for this purpose because R is limited in use to the description of two-ptak separations, so t h a t assumptions such as the constancy of‘ R in a multi-peak separation are necessary. To establish further the value of Plnf as a criterion, a more systematic investigation analogous for example to the work of Grushka on chromatographic peak capacity and the factors influencing it, would be necessary.
ACKNOWLEDGMENT The authors thank H. ~~i~~~and G. ~
~for
i
ableadvice, Received for review January 30, 1973. Accepted June 25, 1973. Work was supported by the “Fonds voor kollektief en fundamenteel onderzoek.”
Thin Layer Chromatographic-Spectrophotofluorometric Analysis of Amphetamine and Amphetamine Analogs after Reaction with 4-Chloro-7-Nitrobenzo-2,1,3-Oxadiazole Fransois Van Hoof and Aubin Heyndrickx D e p a r t m e n t of Toxicology, S t a t e U n i v e r s i t y of Ghent, Ghent,
Belgium
Amphetamine and many other sympathomimetic drugs NBD-Cl was first used by Ghosh and Whitehouse as a are actually of great importance as drugs of abuse, appelabeling agent for amino acids (6). NBD-Cl was applied in tite depressors, and stimulants used by men in sports. former studies for identification of phenylethylamines Several methods have been developed to identify these (7-9) and for quantitative determination of carbamate compounds by spectrophotometry, thin layer chromatoginsecticides ( I O , 11) and dithiocarbamate fungicides (12). raphy, and gas chromatography. Most existing thin layer EXPERIMENTAL chromatographic detection methods suffer from a lack of sensitivity because for most compounds, several microReagents. Pure NBD-CI was obtained from Serva, Heidelberg, Germany. Stock solutions, 1%, were prepared in methyl isobutyl grams are needed for visualization. ketone and methyl n-amyl ketone. Nanogram amounts of sympathomimetic drugs can be The products investigated are: amphetamine phosphate (1detected by gas chromatography only if derivative formaphenyl-2-aminopropane phosphate); pervitine hydrochloride (1tion is combined with electron capture detectors. phenyl-2-methylaminopropane hydrochloride); ethylamphetamAn excellent review of existing methods, for analysis of ine hydrochloride (1-phenyl-2-ethylaminopropanehydrochloride); amphetamine analogs has recently been published ( I ) . ritaline hydrochloride (2-phenyl-a-2-piperidineacetic acid methylester hydrochloride):, lidepran hydrochloride (a-phenyl-2-piperiMiles and Schenk made use of the natural fluorescence of dine methanolacetate hydrochloride); preludine hydrochloride phenylethylamines to assay these compounds in pharma(3-methyl-2-phenylmorpholine hydrochloride); sympatol (p-hyceutical preparations (2). Few attempts have been made droxy-a-(methy1amino)methyl benzylalcohol tartrat,e); effortil to form fluorescent derivatives of amphetamine and relat(a-(ethy1amino)methyl-m-hydroxybenzylalcohol hydrochloride); ed substances (3-5). chlorphentermine hydrochloride (4-chloro-a,a-dimethylphenethylIn our work, 4-chloro-7-nitrobenzo-2,1,3-oxadiazole amine hydrochloride); vasculat (a-(buty1amino)methyl-p-hydroxybenzylalcohol sulfate); P-phenylethylamine hydrochloride (NBD-C1) is used to form fluorescent derivatives. The (1-amino-2-phenyl-ethane hydrochloride); ephedrine hydroreaction between amphetamine and NBD-Cl is shown in chloride (a-1-(methy1amino)ethyl benzylalcohol hydrochloride); Figure 1. hyheptaminol hydrochloride (6-amino-2-methyl-2-heptaminol drochloride); methoxyphenamine hydrochloride ( a - (1-amino NO, ethyl)-2,5 dimethoxy benzylalcohol hydrochloride). Stock solution of all compounds were prepared in distilled water at a concentration of 10 mg base/ml. All solvents used for chromatography were analytical reagent grade quality. A 1M H3B03-NaCI-NazC03 buffer was prepared by adding 370 ml of a 1M NaZC03 solution to 630 ml of a 1M HSBOs--NaCl solution. pH of the buffer solution is 9. Chlorotrimethylsilane was obtained from Merck, Darmstadt, Germany. Test tuhes used for W. Rusiecki and J. Brezenzenski, Diss. Pharm. Pharrnacol., 19. 315
NH I
I
(0) CH,-CHI
CH~
I
Figure 1. Reaction scheme for coupling of amphetamine and NBD-CI (1) K. K. Kaistha. J. Pharm. Sci., 61, 655 (1972). (2) C I . Miles and G. H. Schenk. Anal. Chem., 45, 130 (1973). (3) J. T. Stewart and D. M . Lotti, J . Pharm. Sci., 60, 461 (1971).
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(1967). N. Seller and M. Weichmann, Hoppe-Seyler’s Z. Physiol. Chern., 337, 229 (1964). P. B. Ghosh and M. W. Whitehouse, Biochem. J . . 108, 155 (1968). J. Monforte, R . J. Barth, and I . Sunshine, Clin. Chem.. 18, 11, 1329 (1972). J. Reisch, H. J. Kornrnert. H. Alfes, and H . Mollman, Fresenius’ 2. Anal. Chem., 245, 56 ( 1969). D. Clasing. H. Alfes. H. Mollman, and J. Reisch, Z. Klin. Chem. K l h . B m h e m . , 7, 648 (1969). J. F . Lawrence and R . W. Frei, Anal. Chem., 44, 2046 (1972) R . W. Frei and J. F. Lawrence, J . Ass. Offic. Anal. Chem., 55, 1259-64 (1972). F . Van Hoof and A . Heyndrckx. Med. F a k . Landbouwwet., in press (1973).
~
-
~~
Table 11. M a x i m a in Fluorescence Spectra of NBD-Derivatives
Table I. RF V a l u e s of S y m p a t h o m i m e t i c D r u g s in Different S o l v e n t Systems4 Compound
Amphetamine Pervitine Methoxyphenamine Ethylamphetamine Ritaline Lidepran Preludine Sympatol Effortil Chlorphentermine Vasculat p-Phenylethylamine Ephedrine Heptaminol
A
0.70 0.65 0.76 0.73 0.72 0.10 0.73 0.00 0 .oo 0.78 0 .oo 0.76 0.20 0 .oo
B
0.53 0.47 0.41 0.48 0.48 0.34 0.42 0.13 0.23 0.69 0.37 0.72 0.42 0.16
C
0.65 0.42 0.54 0.42 0.29 0.15 0.23 0.05 0.07 0.29 0.13 .O .30 0.07 0.15
D
Color
0.52 Yellow 0.34 Orange 0.62 Yellow 0 . 6 2 Pink 0 . 5 4 Pink 0 . 2 9 Pink 0 . 4 5 Orange 0 . 0 6 Orange 0 . 1 2 Orange 0.17 Red 0 . 0 5 Orange 0 . 5 0 Yellow 0 . 1 9 Orange 0.10 Yellow
Compound
Amphetamine Pervitine Methoxyphenamine Ethylamphetamine Ritaline Lidepran Preludine Sympatol Effortil Chlorphentermine L'asculat p-Phenylethylamine Ephedrine Heptaminol
a A, 1,2-dichloroethane; B, ethyl acetate-cyclohexane (3:2); C, ethyl acetate-cyclohexane (2 :3): and D, chloroform-tetrahydrofurane (98:Z).
evaporation of ether layers were put overnight in a 5% chlorotrimethylsilane solution in toluene. They were rinsed afterward with methanol and dried at 70 "C. Reaction Procedure. A 10-gl sample of each stock solution is evaporated in a test tube at 80 "C on a warm-water bath under a smooth stream of nitrogen. Then 0.2 ml of a 0.1M NaHC03 solution is added. The mixture is mechanically stirred. Next, 0.2 ml of a 1% solution of NBD-C1 in MIBK is applied on top of the bicarbonate solution. The test tube is stoppered and heated at 80 "C during 30 minutes on a warm-water bath. After cooling. a 10-jtl aliquot of the upper phase was used for chromatography. Chromatography. Thin layer plates (20 cm X 20 cm) coated with a 0.25-mm thick layer of Silica gel GF 254 (Merck, Darmstadt, Germany) were used throughout the study. Solvent systems employed were: 1,2-dichloroethane ( A ) ; ethyl acetate-cyclohexane (3:2) (B); ethyl acetate-cyclohexane (2:3) (C); chloroform-tetrahydrofurane (98:2) (D). Spots were visualized under a Camag TL 900 Universal UV lamp at 350 nm. Instrumental Analysis. All fluorescence measurements were made in situ with an Aminco Bowman spectrophotofluorometer equipped with a thin film scanner. Slit width was kept constant at 0.5 mm. Fluorescence spectra were recorded on an MFE 1620855 x-y recorder. For quantitative work, a Hitachi 159 1-mV recorder was used. An attenuator was placed between photometer exit (50 mV full scale) and the Hitachi 159 recorder entrance (1 mV). For quantitative determinations with NBD-amphetamine, the excitation monochromator was set at 482 nm and the emission monochromator at 523 nm, scan program 2 was used for the thin film scanner; the recorder chart speed was 10 mm/min. Peak heights were plotted against quantities of amphetamine applied on thin layer plates in order to obtain calibration curves. Extraction of Urine Samples. Three 5-ml urine samples were fortified with 5 jtg of amphetamine giving an amphetamine concentration of 1 ppm. A few drops of 2N NaOH were added to obtain an alkaline medium. Extraction was done by mechanically shaking the two 20-ml portions of diethyl ether. The combined ether layers were washed twice with 5 ml of O.OO5N NaOH. The ether phase was dried over anhydrous NazSOa. Complete evaporation of the ether layer was done in a glass, silanized test tube, containing 0.1 ml of 0.1N HC1, on a warmwater bath at 35 "C under a smooth stream of nitrogen. To the residue, 0.2 mi of 0.1M NaHC03 was added and the reaction was carried out as described above. Extraction of Blood Samples. To three 1.0-ml blood samples fortified with 100 ng of amphetamine, 5 ml of a 1M HsBOs-NaCl -NazCOs buffer was added. Extraction was done by shaking twice with 10 ml of ether for 10 minutes. Blood and ether layers were separated by centrifuging for 10 minutes at 2500 rpm. The ether layers were taken from the blood with a Pasteur pipet. The combined ether layers were dried over anhydrous NaZSOd and evaporated in a silanized glass test tube, containing 0.1 ml of 0.1N HCI, at 35 "C under a gentle stream of nitrogen. The evaporation residue was treated as described under the following Fkaction Procedure, but 100 gl of the MIBK phase was applied on a thin layer plate.
Maximum in fluorescence spectrum (in nm)
523 537 535 530 535 535 535 525 545 535 525 540 525 512
RESULTS A N D DISCUSSION Reaction Procedure. The reaction underlying the determination is the coupling of primary or secondary alkylamine functions with NBD-C1. The reaction was carried out in a two-phase (0.1M NaHC03-MIBK) system as the coupling proceeds better in organic solvents, and salts had to be converted -into free bases which are better soluble in
MIBK. The reaction was complete after heating a t 80 "C fcr 30 minutes. Longer reaction times ( u p to 60 minutes) and higher reaction temperatures (up to 100 "C) did not yield more intense fluorescence for amphetamine. For reaction a t 100 "C, a 1% solution of NBD-Cl in methyl n-amyl ketone was used instead of 1% NBD-C1 in MIBK to prevent losses of organic solvent by evaporation. Chromatography. RF values and colors of all compounds are summarized in Table I. RF values for amphetamine in different solvent systems were: 0.70 (A), 0.53 (B), 0.65 ( C ) , and 0.52 (D). Systems B and C offer a better separation t h a n A and D. These solvents should be preferred if only qualitative work has to be done. Some substances with high RF values in systems B and C can be covered by a n excess of NBD-C1, which in all systems concentrates at elevated RF values. For quantitative work, system A was preferred as spots were kept very narrow during chromatography. These spots gave very narrow peaks from which quantitative d a t a could be derived by measuring peak heights, avoiding planimetry. Nevertheless system D yields compact spots for most compounds and can be used for quantitative work as well. Primary amines gave a yellow color in daylight; secondary amines gave orange or pink spots. All compounds, except chlorphentermine, could be visualized under a Camag TL 900 Universal UV lamp a t 350 nm down to 1 ng/ spot. Fluorescence Phenomena. All fluorescent derivatives have a maximum in their fluorescence spectrum between 510 and 545 nm (Table 11). Excitation spectrum maxima were observed a t 482 n m for all compounds, except lidepran (484 n m ) . The similarity in fluorescence spectra makes it impossible to identify a n unknown sympathomimetic drug through its NBD derivative in MIBK solution. Moreover, we found t h a t a 1% NBD-C1 solution in MIBK yields a n intense fluorescence when excitated a t 482 nm. The maximum in t h e NBD-C1 fluorescence spectrum was found at 518 nm. Quantitative Analysis. The precision of the method was controlled by applying 10, 20, 30, 50, and 100-ng
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, FEBRUARY 1974
287
T a b l e 111. Precision S t u d y of A m p h e t a m i n e Quantity amphetamine applied, ng
10 20
30 50 100
Av re1 std dev, %
9.5 4.5 7.4 6.3 5 .O
phetamine was recovered from urine samples. Recovery from blood was 70 f 3%. In order to obtain satisfactory recoveries, addition of 0.1 ml of 0.1N HC1 before evaporation was necessary to prevent loss of amphetamine by volatilization. Coating of glass test tubes with chlorotrimethylsilane improved recoveries, preventing adherence of amphetamine to the glass surface.
CONCLUSIONS amounts of amphetamine on thin layer plates and measuring fluorescence intensities after chromatography in 1,2-dichlomethane. Standard deviations obtained from a t least six measurements at each concentration are summarized in Table III. With each quantitative analysis, 10-ng, 50-ng, and 100-ng amphetamine standards underwent the reaction procedure and were applied to the thin layer plate to obtain correct calibration curves. Linear relationships between peak height and applied quantity were obtained u p to 500 ng of amphetaminel spot. No interferences from coextractives of urine or blood samples were found. Using the extraction procedure described, 83 i 6% am-
The method presented allows a fast identification of most important amphetamine analogs. Only substances lacking primary or secondary alkylamine functions (diethyl propionate) cannot be detected. No interferences from nicotine or other coextractives are encountered. The method is more sensitive than existing TLC detection methods and allows quantitative determination of therapeutic amphetamine levels on 1-ml blood samples.
ACKNOWLEDGMENT The skillful1 technical assistance of J. Pollet was greatly appreciated. Received for review May 31, 1973. Accepted September 21, 1973.
Rapid, Sensitive Gas-Liquid Chromatographic Screening Procedure for Cocaine J. W. Blake, R. S. Ray, J. S. Noonan, and P. W. Murdick Equine Research Center, Ohio State University. Columbus, Ohio 4321 0
Cocaine is an alkaloid which is isolated from t h e leaves of the plant Erythroxylon cdca. The drug may also be prepared semi-synthetically from the acid, ecgonine. Based upon the observations of Nieman, Koller, and Freud, circa 1884, the use of cocaine as a n anesthetic spread rapidly through the medical community. The drug served as a local anesthetic until supplanted to a great extent by Einhorn’s introduction of procaine in 1905. The addictive nature of cocaine was recognized early (I). Cocaine causes feelings of elation in the user. Chronic abuse of the drug may cause mental impairment, loss of appetite, and a tendency to withdraw from society. The pure drug is extensively metabolized in the body. The major metabolite of cocaine is benzoylecgonine (2). Presently used screening tests are for the most part chromatographic techniques. Thin layer chromatography and gas-liquid chromatography are in common use (3, 4 ) . The technique of analysis to be described uses an 0acylated derivative of the drug and employs an electron capture detector to greatly increase the sensitivity of the gas-liquid chromatographic method. (1) (2)
“Drill’s Pharmacology in Medicine.” 4th ed.. J. R . DiPalma. Ed. McGraw-Hill. NewYork, N.Y., 1971, p 190. F. Fish and W. C C. Wilson, J . Pharm. Pharmacol., 21, 135s
(1969). (3) E. G . C. (4)
Clarke, “Isolation and Identification of Drugs.” The Pharmaceutical Press, London, 1969, p 267. Perkin-Elmer Corporation, “Clinical Chemistry Application Study 41 ,” p 6, July 1971.
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* ANALYTICAL CHEMISTRY, VOL.
EXPERIMENTAL Apparatus. The gas chromatograph used was a Beckman GC-5 equipped with a Beckman electron capture detector. The column was a four-feet, 4-mm i.d. glass column packed with 3% OV-1 on sO/lOO mesh Chromasorb GHP provided by Supelco, Inc. Extracts, reduction, derivatization, and washes were performed in Kimble glass culture tubes, 125 mm in length, employing screwon polypropylene caps. Extractions and washes were made by inverting the capped tubes on a “Fbtorack” sold by Fisher Scientific Company. “Dispo” micropipets of 50-pl capacity, purchased from Scientific Products Company, were used to transfer the reducing and derivatizing reagents. The centrifuge used was an International Model HN. Reagents. The sodium tetraborate decahydrate powder was procured from the J. T. Baker Co. Pesticide quality cyclohexane and heptafluorobutyric anhydride were purchased from Matheson, Coleman, and Bell. Pentafluoropropionic anhydride, which provided a better derivative yield, was a product of Pierce Chemical Company. Lithium aluminum hydride was purchased from K and K Laboratories, Inc. The diethyl ether was B and A reagent grade (Allied Chemical). A diethyl ether solution saturated with lithium aluminum hydride provided a reducing reagent. Procedure. To 5.0 ml of aqueous solution containing cocaine was added 1 ml of a saturated sodium tetraborate solution and 2.0 ml of cyclohexane. To determine method sensitivity with urine, cocaine was added to horse urine prior to extraction. The drug was extracted into the cyclohexane phase by rotating on the “Fbtorack” at moderate speed for four minutes. Following centrifugation, the cyclohexane phase was transferred to a clean culture tube. To the cyclohexane phase in the culture tube were then added 50 p l of the LiAlHd-ether solution. Reduction was allowed to pro-
46, NO. 2, FEBRUARY 1974
