ELSEVIER SCIEhCE IRELAND

Forensic Science International 65 (1994) 149-156

Forensic Science

International

Analysis of cocaine and metabolites in brain using solid phase extraction and full-scanning

gas c:hromatography/ion trap mass spectrometry

Antonio HernandezaTb, Wilmo Andollo”, William Lee Hearn*a ‘Dade County Medical Examiner Department, Number One On Bob Hope Road, Miami,

Florida 33136-1133, USA bDepartment of Legal Medicine, Avda. Madrid, II, 18071 Granada, Spain

(Re&ived 1 February 1993; revision received 13 September 1993; accepted 8 October 1993)

Abstract

A methold is described for the determination of cocaine, benzoylecgonine and cocaethylene in the hum,an brain using Clean ScreenTM solid phase extraction cartridges and gas chroma- tography/ion trap mass spectrometry with electron impact and full scan analysis. The proce- dure uses dleuterated internal standards. Run-to-run and within-run coeffkients of variation are < 7% and the sensitivity proved to be 50 rig/g from 1 g of sample. The procedure has been applied to a number of forensic cases involving cocaine intoxication. Cocaine was relatively unstable in brain tissue stored at 4°C when compared to storage at -8OYZ.

Key words: Toxicology; Cocaine; Benzoylecgonine; Cocaethylene; Solid phase extraction; Brain

1. Introduction

A previous study [l] suggested that blood cocaine concentrations change significantly during the interval between death and autopsy. Therefore, it is not pos- sible to draw conclusions about the concentration of cocaine existing at the time of death frolm postmortem data.

* Corresponding author.

0379-0738/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0379-0738(93)01466-5

150 A. Hernandez el al. /Forensic Sri. IN. 65 (1994) 149-156

Another approach is to determine brain cocaine concentrations. Spiehler and Reed [2] stated that brain cocaine concentrations measured in autopsy specimens should be representative of the perimortem drug concentration in that organ, since cocaine is more stable in that lipid-rich environment. Therefore, the brain could be a better matrix than blood for cocaine analysis.

A number of methods for identifying and quantifying cocaine and metabolites in the brain have been described, including high performance liquid chromatography (HPLC) [3] and gas chromatography/mass spectrometry (GUMS) [4,5]. All of these procedures utilize liquid-liquid extraction of the drug prior to analysis. Matsubara et al. [6] reported an extraction method using ExtrelutR solid phase extraction col- umns with analysis by chemical ionization GUMS and selected ion monitoring, but it was applied to plasma and urine samples.

Brain tissue extraction with bonded sorbents has not been extensively reported in the literature. Brain homogenates do not pass easily through tightly packed bonded cartridges. That problem can be addressed by digestion with lipase prior to extrac- tion or by using high-flow cartridges [7].

Two methods have been reported using solid phase extraction [8,9] for the analy- ses of cocaine and metabolites in brain tissue by HPLC. However, GUMS is the pre- ferred technique for drug confirmation in forensic analyses because of the potential for legal challenge. GUMS can provide highly accurate quantitative data for drugs extracted from complex matrices such as brain tissue, but its sensitivity is dependent upon obtaining an extract with minimal matrix-derived impurities.

This paper describes a sensitive method for the isolation and identification of cocaine, benzoylecgonine and cocaethylene in brain tissue using Clean ScreenTM solid-phase extraction and ion trap GUMS analysis. The method was tested by analyzing forensic samples from cases that were benzoylecgonine-positive by im- munoassay screening techniques. In addition the stability of cocaine in brain tissue under refrigerated storage conditions was evaluated.

2. Material and methods

2.1. Instrumentation Extracts were analyzed in a Finnigan Model ITS-40 ion trap GUMS equipped

with a 15 m x 0.25 mm i.d. 5% phenylmethylsilicone (DB 5 MS) capillary column with a 0.25 pm film thickness. The instrument was used in the full scan electron ion- ization mode, scanning m/z in the range 75-450.’ The oven temperature was pro- grammed in the range 115-290°C at lS”C/min, with an initial hold of 1.30 min and a final hold of 2 min. The manifold and transfer line temperatures were 260 and 280°C respectively.

The SPI injector was temperature-programmed in the range 105-29O’C at 180”C/min, with an initial hold time of 0.25 min and a final hold of 11.7 min. The injector was operated in a pseudo on-column injection mode.

2.2. Reagents Methanol, dichloromethane and dimethylformamide (DMF) were HPLC grade

(Burdick-Jackson). Isopropanol, hydrochloric acid, ammonia and sodium phos-

A. Hernandez et al. /Forensic Sei. Ini. 65 (1994) 149-156 151

phates were analytical grade (Mallinckrodt). Toluene was HPLC grade (Fisher Scientific). Triacylglycerol lipase (from porcine pancreas) type VI-S No. L-0382, from Sigma Chemical, was employed in enzymatic digestion.

Clean Screen@ DAU extraction columns, containing 200 mg of a mixture of non- polar and cation exchange packing, were supplied by Worldwide Monitoring Corp, and a Vat ElutTM vacuum manifold was provided by Analytichem International, Inc. Screw cap tubes with round bottoms (16 x 100 mm) were silanized by vapor phase before use.

Elution solvent was prepared by combining 80 ml of methylene chloride with 20 ml of isopropanol, removing 2 ml of this solution, adding 2 ml of fresh ammonium hydroxide, and mixing well.

Cocain’e, benzoylecgonine and cocaethylene reference materials were used to prepare stock standard solutions of 1.0 mg/ml in anhydrous DMF. For each batch of samples, the stock solutions were diluted to yield working solutions containing cocaine, benzoylecgonine and cocaethylene at 0.5, 1, 2, 5, 10, 50 and 100 &ml that were added to brain in the proportion of 100 pl/gm to yield calibrators. The cocaine and benzoylecgonine were obtained from Alltech-Applied Science, and the cocaethylene was synthesized in-house. Deuterated (D3) cocaine, benzoylecgonine and cocaethylene internal standards were obtained from Radian Corp. (100 &ml) in DMF. These solutions were used to prepare a working internal standard contain- ing 2 &ml of each substance.

2.3. Sample collection and storage Postmortem brain tissue, removed at autopsy, was sectioned and stored at -80°C

until anal.ysis. Storage intervals were in the range lo-342 days. Additional paired specimens from the same cases were stored at 4°C.

Blood samples were preserved with sodium fluoride, and stored at 4°C. They were analyzed within 3-9 weeks from the time of collection.

2.4. Sample preparation Brain tissue was added to distilled water (1: 1, w/v) and the mixture was homogen-

ized using a Tissue-TearerTM homogenizer. For preparation of standard curves and for method evaluation, drug-free brain was homogenized and then mixed with benzoylecgonine, cocaine and cocaethylene at concentrations in the range 50-10 000 rig/g.. Brain homogenates were mixed with 100 ~1 of internal standard solution. When the effect of lipase was assayed, 10 000 U of triacylglycerol lipase were added to 2 g of brain homogenate with the internal standard, and the samples were incubated for 2.5 h at 50°C in a heating block. Five milliliters of 100 mM sodium phosphate buffer, pl-l 6.0, was added to tissue homogenate or enzyme digest. The sample was mixed with a vortex shaker and centrifuged at 3500 rev./min for 10 min at room tem- perature. The supernatant was extracted as follows in the next section.

2.5. Extraction procedure Clean Screen columns were positioned in a Vat-Elut system. Vacuum pressure was

adjusted to 3 in Hg (75 mmHg). To prepare the solid phase extraction column, 3 ml of methanol, 3 ml of distilled water and 1 ml of 100 mM sodium phosphate buffer,

152 A. Hernandez et al. /Forensic Sci. ht. 65 (1994) 149-156

pH 6.0, were passed through the column sequentially without allowing the sorbent to dry. Then, the sample supernatant was applied to the column and drawn through under vacuum. The column was washed with 1 ml of 100 mM sodium phosphate buf- fer, pH 6.0, 2 ml of distilled water, 2 ml of 100 mM hydrochloric acid and 3 ml of methanol, and then allowed to dry for 5 min at full vacuum. Finally, 3 ml of elution solvent was added to the column and allowed to pass through by gravity. The eluate was collected in silanized tubes and evaporated to dryness under a gentle stream of nitrogen at 50°C in a heating block.

2.6. Derivatization The residue from evaporation of the extract was dissolved in 200 ~1 of a mixture

(1: 1, v/v) of 1 , 1,1,3,3,3-hexafluoro-2-propanol (Sigma H-8508) and pentafluoropro- pionic anhydride (Pierce, No. 65193), and the tube was capped tightly. The mixture was then heated to 75°C for 30 min in a heating block, cooled to room temperature and evaporated to dryness under a gentle stream of nitrogen at 50°C. The residue was reconstituted with 100 ~1 of toluene, and a 1 ~1 aliquot was injected into the GCIMS.

2.7. Calculations Peak areas at masses 182 AMU (cocaine), 185 AMU (deuterated cocaine), 3 18

AMU (hexafluoro-isopropyl benzoylecgonine), 321 AMU (hexafluoro-isopropyl deuterated benzoylecgonine), 196 AMU (cocaethylene) and 199 AMU (deuterated cocaethylene) were measured. The ratio of peak areas of drugs to deuterated internal standards was used for quantitation. Standard curves were determined for the area ratio versus the concentration of each analyte. Then, area ratios for the unknown were used to calculate the corresponding analyte concentrations.

The calculation of recovery was based on the mean of three different assays by each of the two following methods: (a) by comparing the results of analysis of blank brain mixed with 500 rig/g of each analyte prior to extraction and internal standard prior to derivatization to those from the analysis of extracts of blank brain mixed with analyte and internal standard just before derivatization; (b) calibration stan- dards (50-10 000 t&g) without internal standard were extracted on the solid phase column. After eluting, the internal standard was added to the eluate. Recovery rate was calculated as the difference between the slope of this calibration curve and the normal calibration curve. The reported value is the mean of recoveries calculated by methods (a) and (b).

Table 1 Method evaluation

Compound Correlation Linearity Within-run Between-run Recovery coefficient range (n&z) C.V. (‘%I) C.V. (‘%I) (1%))

Cocaine 0.9984 50- 10 000 5.2 3.1 60

Benzoylecgonine 0.9983 50-10000 5.7 6.9 54

Cocaethylene 0.9976 50-10000 5.2 5.3 54

Tabl

e 2

Post

mor

tem

co

ncen

tratio

ns

of c

ocai

ne

and

met

abol

ites

in f

oren

sic

case

s

Cas

e C

ocai

ne

Blo

od

(mg/

l)

Bra

in

(mg/

kg)

Coc

aeth

ylen

e

Blo

od

Bra

in

(mg/

l) (m

g/kg

)

Ben

zoyl

ecgo

nine

Blo

od

Bra

in

(mg/

l) (m

g/kg

)

Oth

er

drug

s (“

Yo)

C

ause

of

dea

th

I 9.

50

15.2

5 0.

86

1.40

9.

00

1.72

Et

hano

l (0

.05)

C

ocai

ne

OD

” 2

0.27

1.

89

ND

b N

D

1.40

0.

37

Phen

ylpr

opan

olam

ine,

gu

aife

nesi

n Li

doca

ine,

ep

hedr

ine,

ca

nnab

inoi

ds

Etha

nol

(0.1

7)

Etha

nol

(0.1

I), e

phed

rine

Exci

ted

delir

ium

+

fall

3 N

D

0.11

N

D

ND

0.

08

ND

Ex

cite

d de

liriu

m

4 10

.00

18.1

8 3.

50

1.15

7.

00

1.04

5

0.17

0.

36

<O.lO

0.

15

0.60

0.

29

Coc

aine

O

D

Gun

shot

w

ound

(h

omic

ide)

C

ocai

ne

+ m

yoca

rdia

l hy

pertr

ophy

Ex

cite

d de

liriu

m

Arte

riosc

lero

tic

hear

t di

seas

e Ex

cite

d de

liriu

m

Coc

aine

O

D

- bo

dy

pack

er

6 0.

21

0.99

N

D

ND

3.

40

0.92

N

D

7 0.

36

1.58

N

D

8 0.

19

0.24

N

D

ND

N

D

5.80

1.

91

ND

4.

10

1.53

Et

hano

l (0

.01)

lid

ocai

ne

Lido

cain

e D

iaze

pam

, no

rdia

zepa

m,

lora

zepa

m

9 0.

26

1.48

IO

4.

60

17.5

5 N

D

ND

N

D

2.20

1.

32

ND

12

.00

8.24

“OD

, ov

erdo

se.

bND

, no

t de

tect

ed.

E

154 A. Hernandez et al. /Forensic Sri. Int. 65 (1994) 149-156

3. Results

3.1. Efficiency of lipase digestion One blank brain and three more brains with different concentrations of analyte

were assayed to check the effect of the enzymatic digestion of homogenates with lipase. No differences were observed among the different homogenates as passed through the columns, and no column became blocked. Furthermore, the same quan- titative result was obtained either using lipase or without lipase (results not shown). Therefore, the remainder of the experiments were done without using lipase.

3.2. Evaluation of the method Evaluation of the method was accomplished using brain homogenate control sam-

ples with different concentrations of cocaine, benzoylecgonine and cocaethylene (Table 1). Linear responses were obtained for all analytes with correlation coefi- cients of 10.997. Coefficients of variation of < 7% were obtained for both within- day and day-to-day assays.

The limit of detection was found to be 25 t&g, corresponding to signal-to-noise ratios > 5: 1 for the base peak ions. The calculated recovery rates ranged from 54% for benzoylecgonine and cocaethylene to 60% for cocaine.

3.3. Results of analysis of forensic samples The procedure was evaluated by the extraction of postmortem brain tissue, and

results of these analyses are represented in Table 2. Forensic samples that were found to be urine-positive for benzoylecgonine by immunoassay (EMIT@ SYVA) were referred for quantitation by the above procedure. As reported by Spiehler and Reed [2] the cocaine concentrations were higher, and the benzoylecgonine concentrations lower, than in the corresponding blood samples in all cases. However, the observed brain:blood ratios for cocaine and benzoylecgonine differed from the trend noted by those investigators. Overdoses tended to have the lowest cocaine ratios, but the excited delirium cases all had ratios >4. No trend was discernible for the brain:blood ratios of benzoylecgonine.

Table 3 Cocaine stability in brain tissue: comparison of refrigerated (4°C) and frozen (-80°C) storage conditions

Case Storage interval’ (days)

Cocaine Cocaethylene Benzoylecgonine (mg/kg) (mgikg) (mg/kg)

4°C -80°C A (%I) 4°C -80°C 4°C -80°C

7 30 0.809 1.580 -48 NDb ND 3.239 1.910 6 67 0.059 0.987 -94 ND ND I.196 0.921 4 127 0.376 18.182 -98 0.303 I.147 17.305 I .036 2 I44 0.068 1.893 -96 0.027 0.019 1.313 0.368

‘Interval from autopsy to analysis. bND. not detected.

A. Hernandez et al. /Forensic Sci. ht. 65 (1994) 149-156 155

3.4. Stabihty of cocaine in stored brain tissue Four brain tissue samples were each split into two portions, one of which was

stored at 41°C and the other at -80°C. After intervals varying in the range 30-144 days the pairs of samples were analyzed. In the refrigerated sample, the cocaine con- centration had decreased by 48% relative to the frozen sample within 1 month, and over 90% -was lost during 2 months of refrigerated storage. However, traces of co- caine were detectable even after 144 days of storage in the refrigerator (Table 3).

4. Discussiion

The usefulness of the ion trap GUMS to confirm the presence of cocaine and cocaethylene in forensic samples at very low concentrations, using a liquid-liquid extraction, was demonstrated in a previous study [5].

Recently, Browne et al. [8] reported a procedure using solid-phase extraction (silica-bonded C2 columns) and further analysis by HPLC with ultraviolet detection. However, given the characteristic of the sorbent, a lipid digestion step was necessary to break down fatty material in order to avoid obstruction of the column. One year later, the same group [9] improved the former procedure by using high flow sorbents (X-TrackTM, World Wide Monitoring) to obtain rapid passage of the sample through the column. The lipase digestion step was then avoided.

Since confirmation by GUMS is preferred for forensic drug testing, the authors developed a method for the rapid extraction of homogenized brain tissue using Clean Screen (World Wide Monitoring) columns and further analysis by ion trap GUMS. lYhe feasibility of using an ion trap MS for full scan analysis with sen- sitivities below NIDA cut-off limits has been recently documented [lo].

The extraction procedure was very rapid and the resulting extracts were suffici- ently clean for injection onto the GUMS system. The use of 3 ml of elution solvent appeared to elute the majority of the drugs. A second elution using the same amount of elution solvent did not yield higher recovery. To exclude the possible loss of drugs during the methanol washing, the wash fraction from the highest concentration point of the standard curve was analyzed, and only 1% of the initial quantity of co- caine was found. Taking this into account, the recovery, though acceptable, is not as good as desired. Browne et al. [8] reported a recovery for benzoylecgonine and cocaine of 80 and 92%, respectively, using lipase digestion and bond-elut silica- bonded C2 columns. Later, the same group [9] reported a recovery of 34.3, 74 and 78.1% for benzoylecgonine, cocaine and cocaethylene, respectively, using X-trackTTM high flow columns without lipase digestion. In both cases, determina- tions were made by HPLC.

The standard curves were linear for brain tissue extracts over the concentration range 50-10 000 rig/g for all three analytes. The minimum quantitation level was 50 rig/g and the minimum detection level was 25 rig/g..

The results of the authors’ evaluation indicate that the procedure described above can detect and identify cocaine and metabolites within acceptable limits of detection, linearity and precision. When applied to the analysis of actual forensic case samples it has yielded reproducible data. The extraction process is simpler and faster than

156 A. Hernandez et al. /Forensic Sri. Int. 65 (1994) 149-156

liquid-liquid processes and produces cleaner extracts with less instrumental background.

The comparison of refrigerated and frozen storage conditions clearly demon- strated that cocaine is far less stable in refrigerated brain tissue than anticipated. If it is assumed that cocaine is stable in brain tissue stored at -80°C then - 50% was lost in the first month of refrigerated storage, and the drug was almost completely absent by 2 months. Apparently, much of the lost cocaine was converted to benzoyl- ecgonine. If brain tissue is taken for possible cocaine analysis, it should be stored frozen, to -80°C if possible, unless it is to be analyzed within a few days after col- lection.

In the study reported here, the concentration of cocaine in properly stored brain tissue always exceeded that in blood. Therefore, brain analysis may be useful to document recent cocaine use in cases of suspected cocaine toxicity such as case No. 2 of Table 2. The Dade County Medical Examiner Department Toxicology Labora- tory presently includes brain cocaine analysis in the toxicological examination of all cases of probable cocaine toxicity and some other cocaine-positive cases as controls. Analysis of more data may reveal relationships between blood and brain cocaine, cocaethylene and benzoylecgonine concentrations that can be useful for inter- pretation.

5. References

I W.L. Hearn, E.E. Keran, H. Wei and G. Hime, Site-dependent postmortem changes in blood co- caine concentrations. J. Forensic Sci., 36 (3) (1991) 673-684.

2 V.R. Spiehler and D. Reed, Brain concentrations of cocaine and benzoylecgonine in fatal cases. J. Forensic Sci., 30 (4) (1985) 1003-1011.

3 M.A. Evans and T. Moriarty, Analysis of cocaine and cocaine metabolites by high pressure liquid chromatography. J. Anal. Toxicol., 4 (1980) 19-22.

4 D.M: Chinn, D.J. Crouch, M.A. Peat, B.S. Finkle and T.A. Jennison, Gas chromatography- chemical ionization mass spectrometry of cocaine and its metabolites in biological fluids. J. Anal. Toxicol., 4 (1980) 37-42.

5 G.W. Hime, W.L. Hearn, S. Rose and J. Colino, Analysis of cocaine and cocaethylene in blood and tissue by GC-NPD and GC-ion trap mass spectrometry. J. Anal. Toxicol., I5 (1991) 241-245.

6 K. Matsubara, C. Maseda and V. Fukui, Quantitation of cocaine, benzoylecgonine and ecgonine methyl ester by GC CI-SIM after extralut extraction. Forensic Sci. hr., 26 (1984) 181-192.

7 J. Scheurer and CM. Moore, Solid phase extraction of drugs from biological tissues - a review. J. Anal. Toxicol., I6 (1992) 264-269.

8 S.P. Browne, CM. Moore, J. Scheurer, I.R. Tebbett and B.K. Logan, A rapid method for the deter- mination of cocaine in brain tissue. J. Forensic Sci., 36 (6) (1991) 1662-1665.

9 C. Moore, S. Browne, I. Tebbett and A. Negrusz, Determination of cocaine and its metabolites in brain tissue using high-flow solid-phase extraction columns and high-performance liquid chroma- tography. Forensic Sci. Inf., 53 (1992) 215-219.

IO A.H.B. Wu, T.A. Onigbinde and S.S. Wong, Evaluation of full-scanning GC/ion trap MS analysis of NIDA drugs-of-abuse testing in urine. J. Anal. Toxicol.. I6 (1992) 202-206.