Forensic Science International, 53 (1992) 215-219 Elsevier Scientific Publishers Ireland Ltd.

215

DETERMINATION OF COCAINE AND ITS METABOLITES IN BRAIN TISSUE USING HIGH-FLOW SOLID-PHASE EXTRACTION COLUMNS AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

CHRISTINE MOORE, SUSAN BROWNE, IAN TEBBETT and ADAM NEGRUSZ

Department of Pharmaeodynamics, Forensic Toxicology Unit, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60680 (USA)

(Received December 2nd, 1991) (Accepted January 31st, 1992)

SuRmary

A new solid-phase extraction procedure for the determination of cocaine and some of its metabolites in brain tissue, using high-flow co-polymeric sorbents is reported as a substantial im- provement on our recently reported procedure. The recovery of cocaine, norcocaine and cocaethylene was excellent as was the reproducibility of the extraction. The use of high-flow sorbents allowed the easy extraction of tissue without the need for a time-consuming lipase digestion, regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extrac- tion speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and ex- ceptional extract quality. The procedure was successfully applied to rabbit brains spiked with cocaine, benzoylecgonine, norcocaine and cocaethylene.

Key words: High-flow solid-phase extraction; Cocaine; Brain tissue; HPLC

Introduction

The determination of drugs in postmortem tissue samples is of great interest to the forensic toxicologist. Urine, the most desirable sample due to the high con- centration of drugs present is not always available postmortem and blood is often degraded or hemolysed depending upon the time which has elapsed since death. Further, recent studies have shown that postmortem drug levels are frequently site-dependent, with heart blood giving highly elevated concentrations compared to femoral blood [l - 31. Correct interpretation of results is then called into ques- tion and tissue analyses are often required to corroborate or confirm interpreta- tion of data. It has been suggested that drugs which show stability problems (e.g., cocaine, which breaks down rapidly to benzoylecgonine in the blood) can be determined more easily in tissue, with brain being the most common choice due

Correspondence to: Christine Moore, 443 Elgin, Coach House, Forest Park, IL 60130, USA.

0379-0738/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

216

to the large number of receptors therein. Various reports of cocaine concentra- tions in human brain show that levels are much higher than in the blood regardless of the route of administration. Cocaine has been shown to be stable in frozen brain tissue up to three months post mortem [4,5]. Brain tissue has also been suggested as a sampling site for morphine determination following heroin abuse since there is a high concentration of opiate receptors localized in the lim- bit system, primarily in the amygdala, corpus striatum and hypothalamus [6].

Drug extractions from brain tissue have traditionally been carried out using liquid-liquid extraction of brain homogenates. Solid-phase procedures, however, offer such advantages as reduced operator time, decreased solvent volumes and hence decreased solvent disposal costs. Existing solid-phase methods have not yet been widely applied to tissue samples due to the inability of tissue homogenates to pass easily and reproducibly through a bonded sorbent com- pared with the ease of extraction of plasma, serum, saliva and urine. We have recently reviewed solid-phase tissue extraction [7] and have reported a suc- cessful extraction procedure for cocaine and its metabolites from brain which required a lipase digestion procedure prior to solid-phase extra&ion [8].

The current paper improves on the previously reported technique by employ- ing newly developed high-flow solid-phase cartridges which allow the direct passage of brain homogenates and provide a clean extract for final analysis by high-performance liquid chromatography (HPLC) with multiwavelength UV detection. This new solid-phase extraction procedure for cocaine, norcocaine, cocaethylene and benzoylecgonine was applied to rabbit brains spiked with the four named drugs and bupivacaine (as an internal standard). The procedure is in- dependant of the viscosity of the homogenate and sample size. It allows the easy passage of the homogenate through the sorbent and adequate efficiency of ex- traction.

Materials and Methods

Sample collection Brains were taken from female rabbits and stored at - 20°C. Prior to extrac-

tion, they were spiked with cocaine, benzoylecgonine, norcocaine and coca- ethylene at a concentration of 1 kg/g. Bupivacaine was used as an internal standard (0.1 mgll, 20 ~1).

Drug standards and chemicals Cocaine, benzoylecgonine and bupivacaine were obtained from Sigma

Chemical Co. (St. Louis, MO). Norcocaine and cocaethylene were obtained from NIDA (Research Technology Branch, Division of Research). Tris(hydrox- ymethyl)- aminomethane (Tris) was obtained from Bio Rad Laboratories. All chemicals us- ed were analytical grade or better and all solvents HPLC grade (Fisher, Itasca, IL).

Extraction procedure Solid-phase extraction was performed on X-TrackTTM high-flow columns

217

(Worldwide Monitoring, Horsham, PA), containing 500 mg of a co-polymer with dual activity (cation exchange and non-polar interactions, 15 ml capacity).

Tris buffer (0.2 M (pH 6.3) with orthophosphoric acid, 5 ml) was added to spiked rabbit brain (1 g) containing bupivacaine (0.1 mg/l; 20 ~1) as an internal standard. The mixture was homogenised using a high-speed homogeniser for a few seconds. The mixture was then applied to an X-TrackTTM column which had been previously conditioned with methanol (5 ml), water (5 ml) and phosphate buffer (3 ml (pH 6)). The sample was drawn through under vacuum and the column was air-dried. The sorbent was washed with water (3 ml), 100 mM hydrochloric acid (3 ml) and methanol (3 ml). The column was allowed to dry for 5 min under vacuum. Finally, the isolates were eluted with chloroform- isopropanol-ammonium hydroxide (78:20:2, v/v) (10 ml). The eluent was evaporated to dryness without heating. The residue was reconstituted in methanol (0.1 ml) prior to analysis.

Chromatographic equipment and conditions A Perkin Elmer Series 2 pump was used to deliver solvent at a rate of 1.5

ml/min onto a Cl8 ODS rnp Bondapak (30 cm x 3.9 mm i.d.) column (Waters, Milford, MA). A Cl8 guard column (Guard-Pak, Waters) and a Rheodyne injec- tion system with a fixed loop (20 ~1) were also incorporated into the system. A Spectra Physics Focus multiwavelength detector connected to an IBM Personal Data System 2 data system was used to monitor the eluent from the column at 230, 255 and 275 nm and full spectra were recorded over the wavelength range 190- 400 nm. The mobile phase consisted of 0.025 M potassium dihydrogen phosphate-acetonitrile-butylamine (500:125:12.5, v/v). The final pH was adjusted to 2.9 with orthophosphoric acid.

Results and Discussion

Optimisation of elution volume The extraction procedure was very rapid and the resulting extracts were suffi-

ciently clean for direct injection onto the HPLC system without further clean up stages. The use of 10 ml of elution solvent appeared to elute the majority of the drugs although the recovery of benzoylecgonine, though acceptable, was still markedly lower than the other metabolites. The recovery of the drugs from rab- bit brain homogenates spiked with 1 pglg were: 34.3 f 10.1% for benzoylec- gonine; 74.1 + 8.9% for cocaine; 66.1 f 9.1% for norcocaine; 70.8 f 9.6% for cocaethylene and 86.1 f 7.6% for bupivacaine. Similar recoveries were seen with concentrations up to 10 pg/g.

High performance liquid chromatography (HPLC) The HPLC system was linear for spiked brain tissue extracts over the concen-

tration range O.l- 10 pg/ml. The minimum quantitation level of cocaine by HPLC was 100 ng/ml and the minimum detection level 30 ng/ml. The retention times of the compounds were as follows: benzoylecgonine 6.2 min; cocaine 7.9 min; norcocaine 9.1 min; bupivacaine 10.1 min and cocaethylene 12.3 min.

218

Cocaethylene Cocaethylene was included in this study since it is has been found in the urine

of individuals known to abuse both cocaine and alcohol. The trans-esterification of cocaine and ethanol to produce the neuroactive cocaethylene takes place in the liver [9]. Further, cocaethylene has been shown to be equivalent to cocaine in potency and toxicity. The necessity of this assay to include cocaethylene as well as benzoylecgonine and norcocaine was clearly required.

Solid-phase extraction The use of the high-flow columns allows the homogenate to pass easily through

the sorbent. However, the high speed of passage through the phase results in a lower efficiency of extraction compared with solid-phase procedures which use the more common tightly packed sorbents. This is probably due to a reduced time of contact between the drug and the sorbent so that interactions have less time to occur. Because cocaine, norcocaine and cocaethylene are attracted to the co- polymeric sorbent through both non-polar and ionic interactions at pH 6, the importance of the speed of flow through the sorbent is reduced. However, for benzoylecgonine, which is only attracted to the non-polar sections of the sorbent at pH 6 (it becomes ionised after the wash with hydrochloric acid, prior to the methanol wash), the speed of passage through the sorbent takes on a greater im- portance since it only has a chance to interact with one set of functional groups. This hypothesis is supported by the lower extraction efficiency for benzoylec- gonine compared to the other drugs.

In our previous paper, we used C2 solid-phase columns to extract cocaine and benzoylecgonine. This procedure resulted in a higher extraction efficiency for benzoylecgonine (80 f 5%) and cocaine (92 f 6%). The reduction in cocaine ex- traction efficiency using high-flow columns can be explained as above, but the difference in benzoylecgonine extraction efficiency is attributed to other interac- tions. The Cz columns display both polar and non-polar functional groups. The metabolite, benzoylecgonine, is more polar than the parent drug, cocaine and so is held onto the sorbent by both polar and non-polar interactions. A greater percentage of benzoylecgonine is then held onto the Cz sorbent than the high- flow non-polar sorbent. However, the decrease in time required to extract the homogenised brain sample is the most important factor in this procedure. Our previous method required the brain to be incubated for 2.5 h with lipase. This was intended to cleave the long-chain lipids present in the brain which were responsible for physically blocking the solid-phase sorbents, preventing extrac- tion. The use of high speed homogenisation and high-flow solid-phase columns eliminates the need for such a digestion procedure and allows the isolates to be extracted with adequate efficiency and excellent reproducibility. This is a signifi- cant improvement on existing procedures and results in a marked increase in laboratory turnaround time.

Benzoylecgonine is found in limited quantities in the brain and the efficiency of extraction for cocaine is of much greater importance for forensic applications. In our previous paper [8], we reported mean cocaine concentrations from postmortem brain tissue of 32.9 pglg depending upon the region of the brain

219

under investigation. These values were approximately 4-8 times greater than the concentrations of cocaine in the blood. The concentration of benzoylecgonine was substantially lower than that of cocaine in the brain, supporting the previous reports of cocaine stability and suggesting that brain is a better forensic sample than postmortem blood when the presence of cocaine is suspected.

References

T.P. Rohrig and R.W. Prouty, A nortriptyline death with unusually high tissue concentrations. J. Anal. Toxicol., 13 (1989) 303-305. T.P. Rohrig and R.W. Prouty, Fluoxetine overdose: A case report. J. Anal. Toxicol., 13 (1989) 305 - 307. R.W. Prouty and W.H. Anderson, The forensic science implications of site and temporal in- fluences on postmortem drug concentrations. J. Forensic Sci., 35(2) (1990) 243 - 270. V. Spiehler and D. Reed, Brain concentrations of cosine and benzoylecgonine in fatal cases. J. Forensic Sci., 30 (4) (1985) 1003- 1011. L.R. Bednarczyk, E.A. Gressman and R.L. Wymer, Two cocaine -induced fatalities. J. Anal. Toxicol., 4 (1980) 263 -265. E.M. Pare, J.R. Monforte and R.J. Thibert, Morphine concentrations in brain tissue from heroin associated deaths. J. Anal. Toxicol., 8 (1984) 213 -216. J. Scheurer, CM. Moore and I.R. Tebbett, Solid-phase extraction of drugs from biological tissues - a review. J. Anal. Toxicol., in press. S.P. Browne, C.M. Moore, J. Scheurer, I.R. Tebbett and B.K. Logan, A rapid method for the determination of cocaine in brain tissue. J. Forensic Sci., 36(6) (1991) 1662- 1665. W. Lee Hearn, D.D. Flynn, G.W. Hime, S. Rose, J. Cofino and E. Mantero-Atlenza, Coca- ethylene: A unique cocaine metabolite displays high affinity for the dopamine transporter. J. Neurochem., 56 (1991) 698-701.