Table 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
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 up 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-
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 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 the 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 an 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) “Drill’s Pharmacology in Medicine.” 4th ed.. J. R . DiPalma. Ed. McGraw-Hill. NewYork, N.Y., 1971, p 190. (2) F. Fish and W. C C. Wilson, J . Pharm. Pharmacol., 21, 135s (1969). (3) E. G . C. Clarke, “Isolation and Identification of Drugs.” The Pharmaceutical Press, London, 1969, p 267. (4) Perkin-Elmer Corporation, “Clinical Chemistry Application Study 41 ,” p 6, July 1971.
288
*
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” a t 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-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, FEBRUARY 1974
Table I. Elution Data for Electron Capturing Drug Derivative9 Rt
Amphetamine-PFP Amphetamine-HFB Methamphetamine-PFP Methamphetamine-H F B 2- Hydroxymethyltropine-PF P 2- Hydroxymethyltropine- H F B a GLC
HFBA
,
u2c-
I
c
W C ,
UI
I
-CWCH~OHFB I
NCH~
CHOHFB
AH
CHI
- -
I
+
0
I Coho1
derivatives
Figure 1. Reduction and acylation of cocaine to produce an
electron capturing derivative
ceed for three minutes. Then 50 fil of distilled water were added to the cyclohexane, and the mixture was shaken. Next 50 ~l of heptafluorobutyric anhydride (or alternatively pentafluoropropionic anhydride) were added to the cyclohexane phase and allowed to react 3-5 minutes at room temperature. The 2.0 ml of cyclohexane containing the reduced, derivatized moiety were washed in about 6 ml of the saturated tetraborate solution by rotating the cyclohexane and tetraborate *on the “Rotorack.” The cyclohexane phase was transferred to a clean tube from which an aliquot of derivatized drug could be taken for gas chromatographic-electron capture analysis. The derivatized drug was chromatographed isothermally a t an oven temperature of 150 “C. Employing on-column injection, the inlet temperature was 210 “C.Detector temperature was 300 “C. The elution time of the drug derivative was 2 minutes and 36 seconds a t a helium carrier gas flow of 60 cm3/min.
RESULTS AND DISCUSSION The method as presented is a screening procedure, because cocaine as such is not detected, but rather a reduced, derivatized product is detected (See Figure 1). Presently used confirmation tests are necessary. The acylated derivative of cocaine, as well as Ecylated amphetamine and methamphetamine, can be determined under the conditions described above. Relative elution times for 2-hydroxymethyl tropine, amphetamine, and methamphetamine derivatized with heptafluorobutyric and pentafluoropropionic anhydrides are shown in Table I. The gas chromatographic conditions were as noted under Procedure. The HFBA derivative of benzyl alcohol, the reduction product of the benzoyl radical, can be seen on the chromatogram at RT = 0.40 with respect to amphetamine-HFB. The derivative of cocaine noted in Figure 1, 2-hydroxymethyl tropine-HFB, has been verified by mass spectro-
0.83 1.oo 1.44 1.60 1.40 1.92
conditions noted under procedure
metric analysis (5). Also verified by mass spectrometry was the HFBA derivative of benzyl alcohol. Cocaine was added to horse urine a t concentrations of 39 pg/ml, 3.9 pg/m!, 390 ng/ml, and 39 ng/ml. Using the procedure cited, which involves a 2.5-fold concentration factor in extraction, and employing the pentafluoropropionic anhydride derivative, the sample containing 39 ng/ml cocaine in urine gave an average S / N ratio of 14:l for 1.0 pl injected. Therefore, sensitivities of 20-30 ng of drug/milliliter of sample could be achieved without concentration through evaporation procedures. As described, the procedure using the Beckman instrument provides a rapid, sensitive, and relatively specific screening procedure for cocaine, as well as amphetamine and methylamphetamine.
ACKNOWLEDGMENT The authors wish to thank George Maylin of the New York State Veterinary College, Cornel1 University, Ithaca, N.Y., for the mass spectrometric analyses of the drug derivatization products. Received for review February 14, 1973. Accepted August 15, 1973. The work was conducted under a grant from The Ohio State Racing Commission, Columbus, Ohio 43215. (5) G. Maylin. New York State Veterinary College, Cornell University, Ithaca, N.Y.. personal communication,1972.
CORRECTION Annual Subject Index Through an error on our part, two page numbers were transposed in the annual subject index of Analytical Chemistry, December 1973. The material on page 2469 should be in the “S” section where page 2477 is located. The subject matter on page 2477 belongs in the “G” section, where page 2469 is located.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, FEBRUARY 1974
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