Anal. Chem. 2001, 73, 2371-2376
Articles
Analysis of Cocaine, Benzoylecgonine, Codeine, and Morphine in Hair by Supercritical Fluid Extraction with Carbon Dioxide Modified with Methanol William E. Brewer,†,§ Randolph C. Galipo,‡,⊥ Kristen W. Sellers,‡ and Stephen L. Morgan*,‡
Toxicology Department, South Carolina Law Enforcement Division, 4416 Broad River Road, Columbia, South Carolina 29210, and Department of Chemistry and Biochemistry, The University of South Carolina, Columbia, South Carolina 29208
Supercritical fluid extraction (SFE) using CO2 modified with methanol (10%) was employed for the extraction of cocaine from human hair. Extraction conditions were developed by using designed experiments to characterize the effects of pressure, temperature, and percent methanol on the recovery of cocaine from hair. When compared to the conventional acid hydrolysis method for hair analysis, SFE was faster and gave higher recoveries. Amounts of cocaine, benzoylecgonine (cocaine metabolite), codeine, and morphine in a hair standard reference material were determined by SFE-gas chromatography/ mass spectrometry (GC/MS) and found to be in agreement with reported levels. Analyses of hair from forensic case studies are also reported. Supercritical fluid extraction (SFE) has generated considerable interest in the last 10 years as an alternative to traditional extraction techniques. At or above its critical pressure and temperature, a supercritical fluid has the solvating power of a liquid and the diffusivity of a gas. Carbon dioxide (CO2) is commonly used for SFE because of its low critical pressure and temperature (Pc ) 74 atm, Tc ) 32 °C), low toxicity, and low cost. Adjusting the pressure and temperature can increase the solvent strength of supercritical CO2. Because carbon dioxide is nonpolar, the addition of polar modifiers such as methanol may also increase solvating power and improve recoveries of polar analytes not * Corresponding author. E-mail:
[email protected]. † South Carolina Law Enforcement Division. ‡ The University of South Carolina. § Current address: Clemson Veterinary Diagnostic Center, P.O. Box 102406, Columbia, SC 29224-2406. ⊥ Current address: Eastman Kodak Company, Analytical Technology Division, Rochester, NY 14650-2140. 10.1021/ac000871r CCC: $20.00 Published on Web 05/03/2001
© 2001 American Chemical Society
efficiently extracted by supercritical CO2 alone. Supercritical fluids have been used to extract environmental contaminants from soil and sediment,1-3 drugs from animal products4-6 and from human biological matrixes.7-10 Numerous forensic applications of hair analysis have been published in the past few years.10-21 Supercritical fluid extractions (1) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 909-916. (2) Robertson, A. M.; Lester, J. N. Environ. Sci. Technol. 1994, 28, 346-351. (3) Yang, Y.; Gharaibeh, A.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1995, 67, 641-646. (4) Parks, O. W.; Lightfield, A. R.; Maxwell, R. J. J. Chromatogr. Sci. 1995, 33, 654-657. (5) Parks, O. W.; Maxwell, R. J. J. Chromatogr. Sci. 1994, 32, 290-293. (6) Cross, R. F.; Ezzell, J. L.; Richter, B. E. J. Chromatogr. Sci. 1993, 31, 162169. (7) Edder, P.; Staub, C.; Veuthy, J. L.; Pierroz, I.; Haerdi, W. J. Chromatogr. B 1994, 658, 75-86. (8) Liu, H.; Cooper, L. M.; Raynie, D. E.; Pinkston, J. D.; Wehmeyer, K. R. Anal. Chem. 1992, 64, 802-806. (9) Liu, H.; Wehmeyer, K. R. J. Chromatogr. 1992, 577, 61-67. (10) Kintz, P. Drug Testing in Hair; CRC Press: Boca Raton, 1996. (11) Cirimele, V.; Kintz, P.; Majdalani, R.; Mangin, P. J. Chromatogr. B 1995, 673, 173-181. (12) Welch, M. J.; Sniegoski, L. T.; Allgood, C. C.; Habram, M. J. Anal. Toxicol. 1993, 17, 389-398. (13) Goldberger, B. A.; Caplan, Y. H.; Maguire, T.; Cone, E. J. J. Anal. Toxicol. 1991, 15, 226-231. (14) Harkey, M. R.; Henderson, G. L.; Zhou, C. J. Anal. Toxicol. 1991, 15, 260265. (15) Cone, E. J.; Yousefnegad, D.; Darwin, W. D.; Maguire, T. J. Anal. Toxicol. 1991, 15, 250-255. (16) Allgood, C. C.; Sniegoski, L. T.; Welch, M. J. “The Analysis of Human Hair for Drugs of Abuse,” In The 39th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, 1991. (17) Kidwell, D. A. “Analysis of Drugs of Abuse in Hair by Tandem Mass Spectrometry,” In The 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June 5-10, 1988. (18) Sniegoski, L. T.; Welch, M. J. J. Anal. Toxicol. 1996, 20, 242-246.
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of opiates, cocaine, and other illicit drugs from hair have been reported.10,11,21 Hair analyses provide a long-term record of previous drug use and chronic and sporadic uptake can be differentiated by analyzing hair segments. An advantage of hair analysis over urinalysis is that drugs and their metabolites are stable in hair for an extended time.16,17 To detect most drugs of abuse in urine, specimens must be collected within 2-3 days of usage. Hair samples may be useful even for post-mortem cases in which other bodily fluids are unavailable. Differentiation of drugs internally incorporated into hair from external contamination due to environmental exposure is desirable. One possible solution is to identify metabolites whose presence can only be explained by active drug use.15 For example, the presence of benzoylecgonine, the principal metabolite of cocaine, provides presumptive evidence of actual drug use. Sample preparation for hair analysis typically involves extraction with organic solvents, acid or base hydrolysis, or enzymatic digestion. The major disadvantage of these methods is the length of time needed to achieve efficient recoveries. Other issues include tedious sample preparation, poor reproducibility, difficulty in quantitating nonuniformly distributed drugs in hair, and lack of reference materials for all drugs of interest. Edder and co-workers used carbon dioxide to extract (40 °C, 250 atm, 30 min) opiates from 50-mg hair samples of drug addicts.7 The concentration of polar modifiers was varied to improve analyte recoveries. The mean morphine recovery was 93.5% (based on 6 replicates), and detection limits of 30 pg/mg were achieved for morphine, ethylmorphine, and codeine. Cirimele and co-workers used supercritical carbon dioxide modified with methanol/ triethylamine/water (2/2/1, v/v) to extract opiates from hair of drug addicts.11 Extraction recoveries were 61%, 53%, and 96%, for codeine, morphine, and 6-monoacetylmorphine, respectively. Morrison and colleagues extracted cocaine from hair using supercritical carbon dioxide modified with water/triethylamine (85/ 15, v/v).21 Hair samples were extracted statically for 10 min and dynamically for 15 min at 400 atm and 110 °C. Extraction efficiencies of roughly 90% were achieved by adding both water and triethylamine (TEA) to carbon dioxide. Morrison and coworkers21 also found that carbon dioxide that was modified with TEA provided better recovery of cocaine from hair than carbon dioxide with methanol as the modifier under identical conditions. However, benzoylecgonine recoveries using CO2 modified with TEA were about 11% of the values obtained by acid hydrolysis, a deficiency suggested to be a result of poor solubility under the extraction conditions or an inability to desorb benzoylecgonine from hair under these conditions. In this paper, we report a method for extracting cocaine, benzoylecgonine, morphine, and codeine from hair with supercritical carbon dioxide modified with methanol. An experimental design varying pressure and temperature was employed to improve recovery of cocaine from hair. The method was validated by analyzing a commercial hair standard containing cocaine, benzoylecgonine, morphine, and codeine. We have investigated (19) Wang, W. L.; Darwin, W. D.; Cone, E. J. J. Chromatogr. B 1994, 660, 279290. (20) Henderson, G. L.; Harkey, M. R.; Jones, R. Hair Analysis for Drugs of Abuse. Final Report on Grant Number NIJ 90-NIJ-CX-0012 to National Institutes of Justice, Washington, D. C., September 1993. (21) Morrison, J. F.; Chesler, S. N.; Yoo, W. J.; Selavka, C. M. Anal. Chem. 1998, 70, 163-172.
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the use of various derivatizing reagents for the analysis of benzoylecgonine in hair and report excellent results for pentafluoropropionic anhydride (PFPA). The SFE-GC/MS method developed here has been applied to investigate drug levels in hair collected from forensic cases involving cocaine abuse. EXPERIMENTAL SECTION Chemicals and Reagents. Methanol, acetone, and chloroform (reagent grade) were purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Dimethyl sulfoxide (DMSO) was obtained from Fisher Scientific (Fairlawn, NJ). Cocaine (free base form) and benzoylecgonine were provided by Sigma Chemical Company (St. Louis, MO). The internal standards, cocaine-d3, benzoylecgonined3, morphine-d3, and codeine-d3 (0.1 µg/µL acetonitrile solutions) were purchased from Radian International (Austin, TX). Supercritical fluid chromatography grade CO2 with 10% methanol (Scott Specialty Gases, Plumsteadville, PA) was used in all experiments. Hair Standards. The preparation of standards by soaking drug-free hair in solutions of DMSO containing cocaine has been described in the literature.12,18 Hair (0.5-1.5 cm in length) was collected from various parts of the head, rinsed repeatedly with distilled water and methanol to remove contaminants, and dried in air at ambient temperature. Hair samples (4 g) were then soaked in a solution containing 75 mL DMSO and sufficient cocaine (1.0 µg/µL) to yield 1-60 ng of cocaine per mg hair. The beaker was covered with aluminum foil and placed in a fume hood for 4 weeks. DMSO is the solvent of choice because of its ability to penetrate protein matrixes. After soaking, the hair samples were rinsed twice each with 10 mL of distilled water and 10 mL of methanol, and dried overnight in a fume hood. These fortified hair standards were used for optimization experiments. Prior to SFE, the hair samples were ground to a fine powder with a Silamat (Perkin-Elmer, Norwalk, CT) powdering device that consists of a stainless steel capsule, two endcaps, and a stainless steel ball bearing. The hair was inserted into the capsule and shaken vigorously for 2-5 min. To ensure homogeneous sampling, the hair was then placed in a grinding mill and mixed for 5 min. The powdered hair was removed from the grinding mill, transferred to screw-cap vials, and stored at 4 °C. Drug-free hair was also powderized to serve as a blank. Further experiments to validate the use of the SFE-GC/MS method employed a powdered standard reference material for cocaine, benzoylecgonine, morphine, and codeine in hair (reference material 8449) obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). Acid Hydrolysis. Acid hydrolysis was performed by adding 8 mL of 0.1 N HCl to 70 mg of nonpowdered hair in a test tube for 24 h at 45 °C. After 24 h, the mixture was neutralized and buffered with ammonium hydroxide (pH 5-6) before extraction using World Wide Monitoring Clean Screen solid-phase extraction (SPE) columns (United Chemical Technologies, Inc., Bristol, PA). SPE columns were conditioned by rinsing two times with 2.5 mL of hexane, with 3 mL of methanol, and then with 5 mL of phosphate buffer (0.1 N, pH 6). Deuterated cocaine (5 µL of 20 ng/µL standard) was added to the mixture before loading of the column. An additional 4 mL of phosphate buffer was added to the SPE column, and the mixture was poured into the buffer on the column. The mixture was slowly pulled through the column, which was then washed with 2 mL of phosphate buffer, 1 mL of
1.0 N acetic acid, and 2 mL of hexane. After air-drying for 5 min, cocaine was eluted using 2 mL of 2% ammonium hydroxide in methanol. Extracts were evaporated to dryness under nitrogen and reconstituted in 70 µL of 20% methanol in ethyl acetate for GC/MS analysis. Extraction by Sonication. After adding 2 mL of methanol to 70 mg of nonpowdered hair, the solution was sealed in a closed tube and sonicated for 2 h. The test tube was centrifuged and the supernatant methanol was filtered through a column of anhydrous sodium sulfate. Deuterated cocaine (5 µL of 20 ng/µL standard) was added to this extract, which was then dried under nitrogen and transferred to an autosampler vial. The extract was then evaporated to dryness and reconstituted in 70 µL of 20% methanol in ethyl acetate for GC/MS analysis. Supercritical Fluid Extraction. All of the extractions were conducted using a model SFE/50 (Suprex Corporation, Pittsburgh, PA) supercritical fluid extractor. A premixed cylinder of carbon dioxide modified with 10% methanol was employed as the supercritical fluid. Powdered hair samples (60-70 mg) were transferred to a 0.5-mL stainless steel extraction vessel. Both ends of the vessel were packed with 15 mg Celite to prevent contamination of frits. For cocaine optimization studies, 5 µL of a 0.1 µg/µL deuterated internal standard solution (cocaine-d3) was added to hair in the extraction cell. For the NIST reference hair sample analysis, 5 µL of a 0.1 µg/µL deuterated internal standard solution (cocaine-d3, benzoylecgonine-d3, morphine-d3, and codeine-d3) was added. Hair samples were extracted both statically (5-25 min) and dynamically (10-100 min). Supercritical carbon dioxide was depressurized through a stainless steel restrictor into a test tube containing 2 mL of methanol. The test tube was heated to 50 °C in a heating mantle to prevent clogging of the restrictor during depressurization. Extracts for cocaine analysis were transferred to GC autosampler vials, evaporated to dryness under nitrogen, and reconstituted to 70 µL with 20% methanol in ethyl acetate. Vials were capped and vortexed prior to analysis by GC/MS. Extraction vessels and frits were rinsed with distilled water, then sonicated in methanol and acetone after every extraction. Derivatization. Two derivatization procedures were investigated. TMS (trimethylsilyl) derivatives were made by adding 50 µL of ethyl acetate and 50 µL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) to the hair extracts. The mixtures were heated at 70 °C for 20 min, and 2 µL of the solution was injected for GC/ MS analysis. The second derivatization procedure, using pentafluoropropionic anhydride (PFPA), was performed by adding 100 µL of hexafluoropropanol and 175 µL of PFPA and heating for 25 min at 80 °C. All PFPA derivatization mixtures were dried under nitrogen and reconstituted in 70 µL of ethyl acetate. GC/MS Analysis. A model 5890 Series II Plus gas chromatograph coupled to a 5972 series MSD (Hewlett-Packard, Palo Alto, CA) was used for the analysis of all of the extracts. A Restek (Bellefonte, PA) phenylmethyl deactivated guard column (5 m × 0.25 mm) was connected to the head of the analytical column to prevent contamination. Separation of analytes was performed using a 100% poly(dimethylsiloxane) (30 m × 0.245 mm) DB-1 (J & W Scientific, Folsom, CA) fused silica capillary column with a film thickness of 0.25 µm. Injection port and transfer line temperatures were set at 225 °C and 280 °C, respectively. The initial oven temperature was set at 80 °C for 1 min, increased to 120 °C at
50 °C/min, and then ramped to 300 °C at 20 °C/min. A final oven temperature of 300 °C was held for 11 min for a total run time of 22 min. All injections (2 µL) were made under splitless inlet conditions. Helium at a constant linear velocity of 35 cm/sec with electronic pressure control was used as the carrier gas. For the optimization for cocaine analysis, data were acquired in selected ion monitoring (SIM) for ions with mass-to-charge ratios (m/z) of 82, 182, and 303 for cocaine, and 85, 185, and 306 for cocaine-d3. For the analysis of the NIST hair standard, cocaine and codeine were first analyzed underivatized by monitoring ions at m/z 182 and 303 for cocaine, m/z 185 and 306 for cocaine-d3, m/z 162 and 299 for codeine, and m/z 165 and 302 for codeine-d3. After derivatizing with PFPA, benzoylecgonine and morphine were analyzed by monitoring ions m/z 318 and 439 for benzoylecgonine, m/z 321 and 442 for benzoylecgonine-d3, m/z 414 and 577 for morphine, and m/z 417 and 580 for morphine-d3. Quantitation of all of the analytes was performed by external calibration. Experimental Design. A three-factor central composite experimental design22 was used to investigate the effects of extraction pressure, temperature, and static extraction time on the recovery of cocaine from fortified hair. This design consisted of a total of 18 experiments: 15 design points at 5 levels of each experimental factor plus 3 replicates of the center point. The 10-parameter model fitted to the data included up to second-order effects in each factor and all two-factor interactions between the three factors. Forensic Case Samples. Hair samples were provided by a forensic pathology laboratory (Newberry Memorial Hospital, Newberry, SC). Approximately 50-70 mg of powdered hair was transferred to the extraction vessel, and the internal standards (cocaine-d3 and benzoylecgonine-d3) were added. SFE was performed under the conditions developed above for cocaine analysis. Following a methanol extraction, the samples were derivatized using PFPA prior to GC/MS. RESULTS AND DISCUSSION Modifier Experiments. Supercritical carbon dioxide without an added polar modifier was determined to be unsuitable for the extraction of cocaine from fortified hair. For example, using supercritical CO2 at 380 atm and 50 °C, recoveries of cocaine from hair were only 35%. Although supercritical CO2 did remove the drug from hair, the rate of removal was slow, and complete removal of cocaine from hair was not achieved. Morrison and coworkers also found pure CO2 to be ineffective for extracting cocaine from soaked hair, even though they did observe the extraction of drug weakly adsorbed to the surface of hair.21 The modifier type and the amount added play an important role in SFE with CO2. We tested both chloroform and methanol as modifiers for supercritical carbon dioxide to extract cocaine from hair. Recoveries of cocaine from fortified hair extracted with supercritical CO2 modified by adding 50 µL of chloroform to the extraction vessel prior to extraction were roughly 36% at 380 atm and 50 °C. Because these recoveries were similar to those reported for the extraction of hair with supercritical carbon dioxide in the absence of a polar modifier, further work focused on the use of methanol. When methanol (50, 75, 100 µL) was added to hair in (22) Deming, S. N.; Morgan, S. L. Experimental Design: A Chemometric Approach, 2nd ed.; Elsevier: Amsterdam, 1993.
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Figure 1. Response surface fitted to experimental results (based on a full second-order model). Pressure and temperature units are in atm and °C, respectively.
the reaction vessel for the static extraction, the presence of the modifier helped remove cocaine from hair more easily than with unmodified supercritical CO2. The greatest recovery of drugs (2 h) were needed for the complete removal of cocaine from the hair. Continuous modifier flow through the extraction vessel was needed to achieve optimum recoveries of cocaine from hair. One way to provide continual modifier flow during extraction is to use a separate pump for the modifier. Supercritical carbon dioxide and modifier can then be premixed in a mixing chamber prior to flow through the extraction vessel. Another option is to use a premixed cylinder of carbon dioxide/modifier. The instrumental setup described in this paper used premixed carbon dioxide with 10% (v/v) methanol for both static and dynamic extraction conditions Optimization Results. The effects of pressure (169-381 atm) and temperature (51-169 °C) were studied for the extraction of powdered cocaine-fortified hair. Because dynamic extraction time was found to be more important than static extraction time, hair was extracted statically for 5 min in all later experiments. The extraction vessel was flushed with fresh mobile phase (dynamic extraction) until the cocaine was completely removed. Extracts were collected in successive fractions, and recovery was determined by analyzing each fraction by GC/MS. Recovery was considered complete when cocaine was absent in the chromatogram of an extract. Recovery of cocaine from hair increased with increasing temperature (as shown in Figure 1). Optimum recovery of the drug was achieved at a pressure of 300 atm and a dynamic extraction time of 70 min. In Figure 1, the response surface fitted to the experimental results is shown. This plot resembles a parabola, with optimum response at a temperature of 145 °C. Temperature has the greatest effect on the recovery of cocaine from hair. Although the trend in Figure 1 suggests that even higher recoveries might be obtainable at higher temperatures, 145 °C was selected for all further work to avoid thermal degradation of cocaine or metabolites. The optimum extraction conditions were 75 min extraction time (5 min static and 70 min dynamic) at 300 atm and 145 °C. Comparison of Extraction Methods. Recoveries of cocaine from fortified hair standards extracted by acid (HCl) hydrolysis 2374 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001
Figure 2. GC/MS chromatograms of hair that was extracted by SFE using (A) BSTFA and (B) PFPA as derivatizing reagents.
and sonication with methanol were less than the recoveries obtained by extraction with supercritical carbon dioxide modified with methanol (10%). In our laboratory under the conditions employed, about twice the amount of cocaine was detected by GC/MS following SFE (800 pg/mg) than by GC/MS following acid digestion with HCl (350 pg/mg). The SFE method was more efficient than the acid hydrolysis, although the extraction time was much shorter (75 min compared to 24 h). Cocaine was not detected by GC/MS following methanol sonication procedures of the fortified hair. This suggests that cocaine in the fortified hair was not loosely adsorbed to the surface of the hair (following the washing procedures of the hair). Analysis of NIST Hair Standard. The commercial hair standard was analyzed to validate this SFE method. In addition to cocaine, the hair standard contained benzoylecgonine, morphine, and codeine. All four of the analytes were quantitated with this SFE method optimized for cocaine extraction. The GC/MS analysis of benzoylecgonine and morphine requires derivatization. We prepared TMS derivatives of hair extracts, but too much interference was observed with this method. The resulting intense interfering peaks (Figure 2A) were presumed to be due to derivatization of components such as fatty acids. Derivatization with PFPA, however, produced negligible interference (Figure 2B). The results of the SFE method applied to the NIST hair standard are shown in Table 1 and Figure 3. Quantitative results for cocaine, benzoylecgonine, codeine, and morphine that were obtained by SFE are similar to the NIST values. Standard deviations for all four of the analytes are also comparable to those reported for the NIST reference material. Quantitative results from
Table 1. Comparison of SFE-GC/MS Results with Reported Levels for the NIST Hair Standard
b c
analyte
NIST results ng/mg
SFE-GC/MS results ng/mgc
cocaine benzoylecgonine codeine morphine
5.40 ( 0.44a 5.40 ( 0.41a 2.85 ( 0.20b 3.71 ( 0.27b
4.74 ( 0.36 4.32 ( 0.65 2.97 ( 0.32 4.08 ( 0.49
Table 2. Summary of Results from Forensic Case Studies blood (mg/L)
brain (mg/kg)
hair (ng/mg)
benzoylbenzoylbenzoylcocaine ecgonine cocaine ecgonine cocaine ecgonine case 1 case 2 case 3
0 0.40 0.14
0.72 1.90 1.10
0 0.39
0.64 0.61
10.5 0.7 13.7
10.7 0.2 6.0
a Mean ( standard deviation based on 16 replicate measurements. Mean ( standard deviation based on 12 replicate measurements. Mean ( standard deviation based on 3 replicate measurements.
Figure 4. GC/MS ion chromatograms of a forensic case sample extracted by the SFE method. The displayed ion chromatograms of 318 and 321 correspond to PFPA-derivatized benzoylecgonine and benzoylecgonine-d3, respectively, and the ion chromatograms of 182 and 185 correspond to cocaine and cocaine-d3, respectively.
Figure 3. GC/MS chromatograms of the SFE-extracted NIST hair (A, B) standard underivatized and (C, D) derivatized with PFPA. Ion chromatograms of cocaine (182) and cocaine-d3 (185) are shown in A; codeine (162) and codeine-d3 (165), in B; PFPA-derivatized benzoylecgonine (318) and benzoylecgonine-d3 (321), in C; and PFPA-derivatized morphine (414) and morphine-d3 (417), in D.
our SFE-GC/MS method are consistent with the NIST values, considering the number of replicate measurements performed and the experimental uncertainties reported. Case Studies. Results from the hair analysis of three forensic case studies are shown in Table 2. Toxicological results for blood and brain analysis from the SLED Toxicology Laboratory are also shown in the table. The GC/MS chromatogram recorded from the SFE extraction of hair from case 1 is shown in Figure 4. Case 1 involved a death associated with an overdose of meperidine and alcohol. Cocaine was not determined to play a role in this death because only the pharmacologically inactive metabolite (benzoylecgonine) was found in blood and brain tissue and because a lethal level of meperidine was found in the blood. The detection of cocaine in the hair demonstrates the utility of hair analysis in providing a long history of previous drug use. These results suggest that the victim was a chronic user of
cocaine, but was not impaired on cocaine at the time of death. The presence of benzoylecgonine in blood and brain only infers that the victim most probably used cocaine within 48 h prior to death. The quantitation of meperidine in hair was unfortunately not attempted due to a limited amount of sample. It was determined that case 2 involved an acute overdose of cocaine. Interestingly, the hair analysis indicates the presence of only small amounts of cocaine and benzoylecgonine. This result suggests that the victim was not a chronic user of cocaine and, in fact, may have been a naı¨ve user. Case 3 involved a suicide by hanging. The hair analysis suggests that the victim was a chronic user of cocaine. On the basis of the blood and brain analyses, the victim’s judgment was undoubtedly impaired by cocaine at the time of the suicide. Concentrations of cocaine ranging from 0.12 to 5.7 ng/mg, 8.2-178 ng/mg, and 0.4-173 ng/mg have been previously reported in hair of drug users by Harkey and co-workers,14 Cone et al.,15 and Wang et al.,19 respectively. Our results are consistent with the reported literature and demonstrate the utility of hair analysis in providing a long history of drug use. The hair analysis complements blood and tissue analysis and provides useful information for forensic investigations. CONCLUSIONS Physical characteristics (color, thickness, etc.), different ethnic types, and condition of hair are also important factors that must Analytical Chemistry, Vol. 73, No. 11, June 1, 2001
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be considered when fortifying hair with drugs to prepare standards.10 Henderson and co-workers observed incorporation of cocaine in Caucasian hair to be less than incorporation of cocaine in Asian and African hair.20 In our preliminary studies, differences in uptake of the drug into hair was also observed. Future studies should investigate standards prepared from hair with different physical characteristics. Because hair consists mostly of protein and lipids, many fattyacid-type compounds will be extracted by the SFE method. High baseline and interfering peaks may appear in chromatograms of hair extracts. With a preliminary extraction under mild conditions (lower temperature and pressure), fatty acids and other biological compounds characteristic of hair can be removed prior to extraction of the drug. In addition, external contamination by drugs can also be removed by using mild SFE conditions. The internal standard solutions can then be added to hair before the final extraction under SFE conditions that are required to remove illicit drugs. Preliminary work in our lab shows that lower chromatographic backgrounds could be observed after first extracting under mild conditions. Cocaine users may escape drug testing procedures as a result of the metabolism and excretion of cocaine within 72 h of drug use. Hair analyses permit an increased detection window between the time of last drug administration and detection. The method reported here uses supercritical carbon dioxide modified with methanol (10%) to extract cocaine from hair. Recovery of cocaine from fortified hair by SFE was determined by GC/MS to be two times the amount detected by GC/MS following acid hydrolysis of hair from the same batch. In addition, extraction times were much shorter for SFE (70-80 min) than for existing classical techniques (24 h). The addition of the polar modifier methanol (10%) to carbon dioxide was crucial in achieving optimum
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extraction efficiencies, and extraction temperature was determined to be more important than pressure for recovery of the drug from hair. The optimum pressure and temperature were determined to be 300 atm and 145 °C, respectively. The optimized SFE-GC/MS method for cocaine analysis in hair was also applied to the analysis of benzoylecgonine, morphine, and codeine. This method was validated by comparing quantitative results of all four of the analytes with the reported levels from a commercial hair standard and by the successful application of this method to the analysis of hair from forensic case samples. The usefulness of hair analysis in providing a long history of drug use is demonstrated by the case studies reported. ACKNOWLEDGMENT The authors acknowledge the assistance of Dr. Grayson D. Amick of the Toxicology Department (South Carolina Law Enforcement Division, SLED, Columbia, SC) and the support of Kenneth H. Habben (retired Chief Toxicologist, SLED). We also gratefully acknowledge Keene Garvin, M.D. (Forensic Pathologist, Newberry Medical Hospital, Newberry, SC) for collection of the hair samples from forensic cases. Portions of this work were presented at PittCon 97 (Atlanta, GA, March 1997) as paper 382, and at PittCon 99 (Orlando, FL, March 1999) as paper 668. Support for this work was provided in part by Award 97-LB-VX-0006 from the Office of Justice Programs, National Institute of Justice, Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position of the U.S. Department of Justice. Received for review July 27, 2000. Accepted March 8, 2001. AC000871R
