Mini thin-layer chromatography. III. A rapid and sensitive method for the estimation of amphetamine and methamphetamine

H. H. Loh, I. K. Ho, W. R. Lipscomb, T. M. Cho, C. Selewski

Research output: Contribution to journalArticlepeer-review

6 Scopus citations
Original languageEnglish (US)
Pages (from-to)289-293
Number of pages5
JournalJournal of Chromatography A
Issue number1
StatePublished - May 31 1972

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DNS-Cl has been employed successfully in the N-group determination of polypeptide chainsl2 and the method is roe-fo1.d more sensitive than SANGER'S Auoro-dinitrobenzene procedure13. SEILER AND WIECEIMANN~”h ave used TLC to separate a large number of amines as their DNS-derivatives. These authors used regular size Silica Gel plates and their studies concerned only qualitative separation of these amines. Recently several mini TLC methods for drug screening have ‘been pul~lishedlO~ lG. The utilization of mini thin-layer plates is not only more simple and rapid, but these advantages are attcainable without sacrificing sensitivity and accuracy. The use of DNS-Cl allows for the detection of picogram quantities of the DNS-amphetamine derivatives after separation on a mini polyamide plate. The highly fluorescent spots were visualized under UV light. Since d-amphetamine is converted quantitatively (92 %) to DNS-amphetamine, measurement of the fluorescent intensity by thin-layer scanner is a rapid and simple way of quantifying these drugs. The sensitivity of the DNS-amphetamine derivative is extended to the spectrofluorometer where a range of 2 ng may be detected (Fig. 2). By using a radioactive drug with high specific activity, less than 0.2 ng can easily be quantified. The use of polyamide thin-layer plates offers several advantages: (I) The diminutive pieces (3 x 3 cm) used require less than 5 min and 1.5 ml of solvent for chromatogram to develop; (2) both sides of the polyamicle thin-layer plate may be utilized for spotting; (3) a good separation with discrete spots is achieved; and (4) the plate can be re-used after washing. The qualitative analysis of the amphetamines as performed in our procedure enables one to detect picogram quantities of these clrugs in biological fluids. Since DNS-Cl reacts with primary and secondary amines, phenolic hydroxyl groups, thiols, and imidazoles, it is essential to partially purify the amphetamine either by solvent extraction (at pH 13) or by ion-eschange resin paper, in orcler to reduce the background fluorescent spots. Studies relating to the application of this procedure for the determination of other drugs are being continuecl in our laboratories, We thank Mrs. KAY WELCI-I for her help in editing and typing the manuscript and Miss JEAN CARR for making the graph. The financial support of USPHS Grant MH-19768 ancl Project 7-T, Bureau of Research, California Department of Mental Hygiene, is also gratefully acknowledged.

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