Anal. Chem. 1992, 64, 1509-1512
AC RESEARCH
Automatic Continuous-Flow Method for the Determination of Cocaine Marcelina Eisman, Mercedes Gallego, and Miguel ValcBrcel' Department of Analytical Chemistry, Faculty of Sciences University of Cbrdoba, 14004 Cbrdoba, Spain
A new mahod for the indirect determination of cocaine wHh an automatic conlinuous iiquid-liquid extractor is reported. ~mahodinvdve,thefformatknofd#ferentlonpalrsbetween the drug and inorganic complexes, BIId-, Co(SCN)42-, Ni(SCN)42-,Fe(SCN)8", and Reinecke sail, followed by thelr extraction and the d@terminatlonof bismuth, coban, nickel, kon,and chromlum in the organic phase (1,2dlchloroethane) by atomk absorption spedromelry. The optimalpH, complex concentration, and values of FIA variables were determined. The method permitsthe determlnatlonof cocaine over a wide concentration range (1-2000 pg/mL). The method is mod sensitive for Bi14- and mod selective for Fe(SCN)8S-.
INTRODUCTION Benzoylmethylecgonine (cocaine) is a psychotropic drug with a long history of human consumption. It is an ester of benzoic acid and the amino alcohol, ecgonine,which contains a tropine moiety and is chemically,but not pharmacologically, related to atropine.' Its medicinal use is that of a topical local anaesthetic; thus, clinically, its most important mechanism of action lies in its ability to block sodium channel conductance, thereby increasing the threshold required to generate an action potential.2 Immunoassays are ideal for screening urine samples for benzoylecgonine. Available kits for this purpose have been adapted for screening plasma, serum, whole blood, bile, and tissue samples. However, all positive results must be confirmed by using a noninmunological assay, ideally gas chromatography/mass spectrometry (GC/MS). GUMS in its electron impact and chemical ionization variants is the separation technique most frequently used for the analysis of cocaine and its metabolites. Several applications3+ reported so far testify to the remarkable high sensitivity of (1)Deutsch, D. G.,M.AnalyticalAspectsofDrugTesting;John Wiley: New York, 1989. (2)Caplan, Y. H. Abused Drugs Monograph Series. Cocaine; Abbot Laboratories: Irving, TX,1988. (3)Isenechmid, D. S.;Levine, B. S.; Caplan, Y. H. J.Anal. Toxicol. 1988,12,242-245. (4)Mitsui, T.; Matsuoka, T.; Fujimura, Y. Eisei Kagaku 1989, 35, 194-197. J.;Chiarelli, (5)LeBelle,M.J.;Lauriault,G.;Callahan,S.A.;Latham,D. C.; Beckstead, H. J. Forensic Sci. 1988, 33,662-675. (6)Jindal, S. P.;Lutz, T. J. Pharm. Sci. 1989, 78,1009-1014. (7)Hudson, J. C. J. Can. SOC. Forensic Sci. 1989,22,203-218. (8)Duncan, W. P.; Deutsch, D. G . Clin. Chem. 1989,35,1279-1281. 0003-2700/92/0364- 1509$03.00/0
GC/MS compared to liquid chr~matography.~J~ Others techniques such as potentiometryl' and calorimetry12 have also been used for this purpose. Determinations of organic products by extraction of ion pairs with acidic or basic dyestuffs using molecular spectrophotometry have been developed for many years. Ion pairs of alkaloidswith methyl orange,13J4bromothymol blue,ls bromocresol,16 and gallion" are usually extracted into CHC13, and the absorbance of the organic extract due to the ion pair complex is measured. However, the use of metal complexes allows the indirect determination of organic products by atomic absorption spectrometry; the increased sensitivity inherent in this technique has aroused much interest from researchers, as shown by the number of papers on this topic which have appeared in recent years.18 Thus by using Reinecke salt29 C O ( S C N ) ~ ~Dragendorf -,~~ reagent,21 or Ni(SCN)d2-F2alkaloids can be determined at concentrations between 0 and 40 mg/mL by extraction into nitrobenzene or 1,2-dichloroethane. Surprisingly, cocaine has not yet been determined in this way. In fact, there is only a single reference to identification tests for cocaine23by ion pair extraction into chloroformusingiron or copper ion and thiocyanate; the purity of cocaine is estimated by color comparison. Continuous-extraction systems coupled on-line to atomic absorption spectrometers have been used for the indirect determination of surfactants, both cationic and anionic, in ~aters,".~5and of amphetaminesz6 and bromazepam27 in (9)LeBelle, M. J.; Callahan, S. A.; Latham, D. J.; Lauriault, G. Analyst 1988,113,1213-1215. (10)Lurie, I. S.LC-GC 1988,6,1066-1067. (11)Zeng, J. Yaoxue Xuebao 1988,23,767-772. (12)Curini, R.;Zamponi, S.; D'Aecenzo, F.; De Angelis Curtis, S.; Marino, A.; Dezzi, A. Thermochim. Acta 1989,153,ll-26. (13)Chichuev, Yu. A. Farmatsiya (Moscow) 1984,33,70-72. (14)Belikov, V. G.; Karpenko, V. A.; Stepanyuk, S. N. Farmatsiya (Moscow) 1984,33,76-78. (15)Hernhdez, A.; Gutierrez, P.;Thomas, J. Farmaco,Ed. R a t . 1986, 41,300-306. (16)Sane, R.T.; Malkar, V. B.; Nayak, V. G.; Sapre, D. S.; Benawalikar, V. J. Indian Drugs 1984,22,16-19. (17)Mirzaeva, Kh. A.;Ivanova, N. I. Zh.Anal. Khim. 1984,39,16911696. (18)Hasaan, S.S. M. Organic Analysis Using Atomic Absorption Spectrometry; Ellis Horwood: Chichester, U.K., 1984. (19)Minami, Y.; Mitsui, T.; Fujimura, Y.Bunseki Kagaku I981,30, 811-813. (20)Nerin, C.; Garnica, A.; Cacho, J. Anal. Chem. 1986,57,34-38. (21)Nerin, C.; Gamica, A,; Cacho, J. Anal. Chem. 1986,58,2617-2821. (22)Kir, S.;Temizer, A. J.Anal. At. Spectrom. 1989,4, 657-659. (23)Travnikoff, B. Anal. Chem. 1983,55,795-796. (24)Gallego, M.; Silva, M.; Valchcel, M. Anal. Chem.1986,58,22652269. 0 1992 American Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14. JULY 15, 1992
pharmaceuticals and biological fluids. The aim of this work was to apply different previously unused reagents (CO(SCN)~~-, B&-, Ni(SCN)a2-,Fe(SCN)& and Reinecke salt), for the indirect determination of cocaine by atomic absorption spectrometry using a continuousextraction system for simplicity and reduced human participation. EXPERIMENTAL SECTION
Figure 1. Scheme of the contlnuous formation and extraction of ion paks between cocelne and inorganic complexes: SS, solvent segmenter; PS, phase separator; I, injector; r, restrlctor for the aqueous phase; w, waste.
Instrumentation. A Perkin-Elmer 380 atomic absorption spectrometer equipped with a bismuth, chromium,nickel, cobalt, or iron hollow-cathode lamp as required was used. The instrument was operated and the air-acetylene flame was adjusted according to the manufacturer's recommendations. The spectrometer output was connected to a radiometer REC-80 Servograph recorder. The flow system comprised a peristaltic pump (Gilson Minipuls-2), an injection valve (Rheodine 5041),an A10T solvent segmenter and an A-4T-shaped glass separator (Bifok) housing an internal Teflon tube, and displacement bottles for pumping 1,2-dichloroethane. Poly(viny1chloride) pumping tubes and Teflon tubing for the coils were also used. Reagents and Chemicals. 1. Dragendorf Reagent. A standard Bi(NO&.SH20 solution was prepared by dissolving 5 g in 10 mL of HN03 and adding distilled water to 100 mL. A standard KI solution was made by dissolving 50 g of KI in 100 mL of water. The carrier solution was made by taking 0.4 mL of standard Bi(II1) solution and adding 6 mL of standard KI solution and 10 mL 0.1 N HCl to give 100 mL of 4 X lo4 M tetraiodobismuthate(III),BiL-, at pH 2. 2. Tetrakis(thiocyanato)cobalt(ZnReagent. A 1 M standard tetrakis(thiocyanato)cobalt(II) solution was prepared by dissolving 29.1 g of Co(N03)2.6H20and 62 g of NKSCN in 100 mL of water. The carrier solution was prepared by diluting 50 mL of the 1 M cobaltate solution to 100 mL with water (pH 4.5). 3. Tetrakis(thiocyanato)nickel(In Reagent. A nickel(I1) tetrathiocyanatecarrier solution was prepared by dissolving 34.9 g of Ni(N0&6HzO and 73 g of NfiSCN in distilled water to obtain 100 mL of 1.2 M complex (pH 4). 4. Hexakis(thiocyanato)iron(ZZI) Solution. A 0.05 M carrier Fe(SCN),+- solution was prepared by dissolving 2.1 g of Fe(NO3)&H@ and 9.2 g of NKSCN in 100 mL of water (pH 1.8). 5. Reinecke Salt (Ammonium Tetrakis(thiocyanato)diammechromate, CJ&$XV&. A 1 % (w/v) Reinecke salt (Sigma) carrier solution was made in distilled water. 6. Alkaloids. A stock solution of cocaine hydrochloride (Sigma)containing 1 g/L in distilled water was made and stored at 0-4 "C in PVC containers. Aqueous solutions (10 g/L) of the following alkaloids were also used sparteine sulfate, atropine sulfate, pilocarpine hydrochloride,amylocainehydrochloride,procaine hydrochloride, ephedrine hydrochloride, lidocaine hydrochloride. Papaverine hydrochloride, codeine, and bromhexine hydrochloride were dissolved in (1/3) ethanol/water, and stricnine was dissolved in ethanol. Procedure. The manifold used for ion pair (or Reineckate) formation and simultaneous extraction is depicted in Figure 1. First, a cocaine sample of 5-10 mL at pH 1.0 or 3.0 was continuously pumped into the system and mixed with the different carriers. The ion pairs were formed in the coil (70 cm long) and then extracted into 1,2-dichloroethane from a displacement bottle. A fraction of the extract, controlled through another displacement bottle, was isolated in the phase separator (T type, glass-Teflon). The restrictor was chosen in such a way that virtually complete phase separation was obtained. The indirect determination of cocaine was accomplished by injecting microvolumes of the organic extract via injector I into a water stream that was directly aspirated by the nebulizer. A blank measurement was required in a l l instances. The heights of the (25)Madnez-Jimhez, P.;Gallego,M.; Valdrcel, M. Anal. Chim.Acta 1988,215,233-240. (26) Montero,R.; Gallego, M.; Valchcel, M. Anal. Chim. Acta 1991, 252,a3-aa.
(27) Santelli, R. E.; Gallego, M.;Valchcel, M. Talanta 1991,38,12411245.
different peaks (sample and blank) thus obtained were proportional to the cocaine concentration in the sample.
RESULTS AND DISCUSSION The formation of the ion pairs between pure cocaine and the MLnx-complexes (M= Co, Ni, Fe, Cr, or Bi) occurs via the nitrogen atom. The nitrogen of cocaine is protonated in acidic media, which significantly facilitates formation of the ion pair. The mechanism proposed for several alkaloids20.22 can be extended to cocaine according t o the following equation: xcocaine'
+ ML,"-
-
(cocaine),(ML,)
Prior to selecting 1,2-dichloroethane as extractant, methyl isobutyl ketone and chloroform were also tested. With MIBK the efficiency of extraction is negligible. On the other hand, slightly polar solvents such as CHC4 or 1,2-dichloroethane extracted every ion pair fully. 1,2-Dichloroethane was chosen because it produced smaller amounts of HCl in the flame. However, one advantage of the continuous-extractionsyatem used in this work is that the volume to be introduced into the nebulizer is minimal. Five reagent carriers were studied; however, in the preliminary study, the Ni(SCN)d2- method proved to be scarcely sensitive to cocaine. Therefore, the method was not optimized for this ion pair and only a calibration graph was run for comparison with the other ion pairs. Chemical Variables. The variables influencing the system performance were optimized by the univariate method. The concentrations of cocaine used in this study were as follows: 10 pg/mL for BiL-, 20 pg/mL for Co(SCN)r2-,and 50 pg/mL for all other ion pairs. The optimum conditions for the determination of cocaine for each method, the carrier and sample pH, and the concentration of reagent carrier were established as follows. First, with the concentration and pH of the reagent carrier constant, the pH of the sample and the distilled water blank was varied between 0.3 and 8.0. Then, once the optimal sample pH range had been selected, that of the reagent carrier was changed between 0.3 and 7.0. The optimal pH values found are given in Table I and Figure 2. Iodide forms a series of complexes with bismuth whose stoichiometry is a function of the Bi/I- ratio. In order to use Dragendorfreagent solutions, such a ratio was also optimized. Figure 3A shows the influence of the iodide concentration on the ion pair extraction at a constant concentration of bismuth. At the Bi concentration used, lW3 M, an iodide concentration of 0.3-0.5 M, which corresponds to a ratio of 300-500, was required to obtain Bi14-. At lower concentrations, the species BiL- was not prevalent, whereas at higher concentrations, others species such as BiIs2-and Bib3- were prevalent. Thus, a I-/Bi3+ratio of 450 was chosen. The concentration of BiLwas varied between loT4and M. As can be seen in Figure 3B, the ion pair was optimally extractad a t a concentration of 4 X 10-4 M, above which the extraction efficiencydecreased as a result of precipitation of the ion pair being favored and
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, lQQ2 1511
Table I. Experimental Conditions for Determination of Cocaine BibCo(SCN)r2B A A= Bb 3.0 2-4.75 3.0 sample pH 1.5-5.4 4.5 2.7-6.0 2.0 carrier pH 1-2.5 conc of complex, M 4 x 10-4 4 x 10-4 0.4-0.6 0.5 150 150-200 240 injected volume, p L 200-300 300 240-350 300 extraction coil length, cm 120-350 a
B 1.0 1.8 0.05 150 300
Reinecke salt B
A
0.3-2.5 2.8-6.2 0.02SC 100-150 130-430
1.0 4.7 0.02SE 100 300
A optimum range. B: chosen value. % (w/v).
Blli 0.200 0
2
a
m
IL
0
m
m
a o.ioc
I
I
I
I
1
2
3
4
I
5
T
6
PH
Influence of the pH on the absorbance of the cocaine solution (thecarrier pH is kept constant). Forthecocaine concentration used in each case, see Chemical Variables section. Flgure 2.
I w
u 0.100
z
a
m
IL
0
Ln
I
\
0.050
0 3 0 5 07 0 9
2 9 M KI
Influence of the KI concentration on the formation of the cocaine+-Bi,- ion pair (the bismuth concentrationis kept constant at lo3 M). (B) Influence of the Bi1,- concentration on the determination of cocalne (the I-/Bi3+ratio is kept constant at 450). Flgwe 9. (A)
the time of contact with the organic phase in the extraction coil being too short for complete dissolution. Iron forms a series of complexes with thiocyanate whose stoichiometry also depends of the SCN-/Fe ratio. Therefore, the optimal ratio resulting in maximal extraction of cocaine was also determined. For this purpose, the concentration of iron was maintained at 0.05 M while that of SCN-was changed between 0.6 and 2.2 M. The ion pair extraction was optimal at a concentration of 1M or higher. A thyocianate/iron ratio of 24 was chosen for further experiments. The concentration of Fe(SCN)e3-was varied between 0.006 and 0.06 M while the cocaine concentration was kept constant at 50 pg/mL. The absorbance obtained was negligible up to 0.04 M. Above 0.05 M, the signal increased because of the greater extraction of cocaine, so this concentration was selected for subsequent use. The influence of the concentration of Co(SCN)d2-and Reinecke salt on the formation of the ion pairs is shown in Table I, as are the values selected in both instances. The stability of the five reagent carriers was determined by continuously I
Fe(SCN)s3A 0.3-1.0 1.8-2.8 0.05-0.06 150-240 190-350
extracting a known amount of cocaine under the optimal conditions and comparing the atomic absorption signal measured at regular intervals with those of other ion pairs obtained in the same way but with other fresh carrier solutions. The five carrier solutions assayed were stable for at least 1 week. Under the optimal conditions, the absorbance of the blank ranged between -0.020 and 0.040, so blank extraction was required in all five cases. Influence of Flow Injection Variables. The effect of the variables related to the formation of the ion pairs and extraction process, the dimensions and geometry of the reaction coil and extraction coil, and the injected volume were investigated. The tube length between the point of mixing of sample and carrier and the solvent segmentor (reaction coil) had no effect on the signal above 70 cm (0.5-mm i.d.) with any of the ion pairs. At smaller reactor lengths, the sample reagent residence time was too short for the reaction to complete. The extraction coil length was varied from 50 to 450 cm (0.5." i.d.). The optimal interval for each ion pair is listed in Table I. In all cases, extraction was complete for ca. 120 or 240 cm. Above 350 cm, the atomic signal decreased slightly. The extracted sample volume that was injected into the water line exerted a significant effect on the absorbance. Thus, the signal increased with increasing injected volume up to 100 or 200 pL, for Reinecke and Dragendorf varianta, respectively. We chose smaller volumes, in order to avoid the toxic effect of vapors, particularly those of HCl and phosgene. The optimal interval and chosen values of these variables are given in Table I. The effect of the flow rate of the aqueous and organicphase was also studied. The absorbance increased with increasing aqueous/organicphase flow rate ratio. However, ratios higher than 3 had a substantial adverse effect on the performance of the T-shaped separator, which yielded irreproducible measurements as a result of the aqueous phase reaching the flame. We chose a sample flow rate of 1.8 mL/min and an organic phase flow rate of 0.8mL/min for all ion pairs assayed, taking into account the mutual influence of three factors: reproducibility, concentration ratio, and sampling frequency. In all cases, the carrier flow rate was kept at 0.33 times the sample flow rate in order to avoid excessive dilution of the continuously pumped sample. Experimenta involving a membrane phase separator (with Fluoropore membranes) described elsewereZ4were unsatisfactory as the membranes were rapidly damaged by the solvent used (1,2-dichloroethane), so, we chose to use a T-shaped glass-Teflon separator instead. Determination of Cocaine. By using the manifold depicted in Figure 1and the values of the variables given in Table I, several linear calibration graphs were run for cocaine with the differenta reagenta carriers. The features of the calibration graphs (absorbance vs rg/mL) run for cocaine by using the five ion pairs are summarized in Table 11. As can be observed, the sensitivity obtained with the Dragendorf reagent was the highest, while that provided by Ni(SCN)P was the lowest.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, lS92
Table 11. Features of the Calibration Graphs for the Determination of Cocaine method BibCo(SCN)42Reinecke Fe(SCN)s3Ni(SCN)42a
A A A A A
regression eq = 2.25 x io-2x+ 2 x 10-3 = 5.8 x 10-3x + 2 x 10-3 = 2.7 x 10-3x - 2 x 10-3 = 1.5 x 10-3x- 1 x 10-3 = 10-4x 3 x 10-3
+
corr coeff (n = 8) 0.999 0.999 0.999 0.999 0.997
range W m L ) 1-15 3-30 10-100 10-125 200-2000
detn limit (pg/mL) 0.2 1.1 3.1 5 25
RSD (%) 2.6 4.3 4.0 3.2 4.6
A, absorbance; X in pg/mL.
Table 111. Tolerated Limits (pg/mL) for alkaloids Interfering with the Determination of Cocaine by Different Methods. alkaloids
BiL-
ephedrine procaine pilocarpine codeine lidocaine sparteine stricnine atropine papaverine bromhexine amylocaine
>lo0 5 12 1 3 2 4 2 2 2
