair, drugs can persist for extended periods of time, providing information on chronic exposure which can complement the short-term information provided by urinalysis. Evidence for the persistence of drugs in hair is demonstrated by the detection of cocaine and metabolites in the hair of South American Indians3 and Bolivian mine workers4 identified as daily chewers of coca leaves. Because of the potential long-term stability of some drugs in hair, segmental analysis along the hair shaft has been reported to provide information on the history of drug use for an individual by correlation with hair growth rates.5,6 Proponents of hair drug testing argue that sample acquisition is less intrusive and that it is more difficult to evade drug detection with hair analysis compared to urinalysis. Despite these advantages, widespread acceptance of hair as a reliable drug testing medium has been hindered by a number of factors and controversies. Chief among these is our limited understanding of the mechanisms for drug incorporation in hair. The earliest and simplest model for drug incorporation in hair considered the major route of entry to be diffusion of drug molecules from the bloodstream into the rapidly growing cells in the hair follicle, with subsequent entrapment of the drugs as the proliferating hair cells die and fuse to form the keratin fibers of
* Corresponding author: Current address: Department of Chemistry, Trinity College, 300 Summit St., Hartford, CT 06106. † National Institute of Standards and Technology. ‡ Current address: Federal Bureau of Investigation Research and Training Center, Quality Assurance Unit, FSRTC-Building 12, Quantico, VA 22135. § National Medical Services, Inc. | Current address: New York State Division of Criminal Justice Services, 4 Tower Place, Albany, NY 12203-3702. (1) Baumgartner, A. M.; Jones, P. F.; Baumgartner, W. A.; Black, C. T. J. Nucl. Med. 1979, 20, 748-752.
(2) Huestis, M. A. In Drug Testing in Hair; Kintz, P., Ed.; CRC Press, Inc.: Boca Raton, FL, 1996; Chapter 1. (3) Henderson, G. L.; Harkey, M. R.; Zhou, C.; Jones, R. T. J. Anal. Toxicol. 1992, 16, 199-201. (4) Mo ¨ller, M. R.; Fey, P.; Rimbach, S. J. Anal. Toxicol. 1992, 16, 291-296. (5) Strano-Rossi, S.; Bermejo-Barrera, A.; Chiarotti M. Forensic Sci. Int. 1995, 70, 211-216. (6) Nakahara, Y.; Shimamine, M.; Takahashi, K. J. Anal. Toxicol. 1992, 16, 253-257.
The supercritical fluid extraction (SFE) behavior of cocaine and its major metabolite benzoylecgonine (BZE) was investigated and found to be highly dependent upon the chemical nature of the matrix and the manner in which the target drug analytes are incorporated into or on the matrix. The recovery of cocaine from Teflon wool, filter paper, drug-fortified hair, and drug user hair was studied using a variety of CO2/modifier mixtures. Incorporation of a triethylamine (TEA)/water modifier mixture provided dramatic improvements in the recovery of cocaine from interactive matrixes. The results suggest that the SF extractability of cocaine is not limited by analyte solubility; rather, desorption of cocaine from hair binding sites is a rate-limiting step in the SFE process. A displacement SFE mechanism is hypothesized in which TEA (as the triethylammonium cation) competes with cocaine for negatively charged hair binding sites. The dependence of extractability on hair/drug binding interactions allows the differentiation of cocaine present at different discrete sites in hair based on differences in SFE behavior. These findings suggest the potential for distinguishing exogenous (i.e., environmental) from endogenous (i.e., physiological) sources of drugs in hair. In contrast to the results observed for cocaine, SFE recoveries of BZE were poor from all matrixes and under all conditions studied. Its increased polarity, the presence of an additional binding site, and the possibility of multiple charged states suggest that poor BZE recoveries may be due to both poor analyte solubility and failure to desorb the analyte from hair binding sites under the conditions employed.
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Figure 1. Potential routes of entry of drugs in hair.
the hair shaft.7-9 This model, later termed the entrapment model,10 suggests that drugs are incorporated into hair only during its active growth phase and in direct proportion to their concentrations in blood and predicts that segmental analysis along the hair shaft can provide a history of drug use by correlation with known hair growth rates. However, several experimental findings are difficult to explain by the entrapment model and suggest that this model is too simplistic.3,9,10 One major challenge to this model is the finding that parent drug-to-metabolite ratios in hair do not mirror these ratios in plasma. For example, while cocaine is rapidly metabolized in blood, in hair the concentration of cocaine predominates over its metabolite benzoylecgonine (BZE) by factors of 2-10.3,9,11,12 These findings have led to the development of more complex alternative models which describe multiple sources and mechanisms for incorporation of drugs in hair.9,10,13 Although all models and the arguments for and against each cannot be presented here, potential routes of entry proposed by these models are shown schematically in Figure 1 and include (1) diffusion from the blood into the growing cells in the hair follicle, (2) secretion in the sweat, (3) secretion in sebum, and (4) exposure of the hair to drug vapors, particles, or solutions from the external environment. For the purposes of this paper, route 4 will be referred to as environmental contamination. Unlike the entrapment model, these alternative models allow for drug incorporation after hair formation and include the possibility of environmental contamina(7) Baumgartner, W. A.; Hill, V. A.; Blahd, W. H. J. Forensic Sci. 1989, 34, 1433-1453. (8) Harkey, M. R.; Henderson, G. L. Adv. Anal. Toxicol. 1989, 2, 298-329. (9) Henderson, G. L. Forensic Sci. Int. 1993, 63, 19-29. (10) Kidwell, D. A.; Blank, D. L. In Drug Testing in Hair; Kintz, P., Ed.; CRC Press, Inc.: Boca Raton, FL, 1996; Chapter 2. (11) Kintz, P.; Mangin, P. Forensic Sci. Int. 1995, 70, 3-11. (12) Selavka, C. M.; Riker, C. D. In Hair Testing for Drugs of Abuse: International Workshop on Standards and Technology; Cone, E. J., Welch, M. J., Grigson Babecki, M. B., Eds.; National Institutes of Health Publication 95-3727; U.S. Government Printing Office, Washington, DC, 1995, pp 248-276. (13) Wang, W. L.; Cone, E. J. Forensic Sci. Int. 1995, 70, 39-51.
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tion through passive exposure. The acknowledgement of several potential routes of entry for drugs in hair has complicated the accurate interpretation of hair drug findings. The potential for evidentiary false positives caused by environmental contamination of hair has generated a great deal of controversy regarding the ability of laboratories to differentiate reliably endogenous (i.e., physiological) from exogenous (i.e., environmental) sources of drugs in hair. Laboratories engaged in hair drug testing currently employ a wide variety of operationally defined rinsing protocols to remove environmental contamination from the hair surface; however, the significance and effectiveness of these decontamination procedures have been the subject of much debate.10,13-16 Following the rinsing steps, various wet chemical extraction methods (generally acid incubation, alkaline hydrolysis, enzymatic dissolution, or organic solvent extraction) are employed to release “matrix-bound” drugs from the hair.17 The target drug analytes are subsequently isolated from the crude extracts by liquid-liquid extraction (LLE) or solidphase extraction (SPE). These wet chemical procedures, which have been summarized by Selavka and Rieders18 for the determination of cocaine and metabolites, are often labor- and timeintensive, involving multiple manipulations that are not easily automated. The favorable mass transport properties and tunable solvation power of supercritical fluids (SFs) have prompted researchers to investigate supercritical fluid extraction (SFE) as an alternative to currently used liquid solvent-based procedures for drug testing in hair.19,20 Edder et al.21 reported excellent recoveries of opiates from drug-fortified and drug user hair using CO2 modified with a ternary mixture of methanol, triethylamine, and water under subcritical conditions (40 °C, 25 MPa). Morrison and co-workers demonstrated rapid, efficient SFE recoveries of cocaine from drugfortified hair using CO2 with addition of a modifier mixture of triethylamine and water to the hair sample (110 °C, 40.5 MPa)22 and subsequently employed off-line SFE with radioimmunoassay (RIA) detection as a rapid screening tool for detecting cocaine residues in drug user hair.23 Cirimele et al.24 have adapted the procedures of Edder et al. and Morrison et al. for use on a commercial SFE instrument, demonstrating recoveries of opiates, cocaine, and cannabinoids from drug user hair under a single set of SFE conditions. (14) Blank, D. L.; Kidwell, D. A. Forensic Sci. Int. 1993, 63, 145-156. (15) Baumgartner, W. A.; Hill, V. A. Forensic Sci. Int. 1993, 63, 157-160. (16) Blank, D. L.; Kidwell, D. A. Forensic Sci. Int. 1995, 70, 13-38. (17) Mo ¨ller, M. R.; Eser, H. P. In Drug Testing in Hair; Kintz, P., Ed.; CRC Press, Inc.: Boca Raton, FL, 1996; Chapter 4. (18) Selavka, C. M.; Rieders, F. Forensic Sci. Int. 1995, 70, 155-164. (19) Staub, C.; Edder, P.; Veuthey, J. L. In Drug Testing in Hair; Kintz, P., Ed.; CRC Press, Inc.: Boca Raton, FL, 1996; Chapter 5. (20) Maxwell, R. J.; Morrison, J. F. In Handbook of Analytical Therapeutic Drug Monitoring and Toxicology; Wong, S., Sunshine, I., Eds.; CRC Press, Inc.: Boca Raton, FL, 1997; Chapter 5. (21) Edder, P.; Staub, C.; Veuthey, J. L.; Pierroz, I.; Haerdi, W. J. Chromatogr., B 1994, 658, 75-86. (22) Morrison, J. F.; MacCrehan, W. A., Proceedings of the 5th International Symposium on Supercritical Fluid Chromatography and Extraction, Baltimore, MD, January 11-14, 1994, Supercritical Conferences: Cincinnati, OH, 1994; p F-16. (23) Morrison, J. F.; Chesler, S. N.; Reins, J. L. J. Microcolumn Sep. 1996, 8, 37-45. (24) Cirimele, V.; Kintz, P.; Majdalani, R.; Mangin, P. J. Chromatogr., B 1995, 673, 173-181.
Figure 2. Structures and potential charge states of (A) cocaine and (B) benzoylecgonine in aqueous solution.
An ideal extraction method for drugs in hair should not only simplify sample preparation steps and provide rapid, efficient recoveries of target analytes but also accurately address the environmental contamination issue and contribute to a greater fundamental understanding of hair/drug binding mechanisms. A growing body of research in the environmental SFE literature25-32 has demonstrated that the manner in which analytes are incorporated in a matrix has a profound influence on both extraction kinetics and the conditions required for optimum extraction with SFs. The ability of the SF or SF/modifier mixture to overcome analyte/matrix interactions is frequently the rate-limiting step in the extraction process. While this “matrix effect” is often considered one of the limitations of SFE, it can also be exploited to study analyte/matrix bonding mechanisms and to differentiate discrete sites of interaction, since analytes bound by different mechanisms or at different sites within a heterogeneous matrix would be expected to exhibit different extraction behavior. The matrix dependence of SFE, coupled with the existence of a greater number of easily variable extraction parameters for method optimization, suggests the possibility for extraction selectivity “tuning” which is not readily available with conventional liquid solvent-based hair testing methods. The goals of the present study were to (1) investigate the influence of the matrix on the extractability of cocaine (Figure 2A) and its major metabolite BZE (Figure 2B) from hair, (2) elucidate the role of modifiers in improving extractability, and (3) evaluate the potential of SFE as a research tool for studying hair/drug binding mechanisms and distinguishing environmental contamination from active drug use. (25) Alexandrou, N.; Pawliszyn, J. Anal. Chem. 1989, 61, 2770-2776. (26) Alexandrou, N.; Lawrence, M. J.; Pawliszyn, J. Anal. Chem. 1992, 64, 301311. (27) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 909-916. (28) Oostdyk, T. S.; Grob, R. L.; Snyder, J. L.; McNally, M. E. Anal. Chem. 1993, 65, 596-600. (29) Fahmy, T. M.; Paulaitis, M. E.; Johnson, D. M.; McNally, M. E. P. Anal. Chem. 1993, 65, 1462-1469. (30) Yang, Y.; Gharaibeh, A.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1995, 67, 641-646. (31) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1993, 65, 338-344. (32) Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1994, 66, 4005-4012.
EXPERIMENTAL SECTION Chemicals and Reagents. Pure crystalline cocaine hydrochloride and free base cocaine were obtained from Mallinckrodt (St. Louis, MO) and Sigma Chemical Co. (St. Louis, MO), respectively. Benzoylecgonine tetrahydrate was purchased from ADRI Technam (Park Forest, IL). Methanolic solutions of cocaine-d3 hydrochloride (0.098 mg/mL as free base) and benzoylecgonine-d3 (0.1 mg/mL) were obtained from MSD Isotopes (Montreal, Canada) and Sigma Chemical Co., respectively, for use as internal standards for quantification. The silylation derivatization reagent N,O-bis(trimethylsilyl)acetamide (BSA) was supplied in 1 mL ampules from Pierce (Rockford, IL). HPLC-grade methanol and water were obtained from J. T. Baker (Phillipsburg, NJ). Triethylamine (99+%) was obtained from Aldrich (Milwaukee, WI). Samples for Spike-Recovery SFE Experiments. Preliminary SFE experiments designed to optimize fluid composition and collection conditions for cocaine were performed using filter paper and Teflon wool (DuPont, Wilmington, DE) as spiking matrixes. Filter paper disks (1 cm diameter) were cut from Whatman No. 42 filter paper and placed in the extraction vessel to produce a bed ∼0.5 cm thick. A gas-tight syringe was used to deliver a weighed portion of a gravimetrically prepared stock methanolic cocaine free base or cocaine hydrochloride solution to the filter paper bed, resulting in a total spike amount of 6 µg of cocaine. Following evaporation of the spiking solvent, additional filter paper disks were placed in the extraction vessel to produce a total bed thickness of ∼1 cm and the vessel was sealed and subjected to SFE. For Teflon wool spiking experiments, strands of Teflon wool were precut into short segments (3-5 mm), loosely packed in the extraction vessel, and spiked in the manner described above. Hair Samples. Drug-free (blank) hair, drug-fortified hair, and drug user hair were employed in this study. Drug-free hair was pooled from volunteers at NIST and was cryogenically ground33 to a fine powder to produce a homogeneous blank control. Highlevel cocaine-fortified segmented hair (FH-S) and midlevel cocaineand BZE-fortified powdered hair (FH-P) were prepared by soaking segmented hair in a DMSO solution of the drugs (cocaine hydrochloride and BZE tetrahydrate) for a 1 month period. Following removal of the DMSO, the hair was rinsed with methanol, allowed to air-dry, and, for the FH-S samples, extracted with no further pretreatment. For the FH-P samples, the fortified hair batch was cryogenically ground to a fine powder. Hair samples demonstrated as cocaine-positive based on previous tests were provided by private commercial drug testing laboratories. Pooled cocaine-positive control hair (NMS-PPC) from drug users was obtained in powdered form from National Medical Services, Inc. (NMS, Willow Grove, PA). This pooled positive control hair had been rinsed prior to homogenization using methanol and pH 7 phosphate buffer and was extracted with no further pretreatment. Typical hair sample masses for SFE were 30-40 mg. Supercritical Fluid Extraction. SFE was performed using laboratory-assembled instrumentation which was slightly modified from a configuration previously described.23 An Isco Model 100D syringe pump (Isco, Inc., Lincoln, NE) was used for constant pressure delivery of carbon dioxide (SFE/SFC grade, Air Prod(33) Welch, M. J.; Sniegoski, L. T.; Allgood, C. C.; Habram, M. J. Anal. Toxicol. 1993, 17, 389-398.
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ucts, Inc., Allentown, PA) to the extraction system. For introducing modifier to the sample, a Rheodyne Model 7125 rotary valve equipped with a 100 µL sample loop was mounted outside the extraction oven and connected to the entrance end of the extraction vessel. The modifier or modifier mixture was introduced directly to the sample by injection into the CO2 stream upon initiation of the static extraction step. Extraction vessels (1 cm long × 1 cm i.d.; 780 µL internal volume) were constructed from conventional guard cartridge hardware (Upchurch Scientific, Oak Harbor, WA). A GOW-MAC Series 580 gas chromatographic oven (GOW-MAC Instrument Co., Lehigh Valley, PA) was employed for temperature control. A two-way shut-off valve was installed through the oven wall and connected to the exit end of the extraction vessel to allow operation in either the static or dynamic extraction modes. SFE was performed at 40.5 MPa and 110 °C with CO2 as the primary fluid; modifiers and static/dynamic extraction times are specified in the Results and Discussion section. In all cases, a 10 min thermal equilibration period was employed prior to SFE (i.e., no CO2 present) to allow the extraction vessel to pre-equilibrate to the operating temperature. A linear restrictor (90 cm long × 50 µm i.d.) constructed from aluminum-clad fused-silica capillary tubing (Scientific Glass Engineering, Austin, TX) was connected downstream of the shut-off valve for flow control. With this restrictor, fluid flow rates (measured at the pump) during dynamic extraction were reproducible at 1.1-1.2 mL/min (40.5 MPa, 110 °C). Extracted components were collected by immersing the restrictor tip in a test tube containing ∼3 mL of methanol. The test tube was capped with a Mininert valve (Pierce, Rockford, IL) which had its rubber gasket removed to allow insertion of the restrictor. The valve position on the cap was adjusted to a partially closed position sufficient to hold the restrictor in place while allowing adequate venting of CO2. [SAFETY NOTE: While the authors have never experienced any failure in the vented solvent collection system described, the test tube should be placed behind a safety shield to protect against potential shattering due to the rapidly expanding CO2.] Appropriate deuterated internal standards were spiked into the collection test tubes prior to SFE. The collection tube was held in a heating block maintained at ∼35 °C to prevent freezing or plugging at the restrictor tip. Acid Incubation/Solid-Phase Extraction. For comparison with SFE results, selected samples of drug-fortified hair were subjected to acid incubation followed by SPE. Additionally, some hair samples that had been previously extracted by SFE were subjected to acid incubation/SPE to determine whether any additional drug could be recovered. Hair samples (30-40 mg) were weighed into vials with Teflon-lined screw caps, spiked with deuterated internal standards, and incubated with 2 mL of 0.1 mol/L HCl at 45 °C for ∼24 h. The acid extract was subsequently adjusted to pH 6 with 1.0 mol/L KOH, followed by addition of 2 mL of 0.1 mol/L phosphate buffer (pH 6.0). Cocaine and BZE were isolated from the crude extracts using Bond Elut Certify SPE cartridges (Varian, Harbor City, CA) according to the manufacturer’s instructions for urine samples. GC/MS Analysis of Extracts. SFE and SPE extracts (containing deuterated internal standards) were transferred to conical test tubes and concentrated under a gentle stream of nitrogen to a volume of ∼300 µL. This volume was transferred to a 0.3 mL 166
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Reacti-Vial, evaporated to dryness, and reconstituted with ∼30 µL of the silylation reagent BSA, which reacts with the carboxylic acid group of BZE (Figure 2B) to form a volatile trimethylsilyl (TMS) derivative. The vials were tightly capped with Mininert valves and mixed on a vortex mixer prior to analysis. Quantification of cocaine and BZE in extracts was performed by gas chromatography/mass spectrometry (GC/MS) using internal standard calibration with a four-point linear calibration plot. Calibration standards were prepared (by mass) in methanol from gravimetrically prepared cocaine hydrochloride and BZE tetrahydrate stock solutions and spiked (by mass) with the internal standard solutions (cocaine-d3 hydrochloride and BZE-d3). The calibration standards were evaporated to dryness and reconstituted in BSA prior to analysis. Drug concentrations in the calibration standards were chosen to bracket the expected drug concentrations in the extracts. GC/MS was performed using a Hewlett Packard (HP) 5890 gas chromatograph interfaced to a HP 5971 mass-selective detector. Manual injections (1-2 µL) of derivatized extracts and calibration standards were made in the splitless injection mode onto a 30 m × 0.25 mm i.d. DB-5ms capillary column with a 0.25 µm film thickness (J & W Scientific, Folsom, CA). Helium was employed as the carrier gas at a head pressure of 103 kPa (15 psi). The oven temperature was programmed from 150 (1 min hold) to 250 °C at a rate of 25 °C/min and then increased to 290 °C at a rate of 10 °C/min (3 min final hold). The injector and transfer line were maintained at 250 and 280 °C, respectively. Selected ion monitoring was performed at m/z 182 and 185 for cocaine and cocaine-d3, respectively, and m/z 240 and 243 for BZE and BZE-d3 (as the TMS derivatives), respectively. RESULTS AND DISCUSSION Cocaine Spike-Recovery Experiments from Filter Paper. Initial spike-recovery experiments were performed from filter paper to investigate the SF solubility and system collection efficiency for cocaine while avoiding potential matrix interactions encountered in hair samples. A variety of fluid/modifier compositions were studied, and in all cases, SFE was performed at 40.5 MPa and 110 °C with a 10 min static period followed by a dynamic period sufficient to allow 15 mL of CO2 to pass through the extraction vessel (i.e., 12-14 min). Modifier (100 µL) was introduced in the manner described in the Experimental Section. The recovery results shown in Figure 3 illustrate that pure CO2 was inefficient for the recovery of free-base cocaine from the filter paper substrate (
