Extraction, quantitative gas chromatographic determination and gas chromatographic]mass spectrometric detection of ecgonine for identification of cocaine and its metabolites in urine* S. Goenechea 1, G. Rficker 2, M. Neugebauer 2, and U. Zerell 1 Institut ftir Rechtsmedizin der Universitit Bonn, Stiftsplatz 12 g Pharmazeutisches Institut der Universitit Bonn, Kreuzbergweg 26, D-5300 Bonn 1, Federal Republic of Germany Extraktion, quantitative gas-chromatographische Bestimmung und gas-chromatographischer massenspektrometrischer Nachweis yon Ecgonin zur Identifizierung yon Cocain und seinen Mctaboliten in Urin Zusammenfassung. Es wurden quantitative Untersuchungen zur Extraktion yon Urin durchgeffihrt, dem Cocain, Benzoylecgonin, Ecgoninmethylester und Ecgonin zugesetzt wurden. Die Summe dieser Verbindungen kann fiber eine vorgeschaltete Hydrolyse zu Ecgonin indirekt bestimmt werden. Nach Hydrolyse wird Ecgonin an einem Kationenaustauscher isoliert und der Extrakt an einem Anionenaustauscher gereinigt. Die quantitative Bestimmung erfolgt gaschromatographisch nach Silylierung mit MSTFA. Dabei betr/igt die Wiederfindungsrate 77% bei Konzentrationen von 150 gg Ecgonin/ml Urin. Der qualitative Nachweis von Ecgonin durch GC/MS ist bis zu einer Nachweisgrenze yon 20 ng/ml m6glich. Damit eignet sich die Methode zum Nachweis einer Cocain-Einnahme im Urin. Summary. Quantitative extraction studies from urine were carried out by addition of cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine to urine samples. After hydrolysis to ecgonine the compounds were analyzed together. Ecgonine was isolated by a cation-exchange resin and purified by an anion-exchange resin. The quantitative determination was performed by GC after silylation with MSTFA. The recovery was 77% at a concentration of 150 gg ecgonine/ml urine. A qualitative determination of ecgonine by GC/MS was possible up to the detection limit of 20 ng/ mi. The method can be applied for the detection of cocaine abuse.

is difficult [4]. As late as 1978 ecgonine methyl ester was identified as a prominent urinary metabolite of cocaine in man by T. lnaba [5]. J. Ambre [6] suggested its detection in urine as a good indicator of cocaine abuse. Data on the concentration of the other identified metabolites of cocaine are not given in the literature [4, 5, 7 - 9 ] . Quantitative extraction studies have only been described for benzoylecgonine [2, 4, 10, 11]. A hydrolysis of cocaine and its metabolites to ecgonine as a stable endproduct has not been used as a marker of cocaine abuse. In this study a method for the hydrolysis of cocaine, benzoylecgonine and ecgonine methyl ester to ecgonine and a gaschromatographic and mass spectroscopic quantitative method for determination of ecgonine in human urine are described.

Experimental Reagents Cocaine hydrochloride was supplied by E. Merck, Darmstadt. 1-Nitro-4-phenoxy-benzene was provided by Aldrich Chemie, Steinheim, and recrystallized from ethanol. N-Methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) was obtained from Macherey-Nagel & Co., Dfiren. Silyl-8column conditioner was purchased from Pierce GmbH, Rodgau. Strongly acidic cation-exchange resin SP 1080, 0.1-0.25 mm (60-150 mesh ASTM) macroporous Na +form p.a. (Art.-No. 5258) was supplied by E. Merck, Darmstadt. Strongly basic anion-exchange resin MP 5080, 0.1-0.25 mm ( 6 0 - 1 5 0 m e s h ASTM) macroporous C1-form p.a. (Art.-No. 5256) was provided by E. Merck, Darmstadt. The water used was bidistilled and all other chemicals used were of analytical grade quality. The urine used for the extraction studies was collected over a period of 24 h.

Introduction Unchanged cocaine excretion accounts for less than 20% of the cocaine dose administered to humans [ 1 - 3]. For that reason the detection of the metabolites of cocaine promises an analytically more sensitive evidence of cocaine abuse. Up to 1978 only ecgonine and benzoylecgonine were regarded as major metabolites and urinary constituents. Due to their high water solubility, however, their extraction from urine * From the dissertation by U. Zerell, University of Bonn, 1986 (in preparation) Offprint requests to: G. Riicker Fresenius Z Anal Chem (1986) 323:326-329 9 Springer-Verlag 1986

Synthesis of metabolites Ecgonine hydrochloride (according to [12]): A solution of 1.2 g cocaine hydrochloride in 30 ml 2.4% hydrochloric acid was refluxed for 9 h. The reaction mixture was cooled and extracted 4 times with 15 ml ether to remove benzoic acid. The aqueous phase was evaporated to dryness after addition of a few drops of toluene on a rotatory evaporator and the crude product was recrystallized from ethanol. Yield 0.67 g (85%), m.p. 236~ ([13]: 246~ The PMR-data agreed with [4]. MS: m/z (%) = 185(M .+, 12) 168(10), 141(9), 124 (25), 97 (55), 96 (84), 83 (55), 82 (100).

@ igiF ala beileH Ecgonine methyl ester (according to [14]: An etheric Silyloted ecgonine solution of diazomethane (about 60%) was added to 1.0 g 1-Nitro-4-phenoxy-benzene ecgonine hydrochloride in 100 ml methanol until achieving a yellow colouring. After stirring for I h at room temperature the solvent was removed in vacuo. The hydro5.2 chloride was isolated after treatment with a methanolic solution of hydrochloric acid. Yield 0.86 g (81%), m.p. 215~ ([13, 15]: 210-212~ 222~ The spectroscopic data (IR, PMR, MS) agreed with the literature [4, 13, 16]. Benzoylecgonine (according to [17]): A solution of 0.8 g cocaine hydrochloride in 60 ml buffer solution pH 7.2 (Ph. Eur. II) was refluxed for 30 min. The reaction mixture was cooled and extracted 4 times with 15 ml ether and then perforated with chloroform at pH 7.2 for 9 h. The organic 2 phase was evaporated to dryness and the hydrochloride was isolated after treatment with a methanolie solution of 9 retention time [mini hydrochloric acid. Yield 0.53 g (69%), m.p. 216~ ([18]: Fig. 1. Gas chromatograrn of a silylated urine extract 220~ The P M R data agree with [4]. MS: m/z ( % ) = 289(M +-, 7), 184(2), 168(29), 124(47), 105(19), 97(6), 96(9), 83(12), 82(26), 77(15), 36(100). theoreticol

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Instrumentation and procedures Gas-chromatograph: Packard, model 428 with flame ionization detector (FID). Column: Glass, 2 m x 2 m m i.d. Stationary phase: 10% OV-101 on Chromosorb W HP 80/ 100mesh. Carrier gas: N2 55ml/min. FID-gases: Air 200 ml/min, H2 30 ml/min. Column conditioning: The new packed column was heated at 300 ~C for 2 h. Then 50 ~tl silyl8-column conditioner was injected at a oven temperature of 150~ The column was heated again at 300 ~ until a stable base line was achieved. Injection temperature: 300 ~C. Detector temperature: 300 ~C. Temperature programme: 150~ (1 m i n ) - 3 0 0 ~ (1 min), 18~ Injection volume: I t d . Peak integration: Apple I I e personal computer, Adalab A/D converter; the chromatochart programme was supplied by Heyden & Son GmbH, Rheine. Gas chromatograph/mass spectrometer: Model 1020B (Finnigan/MAT), direct coupling. Column: Fused silica capillary column, 30 m length, 0.32 m m i.d., DB-5, 0.25 grn filmthickness (J + W Scientific Inc., Rancho Cordova, USA) Carrier gas: Hz, pressure 12 psi, ~ = 60 cm/s. Split injector: Temperature 250~ split ratio: 1:20 (20 s closed), manifold temperature: 70 ~C; interface temperature: 280 ~C; source temperature: 180 ~C, ionisation energy: 70 eV. Temperature programme: 150 ~C (1 m i n ) - 300 ~C (1 min); 18 ~C/rain; injection volume: 2~tl. Baker-10 extraction system was purchased from J. T. Baker, Grol-Gerau. 6 ml and 3 ml polyethylene extraction columns were obtained from J. T. Baker, Grol3-Gerau. 1 ml conic derivatization tubes (N 1 2 - 1 ) were provided by Macherey-Nagel&Co., D/iren.

Preparation of extraction columns The 6 ml columns were filled with 2 g cation exchange resin, the 3 ml columns with 0.5 g anion exchange resin. The packing material used was glass wool. The columns were rinsed for regeneration and conditioning as follows: 6 ml columns (cation exchange resin): I time with 4 ml water, 4 times with 4 ml 10% hydrochloric acid, 4 times with 4 ml water, 1 time with 4 ml methanol, 2 times with 4 ml water. 3 ml columns (anion exchange resin): 1 time with 3 ml water, 4 times with 3 ml 10% sodium hydroxide solution, 4 times with 3 ml water, 1 time with 3 ml methanol, 2 times with 3 ml water.

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Hydrolysis, extraction and derivatization Cocaine hydrochloride, benzoylecgonine hydrochloride and ecgonine methyl ester hydrochloride were added to 1 ml urine in amounts equivalent to 100 ~tg of the bases. The urine was adjusted to p H 13 with 0.1 ml 10% sodium hydroxide solution and then refluxed for 30 min. After cooling to room temperature the solution was adjusted to pH 6 - 7 with 10% hydrochloric acid and transferred to an 6 ml extraction column filled with cation exchange resin. After rinsing 3 times with 4 ml water the column was treated 5 times with 4 ml 25% ammonia and the eluate passed through a 3 ml extraction column filled with anion exchange resin. The column was rinsed to neutral pH with 6 times 3 ml water and eluted 2 times with 2 ml 10% hydrochloric acid. The acidic solution was transferred to a 100 ml flask with about 10 ml methanol and, adding a few drops of toluene, evaporated to dryness in vacuo at 60 ~C. The residue was dissolved in 2 ml methanol, transferred to a 5 ml flask, rinsed 327

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with 2 ml methanol and evaporated to dryness again. The residue was dissolved in 150 gl methanol, transferred to a derivatization tube and evaporated under nitrogen in a water bath at 40 ~C, followed by a 100 ~tl methanol rinse and evaporation to dryness again [residue (a)]. After adding 150 gg 1-nitro-4-phenoxy-benzene in 75 gl methanol the solvent was removed again. 200 gl M S T F A were added and the extract heated in a water bath at 70~ for 20 rain. The tube was shaken several times until a clear solution was achieved. After that the gas-chromatographic analysis was performed. Figure 1 shows a typical chromatogram. Calibration curve, detection limit

Solutions of ecgonine hydrochloride in methanol containing 20, 50, 100, 150, 200 and 250 gg of the base were added to the residue (a) of 1 ml hydrolyzed and extracted blank urine prepared as described above. The solution was evaporated to dryness under N2 in a water bath at 40 ~C. 150 ~tg 1-nitro4-phenoxy-benzene in 75 gl methanol was added and the solvent removed again, followed by derivatization. The calibration sample of 150 pg ecgonine was analyzed six times, all other samples two times. Peak integration was carried out by integration o f the area with base line control on screen. After calculating the mean absolute response factors (abs RF, n = 6) .for silylated ecgonine (E) and standard (St) the concentrations of the calibration samples were calculated according to the following formular: concentration E 328

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The mean concentrations achieved were plotted against the theoretical concentrations. The calibration curve (Fig. 2) was linear over the range of 2 0 - 2 5 0 pg/ml, the detection limit proved to be 20 gg/ml.

Results and discussion Due to its betaine structure, ecgonine cannot be extracted from aqueous solutions by organic solvents, not even in the range of its isoelectric point at p H 6.9. The separation from urine was achieved by cation exchange resin. For extraction of 1 ml urine the amount of ion-exchange resin calculated for 4 times of the capacity was used. To separate impurities it was necessary to clean the extract with an anion exchange resin. Otherwise a precipitate appeared, causing a loss of silylated ecgonine. GC-determination was performed according to J.M. Moore [19]. Conditioning of the column with 50 gl silyl-8-column conditioner at 300~ before each measuring series is necessary in order to achieve a satisfying reproducibility. The coefficient of variation determined by 6 measurements of the same calibration sample (150 tig) was 0.53%. Because the absolute response factors for silylated ecgonine and standard differed up to 10% after each column conditioning, the response factors had to be calibrated before each measuring series again, l-Nitro-4-phenoxybenzene was chosen as internal standard due to its good solubility in M S T F A and its similar response factor to silylated ecgonine.

OrigJnaJarbeten Recovery studies" Six urine samples with 150 ~tg ecgonine/ml urine were extracted and analyzed as described above. The rate of recovery was 77.1% o f the theory with a coefficient of variation of 1%. The stability of ecgonine was analyzed in a concentration of 150 gg/ml urine, when the acidic solution was evaporated to dryness after the extraction. The recovery was 98.7%. To test the hydrolysis procedure, six urine samples with 100 ~tg cocaine, 100 gg benzoylecgonine and 100 gg ecgonine methyl ester per milliliter were analyzed. The recovery was 90.4% of the theory with a coefficient of variation of 1.9%. The stability of 218.1 ~tg ecgonine/ml urine was analyzed at p H 13. The recovery was 100.9% o f the theory.

Qualitative GC/MS determination, detection limit In concentrations of less than 20 ~tg/ml urine ecgonine can be identified as silylated derivative by G C / M S with a capillary column. A complete mass spectrum of the silylated compound is shown in Fig. 3. Important mass spectroscopic fragments are at m/z (%) = 329(M +., 2), 314(3), 97(43), 96(51), 83(100), 82(91). Qualitative determination of ecgonine by multiple ion detection is possible up to the detection limit of 20 ng/ml.

2. Kogan M J, Verebey KG, De Pace AC, Resnick RB, Mul6 SJ (1977) Anal Chem 49:1965-1969 3. Ambre J, Fischmann M, Ruo T (1984) J Anal Toxicol 8 : 2 3 25 4. Mul~ S-J (1976) Cocaine: chemical, biological, clinical, social and treatment aspects, vol 20. CRC Press, Cleveland, Ohio, pp 20, 2 6 - 6 9 5. Inaba T, Stewart DJ, Kalow W (1978) Clin Pharmacol Therapeut 23: 547- 552 6. Ambre JJ, Ruo T, Smith GL, Backes D, Smith CM (1982) J Anal Toxicol 6: 2 6 - 29 7. Smith RM, Poquette MA, Smith PJ (1984) J Anal Toxicol 8:29-34 8. Smith RM (1984) J Anal Toxicol 8 : 3 5 - 3 7 9. Smith RM (1984) J Anal Toxicol 8 : 3 8 - 4 2 10. Matsubara K, Maseda C, Fukui Y (1984) Forensic Sci Int 26:181 - 192 lt. Garrett ER, Seyda K (1983) J Pharm Sci 72:258-271 12. Bell MR, Archer S (1960) J Am Chem Soc 82:4642-4644 13. Singh SP, Kaufmann D, Stenberg VJ (1979) J Heterocycl Chem 16:625-63l 14. Jane I, Scott A, Sharpe RWL, White PC (1981) J Chromatogr 214:243-248 15. Findlay SP (1954) J Am Chem Soc 76:2855-2862 16. Lowry WT, Lomonte JN, Hatchett D, Garriot JC (1979) J Anal Toxicol 3 : 91 - 95 17. Einhorn A (1888) Chem Ber 21:47--51 18. Beilsteins Handbuch der Organischen Chemic, EIII/IV, Bd. 22/ 3. Springer, Berlin Heidelberg New York (1979), S 2098 19. Moore JM (1974) J Chromatogr 101:215-218

References 1. Fish F, Wilson DC (1969) J Pharm Pharmacol Supp121 : t 35 S 138S

Received July 10, 1985

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