Simultaneous Determination of Cocaine and Benzaylecgonine in Urine by Gas-Liquid Chromatography David
L. von
Minden" and Nicholas A. D'Amato
Laboratory Service, Naval Regional Medical Center, Portsmouth, Virginia 23708
A procedure for the simultaneous determination of cscaine and Its principal metabolite, benroylecgonlne (BE), In urine by gas-liquid chromatography (3 % SP-2250-DA or 3 % SE-30, FID) is presented. The method Involves extraction of the two compounds from urlne into ethanol-chloroform (25175, v/v) followed by propylation of BE using an organic base and 1-lodopropane. The recovery from blologlcal samples of 99 and 80 % for cocaine and BE, respectively, and 98 % conversion of BE Into Its n-propyl ester provlde detection limits of 0.2 pg/rnL in 5 mL of urlne. The alkylation conditions were optimized.
The application of gas-liquid chromatography (GLC) to the analysis of cocaine and its metabolites in urine presents several problems to the analyst. The primary difficulty is that cocaine, a lipophilic drug, is extensively metabolized in man ( I d ) , with less than 1.0% of the total amount of drug and metabolites being unchanged cocaine (2, 4 ) . Another serious problem is that the primary metabolite, benzoylecgonine (BE),is highly soluble in water and is not readily extracted into many organic solvents ( 4 , 7, 8). Finally, in order to analyze B E by GLC, i t must first be converted into a volatile derivative ( I , 4 , 7, 8). Critical reviews on the numerous analytical techniques for the detection and quantitation of cocaine and BE have been published (7,9).Fish and Wilson ( I ) , Koontz et al. (8), and Wallace et al. ( 4 ) have developed GLC procedures which incorporate methylation of the extracted BE into cocaine prior to GLC analysis. The nonquantitative method of Koontz et al., requiring an elaborate thin-layer chromatographic purification step, provides for the detection of 1r g / m L of B E in 10 mL of urine. T h e continuous liquid-liquid extraction method of Fish and Wilson affords linear recoveries of BE from urine over the range of 1-14 gg/mL. Quantitative data (percent recovery, standard deviation, or limit,s of detection) were not presented in either of these two procedures. The method of Wallace et al. provides for the quantitative determination of cocaine and B E (93 and 65% recovery, respectively) over the range of 0.25-50 rg/mL in 5 mL of urine, but requires two separate extractions and the running of two separate chromatograms. The methylation of B E is accomplished by heating in methanolic sulfuric acid. T h e GLC procedure described in this report allows for the simultaneous determination of both cocaine and B E in urine with quantitative recoveries over the range of 0.20-100 %/mL in 5 mL of specimen. The method utilizes a modification of t h e excellent solvent extraction technique of Wallace e t al., propylation of the extracted metabolite by the use of an organic base and 1-iodopropane (IO),back-extraction followed by a clean-up step, and subsequent analysis of cocaine and its analogue by a gas chromatograph equipped with a dual flame ionization detector and a reporting integrator. T h e procedure is not applicable to the analysis of cocaine or B E in serum.
EXPERIMENTAL Materials. Cocaine was purchased from Applied Science Laboratories, State College, Pa. Benzoylecgonine was obtained from Technam, Park Forrest South, Ill. The following items were 1974
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
purchased from Eastman Organic Chemicals, Rochester, N.Y.: 1-iodopropane, 1-iodopentane, trimethylphenylammonium hydroxide (0.1 M in methanol), tetramethylammonium hydroxide (25% in methanol), and spectrograde chloroform, n-butylacetate, and NJV-dimethylacetamide. All chemicals were checked for their purity and used without further purification. Apparatus. Gas chromatographic determinations were carried out on a Perkin-Elmer Model 900 gas chromatograph equipped with dual flame ionization detectors and a Hewlett-Packard Model 3380A reporting integrator. The silanized glass columns (1.8-m x 2.0-mm i.d.) were packed with either 3% SP-2250-DA or 3% SE-30 on 100/ 120 mesh Supelcoport (Supelco, Bellefonte, Pa.). Injector and detector temperatures were 250 "C. The column temperature was either 230 "C for 1 min and then programmed at 12 OC/min to 260 "C (3% SP-2250-DA) or 200 OC for 1 min and then programmed at 6 OC/min to 230 O C (3% SE-30). Nitrogen was used as the carrier gas with a flow rate of 50 mL/min. The hydrogen and air flow rates were adjusted to give optimum sensitivity. Low resolution mass spectra were recorded on a Varian MAT 112 S mass spectrometer with sample introduction by direct probe. The ion source temperature was 220 OC. The accelerating voltage was 0.8 kV, while the ionizing voltage was 80 eV. High resolution mass spectra were recorded at,R = 20000 using a Varian MAT 731 mass spectrometer with sample introduction by direct probe. The ion source temperature was 220 OC. The accelerating voltage was 3 kV, and the ionizing voltage was 100 eV. Elemental compositions were derived using the peak-matching technique, with maximum errors of 10.4 millimass unit. Organic Base (Derivatizing Reagent). One part of the tetramethylammonium hydroxide reagent and 20 parts of the trimethylphenylammonium hydroxide reagent are added to 200 parts of N,N-dimethylacetamide. Synthesis of the Internal Standard ( n-Pentylbenzoylecgonine, PeBE). To a solution containing 100 mg of BE in 100 mL of the organic base was added 1 mL of 1-iodopentane. The reaction mixture was stirred at 50 OC for 1h, after which it was terminated by the addition of 100 mL of 0.75 M Na2C03/NaHC03 buffer (pH 9.6). The product was extracted with three 25-mL portions of CHC13, and the combined extracts were washed with three 50-mL portions of the carbonate buffer. The organic phase was dried over anhydrous Na2S04and concentrated in a rotatory evaporator. The gum obtained was crystallized from ethanol with a 75% overall yield. Analysis by GLC showed the material to be homogeneous. An exact molecular mass of 359.2096 (required for CZ1Hz9NO4, 359.2097) was obtained. Principal mass spectral peaks, m / e (rel. int.): 359 (52) M+.; 272 (21) M - OC5Hll; 254 (25) M - C,H,CO; 238 (100) M - CsHbCOz. Synthesis of n -Propylbenzoylecgonine (PrBE). This compound was prepared using the above procedure. Overall yield was 78%. An exact molecular mass of 331.1780 (required for C,&fi04, 331.1784)was obtained. Principal mass spectral peaks, m / e (rel. int.): 331 (52) M+.; 272 (24) M - OC,H,; 226 (16) M - C~HSCO; 210 (100) M - C6H5C02. Optimization of the Alkylation Reaction. Influence of the Organic Base Reaction Time. To tubes containing 50 pg of cocaine and BE, 500 pL of the organic base was added. After a reaction time of 30 I, 1, 2, 5, 15, 30, and 45 min, respectively, 10 p L of 1-iodopropane was added and each mixture was allowed to stand for an additional hour. The reaction was then stopped by the addition of 5 mL of the carbonate buffer. The reaction products were extracted into 3 mL of CHC13containing 50 pg of the internal standard (PeBE). The aqueous layer was aspirated and the organic phase was taken to dryness under a stream of nitrogen in a 60 "C water bath. The residue was dissolved in 20
Table I. Recovery of Benzoylecgonine from Urine Added to urine, P g/mL 0.25 0.50 1.0 2.0 4.0 8.0 20.0
Amount determined, P g/mLa 0.21 0.39 0.76 1.63 3.14 6.47 15.91
0.04 & 0.04 i 0.05 i: 0.11 0.09 i 0.23 i: 0.66 i:
*
Mean of quintuplet determinations deviation.
Recovery, % 84.0 78.0 76.0 81.5 78.8 80.9 79.6 Av 80.5 i:
standard
pL of CHC13,and 1pL was injected on the top of the GLC column. Peak area ratios of cocaine and PrBE to PeBE were plotted vs. organic base reaction time. Influence of t h e 1-Iodopropane Reaction Time. Another but similar experiment was performed as follows: the organic base reaction time was fixed at 1 min; after the addition of l-iodopropane, the reaction was stopped at 30 s, 1, 2, 5, 10, 15, 30, and 45 min, respectively, by the addition of 5 mL of the carbonate buffer. Peak areas of PrBE to PeBE were plotted vs. l-iodopropane reaction time. Influence of &action Temperature. The above experiment was duplicated at 50 "C. Peak areas of PrBE to PeBE were plotted vs. 1-iodopropane reaction time. Procedure. Five mL of urine, 0.2 mL of the carbonate buffer, and 25 mL of 25% (v/v) ethanol in CHC13 containing 25 pg of PeBE were added in sequence to a 40-mL screw-cap, conical glass centrifuge tube. The tube was stoppered, vortexed for 2 min, and centrifuged briefly (2000 rpm for 4 rnin). The aqueous solvent was aspirated, and the organic solvent was evaporated to dryness at 60 "C under a stream of nitrogen. Propylation was accomplished by adding 0.5 mL of the organic base followed by brief vortexing. After 1 min, 10 pL of 1iodopropane was added and the tube was placed in a 50 "C water bath for 15 min. The reaction was terminated by the addition of 5 mL of 0.2 N H2S04,and the solution was washed with two 5-mL volumes of spectrograde n-butylacetate (1min vortexing followed by brief centrifugation and aspiration of the organic layer). Two mL of the carbonate buffer and 2 mL of spectrograde CHC13 were added. The mixture was vortexed for 1 min, after which the tube was centrifuged briefly and the aqueous layer was aspirated. After evaporation of the organic solvent under a stream of nitrogen at 60 "C, 20 p L of spectrograde CHC13was added to the tube. One gL of the mixture was then subjected to GLC analysis. Quantitation was accomplished by the Hewlett-Packard 3380A reporting integrator in the Internal Standard mode, utilizing a standard in urine of 5.0 and 10.0 pg/mL of cocaine and BE, respectively. RESULTS AND DISCUSSION E x t r a c t i o n P r o c e d u r e . The extraction procedure of Wallace e t al. ( 4 ) was employed because of its relative ease, rapidity, and high recovery (65%) of BE. However, experimentation revealed that minor changes in the technique [substituting 25% (v/v) ethanol in CHC13 and utilizing a carbonate buffer] resulted in a significant improvement in the recovery of BE from aqueous media. Analysis of urine specimens to which 0.25 to 20.0 pg/mL of B E or 0.25 to 20 pg/mL of cocaine were added revealed mean recoveries of 80 and 99%, respectively (see Tables I and 11). The 80% recovery of B E represents a 23% improvement over the procedure of Wallace et al. Possible interference from additional urine constituents being extracted with the increased amount of ethanol in the extracting solvent [Wallace et al. used 20% (v/v) ethanol in CHC13] was minimized by the back-extraction and clean-up step. I n t e r n a l Standard. The n-pentyl ester of B E was selected as the internal standard on the basis of relative retention time
Table 11. Recovery of Cocaine from Urine Added to urine, IJ g/mL 0.25 0.50 1.0 2.0 4.0 8.0 20.0
Amount determined, Pg/mLa 0.26 0.51 0.99 1.96 3.90 8.08 19.36
t i:
i: i: i:
i: i.
Recovery, %
0.04 0.05 0.04
104.0 102.0 99.0 98.0 91.5 101.0 96.8 Av 98.9
0.16 0.31 0.30 0.94
a Mean of quintuplet determinations viation.
L
i:
standard de-
.
3,"
I5 T
;'E
*4,
45
NJ'S,
Figure 1. Alkylation: Influence of the organic base reaction time. B: benzoylecgonine; A: cocaine
t o cocaine and PrBE, sensitivity, and its chemical and structural similarity to cocaine. The n-butyl homologue of cocaine was rejected as an internal standard because of the interference of the codeine peak under the conditions of the procedure. Alkylation Reaction. The alkylation procedure is a modification of the method of Greeley (10). Optimal conditions for the reaction were determined experimentally. The use of two organic bases, trimethylphenylammonium hydroxide and tetramethylammonium hydroxide, was necessitated due to the fact that the reaction time of the former was exceedingly long (>1 h) while the exclusive use of the latter resulted in the hydrolysis of cocaine, BE, and PeBE. Under the reaction conditions of the procedure, essentially no hydrolysis was observed. The rate of alkylation is dependent upon the organic base reaction time, the 1-iodopropane reaction time, and the reaction temperature. The influence of the organic base is shown in Figure 1. The rate of hydrolysis of cocaine is the limiting factor in the reaction time of the organic base, with a rapid decrease in cocaine concentration after 2 min; therefore, a n organic base reaction time of no more than 1min is necessary for quantitation of cocaine. Results on the optimization of the second step of the derivatization, namely the l-iodopropane reaction time and temperature, are shown in Figure 2. The optimum is reached after 30 rnin a t 25 "C and 10 rnin a t 50 "C, and remains constant up to 1 h. Utilizing the conditions developed-reaction times of 1 min for the organic base and 15 min a t 50 "C for 1-iodopropane-a linear response for BE was demonstrated in the 0- to lOO-pg/mL range with a 98 f 2% conversion to PrBE; likewise, a linear response for cocaine in the same range was observed. Chromatography. The gas chromatographic properties of cocaine, PrBE, and PeBE were excellent. Both 3% SE-30 and 3 % SP-2250-DA columns were utilized with no statistical differences observed. Typical chromatograms are shown in Figures 3 and 4. ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1975
I5
30
45
TIME (MINUTES)
Figure 2. Alkylation: Influence of reaction temperature and alkyl iodide reaction time. 0: 25 O C : W: 50 O C
C 2 4 6 8 1 C -IME
C 2 4 6 8 1 C T ME
Figure 4. Gas Chromatograms obtained from human urine extracts analyzed on 3% SE-30. A , urine to which 1.0 yglmL of cocaine and benzoylecgonine were added. 5,drug-free urine. Peak a , cocaine; Peak b n-propylbenzoylecgonine; Peak c, n-pentylbenzoylecgonine (the internal standard)
Table IV. Precisiona and Accuracyb in the Determination of Cocaine in Urine Amount added, uJg/mL 0.39 0.78 i 2 4 6 C C
-1v:
: ' _ 3 6 8 :
1.56
-ll'E
Flgure 3. Gas chromatograms obtained from human urine extracts analyzed on 3% SP-2250DA. A , urine to which 1.O yg/mL of cocaine and benzoylecgonine were added. 5,drugfree urine. Peak a: cocaine; Peak b, n-propylbenzoylecgonine; Peak c , n-pentylbenzoylecgonine (the internal standard)
0.78 1.56 3.12 6.25 12.5 25.0 50.0
12.5
0.41 t 0.03 (105.1%) 0.81 t 0.06 (103.8%) 1.52 i 0.10 (97.4%) 3.19 t 0.16 (102.2%) 6.14 t 0.23 (98.2%) 12.56 i 0.39
(100.5%) 24.15 t 0.57 (96.6%)
hlean Coefficient of Variation ( C V ) = 5.1%. Coefficient of Determination ( r 2 )= 0.999 (from least Mean of quintuplet determinations squares line). standard deviation.
Amount determined, pg/mLC 0.83 t 0.03 (106.4%) 1.68 t 0.12 ( 107.7 %) 3.28 t 0.16 (105.1%) 6.36 t 0.29 (10 1.8%) 11.78 r 0.36 (94.2%) 25.03 t 0.33 (100.1%) 48.95 t 0.89 (97.9%)
Mean Coefficient of Variation (CV) = 3.8%. Coefficient of Determination ( r 2 )= 0.999 (from least squares line). Mean of quintuplet determinations standard deviation. +_
Analytical Sensitivity, Reproducibility, a n d Accuracy. The procedure has a sensitivity of 0.2 yg/mL of cocaine or 1976
6.25
25.0
Table 111. Precisiona and Accuracyb in the Determination of Benzoylecgonine in Urine Amount added, g/mL
3.12
Amount determined,c u g/mL
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
BE in a 5-mL specimen. Duplicate determinations of urine of 10 individuals not receiving cocaine indicated a urine blank of 0.10 & 0.15 (std. dev.) pg/mL and 0.05 f 0.10 yg/mL for cocaine and BE, respectively. Quantitation was based on electronically integrated peak area ratios, with a mean coefficient of determination (CV), a measure of precision, of 5.1% for cocaine and 3.8% for BE. The coefficient of determination ( r 2 ) ,a measure of accuracy, was 0.999 for both cocaine and BE (Tables 111 and IV). Specificity. In vitro data indicate that the benzodiazepines, common antihistamines, chlorpromazine, methadone, propoxyphene, amitripyline, amphetamines, barbiturates, caffeine, nicotine, and methaqualone do not interfere with this assay. On the SE-30 (SP-2250-DA) column, the PrBE has a retention time within 0.6 (1.2) min of codeine and PeBE has a retention time within 0.5 (0.4) min of morphine, but both can be resolved from these potentially interfering substances. Chromatographic peaks indicative of drugs or
drug metabolites were not observed in any of the 118 urine drug screen specimens or the 15 patients receiving cocaine anesthesia for rhinoplastic or septoplastic surgery. Applications t o Biological Samples. Analysis of urine specimens obtained from adult patients receiving cocaine anesthesia (250 mg cocaine hydrochloride topically applied to nasal mucosa) for rhinoplastic or septoplastic surgery revealed results that were not significantly different from those reported in an earlier report ( 4 ) . The observed concentration range for the initial 8-h pooled specimens for cocaine and BE were 0 to 0.3 pg/mL and 8 t o 136 kg/mL, respectively. Of the 118 urine drug screen specimens, screened positive for BE by radioimmunoassay (RIA), 105 were confirmed positive by the GLC procedure, with concentrations ranging from 0.2 to 18 wg/mL for BE while cocaine concentrations did not exceed 0.1 pg/mL.
ACKNOWLEDGMENT The authors express thanks to Ralph A. Nelson, Naval Regional Medical Center, for providing urine specimens from
post-surgery patients who had received cocaine anesthesia. The authors are extremely grateful to James A. McCloskey and Pam F. Crain, Department of Biopharmaceutical Sciences, University of Utah, for performing the mass spectral analyses.
LITERATURE CITED (1) F. Fish and W. D. C. Wilson, J . Pharm. Pharmacol., Suppl., 21, 1355, (1969). (2) L. A. Woods, F. G. McMahon. and M. H. Seevers, J. Pharmacol. f x p . Ther., 101, 200 (1951). (3) A. R. McIntyre, J. Pharmacol. f x p . Ther., 57, 133 (1936). (4) J. E. Wallace, H. E. Hamilton, D. E. King, D. J. Bason. H. A. Schwertner, and S. C. Harris, Anal. Chem., 48, 34 (1976). (5) M. L. Bastos, D. Jukofsky, and S. J. Mug, J. Chromatogr.,89,335 (1974). (6) N. N. Vahnju, M. M. Baden. S. N. Valanju. D. Mulligan, and S. K. Verma. J . Chromatogr., 81, 170 (1973). (7) M. L. Bastos and D. 6. Hoffman, J. Chromatogr. Sci., 12,269 (1974). (8) S.Koontz, D. Besemer, N. Mackey, and R. Phillips, J. Chromatogr.,85, 75 (1973). (9) P. I. Jatlow in “Cocaine: Chemical, Biological, Social and Treatment Aspects”, S. J. Mule, Ed.. CRC Press, Cleveland, Ohio, 1977,pp 59-70. (IO) R. H. Greeley. Clin. Chem. (Winston-Sabm, N . C.),20, 192 (1974).
RECEIVED for review May 10, 1977. .Accepted August 1, 1977.
Reconstruction of Gas Chromatograms from Interferometric Gas ChromatographyAnfrared Spectrometry Data J. A. d e Haseth and T. L. Isenhour* Department of Chemistry 045A, University of North Carolina, Chapel Hill, North Carolina 27514
A method has been devised by which gas chromatograms are directly reconstructed from single scan GC/IR interferograms. The Gram-Schmidt vector orthogonalization process is used: first, to define from background interferograms a basis which adequately describes the inherent instrumental and collection characteristics; then second, to orthogonalize ail nonbasis single scan interferograms to that basis and measure each orthogonal component. The orthogonal component is a direct function of the total infrared absorbance of the entire spectral range with respect to the basis. The reconstructed gas chromatograms, belng derived directly from the single scan interferometric data, precisely indicate which interferograms best represent sample spectra. I t has been found that the reconstruction of G W I R chromatograms is computationally more economical than transforming the interferograms individually and searching for nonbackground spectra.
T h e utilization of gas chromatography/infrared spectroscopy (GC/IR) has given the analytical chemist a powerful technique for the qualitative analysis of mixture components. Various methods ( 1 , 2) have been employed for the construction of GC/IR spectrometers. In continuous flow gas chromatography systems where GC peaks are eluted within a few seconds, conventional dispersion spectrometers are unable to complete a scan within the time constraints of the experiment. Rapid scan Fourier Transform spectrometers can collect single scan interferograms fast enough to record the spectra of GC effluents; however, the resulting data are a collection of single scan interferograms that gives no apparent indication which interferograms are to be transformed to yield the effluent IR spectra.
T o conclusively locate the interferograms of spectra of GC effluents it is necessary to transform all interferograms in the collection, plot their respective spectra, then visually discriminate between background and effluent. Clearly, this is a tedious and time consuming process. Attempts have been made to more directly locate those interferograms which correspond to spectra of GC effluents and hence save time and labor. One method is to calculate the overall absorption power of each interferogram and determine which interferograms have been attenuated by infrared active chemical components in the light path. This method, which is analogous to measuring the total ion current (TIC) in gas chromatography/mass spectrometry (GC/MS), does not produce reliable results unless the interferogram intensity is attenuated by a t least 10% (3). Unfortunately, most GC/IR absorbances are well below this threshold. Another solution is to run the GC experiment twice, once in the GC/IR mode, the other time eliminating the GC/IR cell and measuring the response with a standard GC detector. All that remains is t o map the GC trace onto the series of single scan interferograms and transform only those that correspond to sample effluents in the light pipe. In practice, this is not always easily achieved. Operating conditions often cannot be exactly duplicated and volume changes between the IR cell and GC detector systems offset the two data sets. Stream splitters may be useful for simultaneously recording the interferograms and the gas chromatogram. Although this method is more reliable than separately recording the two data sets, there is still a lack of precision in identifying specific sample interferograms. This paper presents a method by which the gas chromatogram may be directly reconstructed from the interferometric data, thereby identifying those interferograms to be transformed to yield mixture component spectra. The method uses ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1977
