Quantitation of Cocaine and Its Major Metabolites in Human Saliva ...

48 downloads 173 Views 839KB Size Report
Abstract I. An analytical method for the simultaneous determination of cocaine (COC) and its major metabolites, ecgonine methyl ester. (EME) and ...
Journal of Analytical Toxicology, Vol. 27, July/August 2003

Quantitation of Cocaine and Its Major Metabolites in Human Saliva Using Gas Chromatography-Positive Chemical Ionization-MassSpectrometry(GC-PCI-MS)* P. C~mpora, A.M. Bermejo t, M.J. Tabernero, and P. Fernandez

Institute of Legal Medicine, Forensic ToxicologyService, Faculty of Medicine, Universityof Santiago de Compostela,Spain

Abstract I An analytical method for the simultaneousdetermination of cocaine (COC) and its major metabolites, ecgonine methyl ester (EME) and benzoylecgonine(BEG), in saliva was developed. The method involves liquid-liquid extraction in ToxitubesA| derivatization with 99:1 (v/v) N,O-bislrimelhylsUyltrifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) and gas chromatography-massspectrometry (GC-MS) determination. The detector responsethus obtained is linear over the range 25-1000 ng/mt, with a reproducibility better than 93% and a recovery close to 100% for the three analytes. The limits of detection achieved were 0.9 ng/mL for EME, 2.2 ng/mL for cocaine, and 0.2 ng/mL for BEG, and the limits of quantilation were 3.0 ng/mL for EME, 7.4 ng/mL for cocaine, and 0.8 ng/m/for BEG. The proposed method was applied to 48 saliva samplesfrom cocaine users, 96% of which were positive for the drug and/or its metabolites. Saliva is thus a suitable biological fluid for determining cocaine, EME, and BEG by GC-PCI-MS.

can be more therapeutically meaningful (4,5). As a rule, drug concentrations are lower in saliva than in urine, so the former type of sample calls for more sensitive analytical methods (5,6). Besides, the natural variation of pH in saliva (7), the method used to collect it (3,5), and its potential contamination with drug residues in the oral or nasal cavities can result in altered levels (1,8). In this work, a gas chromatography-positive chemical ionization-mass spectrometry (GC-PCI-MS) method for the determination of cocaine and its major metabolites (ecgonine methyl ester [EME] and benzoylecgonine [BEG]) in saliva using their deuterated analogues as internal standards was developed.

Experimental Malerial

Introduction Saliva has major advantages over blood or urine samples in the analysis of drugs (1--4). Saliva is easily collected and handled without the need for skilled personnel. Although tampering with samples can never be completely excluded, the occurrence of adulteration is much less frequent than with urine, so the analytical results are normally more reliable (2). The drug concentration in saliva is thought to represent the free fraction, whereas the concentration in plasma represents the combination of free and protein-bound drug (2,5). In plasma, the extent of protein binding of the drug varies with the patient's age and health condition, as well as with the presence of other compounds potentially capable of displacing the drug from protein binding sites; consequently, the drug concentration in saliva 9Partial resu[t~of this work were presenled at lhe XIV Spanish Congressof Toxicology (Murcia, September 2001 ). t Aulhor to whom correspondence should be addressed:Ana Maria Bermejo. Institute of Legal Medicine, Forensic Toxicology Service, Facuhy of Medicine C/San Francisco, s/n. 15782Sanliago de Composlela. Spain. E-mail: [email protected].

270

Cocaine, BEG, EME, and their respective deuterated analogues were obtained from Radian International. Methanol, high-performance liquid chromatography (HPLC)-grade acetonitrile, BSTFA, and TMCS were supplied by Merck. Toxitubes A were purchasedfrom Dipesa,and the Salivette| which was used to collect the real samples, from Sarstedt. Workingstrength solutions (0.01 mg/mL) were preparedfrom 1-m~mL standards in methanol (BEG and BEG-d3)or acetonitrile (COC, COC-d3, EME, and EME-d3). Extraction and derivatization

A volume of i mL of saliva was mixed with 20 pL of the working solutions of the deuterated derivatives, stirred for 10 min to ensure homogeneity, poured into a Toxitube A| that was mechanically shakenfor 10 rain to extract the analytesinto the organic layer, and centrifuged at 5000 rpm for 8 min. The organic layer was then transferred into a conical glass tube and evaporated to dryness in a thermostated bath at 60~ under a nitrogen stream. The dry residue was reconstituted with 40 pL of 99:1 (v/v) BSTFA/TMCS and heated at 100~ for 20 rain to transform the cocaine metabolites into trimethylsilyl-

Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

Journal of Analytical Toxicology, Vol. 27, July/August 2003

derivatives (TMS). Finally, the solution was kept at room temperature for 10 min, and a 2-1~Laliquot was injected into the chromatograph.

Results

Toxitubes A provide a rapid, simple, inexpensive means for extracting cocaine, BEG, and EME. The temperature programme used ensured good chromatographic resolution of peaks (Figure 1). The mass spectra for each drug and its deuterated analogue obtained by injecting standard solutions in the scan mode (Figures 2-4) were used to select the characteristic ions according to their abundances. So, the proposed method presents a high specificity as a perfect differentiation between the studied analytes and other cocaine-related compounds (cocaethylene, egnonine ethyl ester, norcocaethylene, and norcocaine) is obtained. The quantifier ions selected were those at m/z 272 for EME-TMS, 275 for EME-TMS-dg,304 for COC, 307 for COC-d3, 362 for BEG-TMS and 365 for BEG-TMS-d3,all corresponding to one mass unit more than the molecular ion (MH+). The detector response was linear over the range 25-1000 ng/mL of each analyte. The equations and correlation coefficients obtained were as follows: EME-TMS,y = 6.3499x + 0.0281, R = 0.9961; cocaine, y = 3.8694x + 0.0946, R = 0.9952; and BEG-TMS,y = 8.7089x - 0.1435, R = 0.9993. According to the criteria of Bressolle et al. (10) about validation of chromatographic methods, linearity of the method was demonstrated by showing that the slope of the linear calibration curve was statistically different from 0, the intercept

Chromatographic conditions Chromatographic analyses were carried out on a Hewlett-Packard 6890 GC interfaced to a model 5973 mass selective detector, also from Hewlett-Packard. Chromatographic elution was done with a 12-m x 200-]~m i.d. capillary column internally coated with a 0.33-1~m thick film of 5% phenylmethylsiloxane. The injector temperature used was 250~ A volume of 2 ~L was injected by hand in the splitless mode, and a purging time of 0.75 min was used. The flow-rate of reactive gas (methane) used for PCI was 20 mL/min. The temperature program was started at 90~ (1 rain), followed by a 20~ ramp to 180~ a second, 5~ ramp to 240~ and a third, 30~ ramp to 290~ The mass selective detector was kept at 320~ the ion source at 250~ and the quadrupole at 106~ The selective ion mode (SIM) was used to monitor the ions selected for each compound studied. The instrument liner and septum were replaced every two weeks.

Saliva collection Saliva used to prepare the calibrators was collected without stimulation (i.e., only in the amounts naturally produced by the individuals) for 3 min and stored in Ion ~ 0 (271.70 to 272,70~ 150-3,D COC-d 3 polyethylene flasks. Real samples were ob11 ion 27500 (274.'r0 to 215.70); 150-3.D ~n 3~. nn r to 3D4.70~r 1SO-3D tained from chronic users in a drug 9n 30t 'nn t'3W.TO to 307.;'0"; t SB-3.D k~ 362 nn (381'?0 t~ 382'70~ lS0"3D COC detoxification program of the Drug Ad~"J ~6,OG (384,YD to 3E5.?0~ I SO-3D ;'B dict Health Care Unit of Galicia (NW Spain), with the aid of Salivette and EME 1 ~ BEG-d 3 TMS frozen at -20~ (or only refrigerated at L 1~,~9 BEG TMS 4~ if they were to be analyzed on the collection day). This commercial device con12~O tains a cotton-bud that is introduced into the patient's mouth for 2-3 min until it is Figure 1. Chromatogram obtained from the simultaneous injection of EME-drTMS, EME-TMS,COG soaked with saliva, and then it is trand~, COC, BEG-drTMS, and BEG-TMSstandards. [Analyte d0] = 150 ng/mL. [Analyte d3} = 200 ng/mL. ferred to a collecting glass to be analyzed. tOWO,

[

0

i

.M

.

9

!

.

.

.

.

!

.

.

Procedure

Drug-free saliva was mixed with appropriate volumes of 0.01 mg/mL standards of the three analytes to prepare solutions containing them, at nine different concentrations (25, 50, 100, 150, 200, 250, 300, 600, and 1000 ng/mL), in addition to the respective deuterated analogues (at 200 ng/mL) as internal standards. Calibration curves were obtained by plotting the ratio of the area under the chromatographic peak of the quantifier ion for each analyte to that for its deuterated analogue (drug/drug-d3) against concentration (including 0,0). Each point in the curve was obtained in triplicate. Limits of detection (LOD) and limits of quantitation (LOQ) were determined according to the method proposed by Nix et al. (9).

45 $7

o-.~,

73

L

....

9~. tl1

(~o . . . .

1,5[ L

t~13Q14~158

,~ . . . .

2"L L ' ' '~o . . . .

196 207 217 227

~-'

2-

~5

t L~,~ ~o . . . .

~

~3. ....

m/z

,Iooom

EME "rIMS

~

82

' -

6b

,l

. . . .

'

le2

;

t

'

-

;

'

'

"L5 '"

'

'

'

'

312 , [ 328

3~

3•i0

m/z

Figure 2. Mass spectra for EME-drTMS and EME-TMSstandards, obtained in the scan mode. 271

Journal of Analytical Toxicology,Vol. 27, July/August2003

centration (N = 11). The accuracy was expressed as percent of the difference between the measured concentration and the true value for the same concentration (N = 11). The proposed method was applied to 48 samples from cocaine users under a detoxification treatment by the Drug Addict Health Care Unit of Galicia. The urine i from studied individuals was previously analyzed by using fluoroinmunoassay, and positive results were always obtained. Careful examination of the 46 positive samples revealed that 19 contained EME