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Joseph
Almog
KEYWORDS: Thin Layer Chromatography, TLC, Spray Reagents, Ninhydrin, Illicit Drugs Introduction Since the discovery by Dutt and Teo1 that spraying thin layer chromatographic (TLC) plates bearing drug spots with ninhydrin produces a variety of colors that can distinguish between many drugs, this reagent has been intensively used in this laboratory. The colors that are produced with ninhydrin, when correlated with the specific migration values (Rf) for each spot on specific TLC plates and using select solvent systems, greatly enhance the specificity of TLC for various drugs. In forensic laboratories, the main use of ninhydrin as a spray reagent has been for detection of fingerprints, especially on porous surfaces such as paper and cardboard.2-4 However, despite its great utility, research has continued to develop even more sensitive or selective reagents. Over the last two decades a significant number of ninhydrin analogous and similar, electron deficient compounds have been synthesized and evaluated as fingerprint reagents. Some of these new reagents have displayed superior properties versus ninhydrin in their sensitivity to amino acids and latent fingerprints, particularly in the fluorescence mode.2-7 The aim of the present study was to evaluate some of these new fingerprint detection reagents for drug detection on TLC plates. The development of new, more intense, or fluorescent colors for various drugs would increase the overall specificity and sensitivity of drug-screening TLC. Such reagents could also discriminate between drugs that produce the same color with ninhydrin. Experimental Drugs The controlled substances examined in this study included the following pharmaceutical and illicit drugs: Cocaine HCl and morphine HCl (Merck, Germany), diazepam, flunitrazepam, codeine phosphate, and methadone HCl (Teva, Israel), lysergic acid diethylamide (LSD) (Sigma, Israel), amphetamine (Assia Chem Laboratory, Israel), heroin base, opium, and 3,4 methylenedioxymethamphetamine (MDMA) HCl (from DIFS case files), and methamphetamine, 3,4-methylenedioxyamphetamine (MDA) HCl, and 3,4-methylenedioxyethylamphetamine (MDEA) HCl (synthesized at DIFS). Similar aliquots (same concentration) of each drug were deposited on TLC plates for comparison. Imaging Reagents Twenty-four potential imaging reagents were tested (see Table 1, for names, sources, and structural formulas). Like ninhydrin, all the compounds that were studied are molecules with electron-deficient cores. Also like ninhydrin, most of them possess the indane-dione skeleton; the remainder have quininoid or cyclobutenedione type structures. All reagents were dissolved in 95% ethanol to reach testing concentrations from 0.5 - 10%. TLC, Elution Solvents, and Spray Reagents TLC was carried out on standard silica gel plates (10 x 20 cm) containing a fluorescent indicator (254 nm) on aluminum support (Macherey-Nagel, Germany). A dioxane:xylenes:ethanol:ammonia (40:30:5:5) solvent mixture was used as the mobile phase in the developing tank. After the solvent elution, the plates were dried in an oven at 120°C for 3 - 4 minutes, then cooled to room temperature. The plates were then sprayed with the reagent solution, then heated again for 10 minutes. The colors of the spots as well as background interferences were immediately recorded and photographed. Methods 1st Stage At the first evaluation stage, all 24 reagents were tested on TLC plates against five basic drugs: Heroin, cocaine, MDMA, diazepam, and flunitrazepam. At this stage the plates were not processed in the solvent system; rather, the drugs were spotted on the plates and the spots were treated with the reagents (5 - 10% w/v) via direct application using a pipette or cotton swab. When a color reaction was noted using these initial reagent concentrations, a lower concentration solution (0.5%) of the target reagent was attempted. 2nd Stage At the second evaluation stage, only those reagents that had produced colored spots with at least one drug were investigated. At this stage, the selected color reagents were evaluated for all 14 of the above listed target drugs. In addition, in the second stage, each TLC plate bearing the drug spots was eluted using above specified the TLC solvent system, then sprayed with the reagent solution, then heated to 120°C. The results were compared versus those obtained by the ninhydrin solution routinely used in the laboratory. 3rd Stage In the third stage, experimental parameters were optimized for the successful color reagents identified at the second stage. The principal optimization parameters were reagent concentration and color development temperature. Ethanolic solutions of six concentrations (0.5, 1, 2, 3, 4 and 5% v/w) were prepared for each one of the successful reagents. Each successful reagent at each given concentration was tested against each drug that it had displayed a colored spot with in Stage 2, and after elution evaluated at different development temperatures (80, 100, 120, 130, 140, 160 and 200°C). It was noted that while high reagent concentrations produced more intense colors, they also usually resulted in development of significant background colorations. High temperatures had a similar effect. Colors developed and background interferences were recorded for each set of experiments. Table 1. Names, Sources, and Structural Formulas for Imaging Reagents
Five of the twenty-four reagents examined at the first evaluation stage yielded a color reaction with at least one drug (see Table 2). These were reagents E (mixture of 5-methoxy-1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene) malononitrile and (2Z)-2-(5-methoxy-1,3-dioxo-1H-inden-2(3H)-ylidene) propanenitrile, F (3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile, H (4-chloro-7-nitro-2,1,3-benzoxadiazole), K (5,6-dimethoxyninhydrin), and O (5-methoxyninhydrin). Seven of the twenty-four compounds gave no visible reaction, and the remainder were rejected because of the development of intense background coloration. At the second stage, the five preliminarily successful reagents listed above were evaluated for all 14 drugs. The results are summarized in Table 3, and are detailed below:
Of the five above reagents, H and K showed better performance versus the other three, and were therefore selected for further investigation. Optimization trials were carried out with both H and K at various concentrations and color development temperatures. The optimized parameters for H are: A 3% solution (w/v) with color development at 120°C. Under these conditions, opiates (heroin, morphine) can also be detected. The optimized conditions for K are: A 0.5-1% solution (w/v) with color development at 120°C. Under these conditions, only amphetamines show strong color reactions. It is noted for comparison that ninhydrin is typically utilized as a 10% solution. Conclusions 4-Chloro-7-nitro-2,1,3-benzoxadiazole and 4,6-dimethoxyninhydrin both show good potential as spray reagents for drugs on chromatographic plates. Both reagents show some advantage over ninhydrin in their reactivity, developing more intense colors at lower reagent concentrations. Furthermore, 5,6-dimethoxyninhydrin also produces two different colors with different amphetamines: Purple spots are formed by methamphetamine, MDMA, and MDEA, and milky yellow spots are formed by amphetamine and MDA. A mechanistic study of these color formation reaction may lead to a rational design of even better reagents of this family. Table 2. Results Correlated Against Structures.
Acknowledgments: The authors are indebted to Dr. Antonio A. Cantu, Chief Chemist, US Secret Service, to Professor Robert C. West of the Chemistry Department, University of Wisconsin, Madison, and to Professor E. Roland Menzel, Director of the Center for Forensic Studies, Texas Tech University, Lubbock, for kindly providing them with some of the compounds for testing. The authors also gratefully acknowledge Ms. Lital Cohen for her technical assistance. References
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