Author Institution: Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522

Day, Richard A.

Goldman, Margaret

The Ohio Journal of Science

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190 J. G. HUMPHREYS Vol. 67A TEST FOR PENICILLINS ON CHROMATOGRAMS AND IN SPOTTESTS1-2. A simple procedure for detecting small amounts of a penicillin on apaper chromatogram or in a spot test is described in this paper. A simple testhas previously been described (Day, 1957) for penicillins, including a totally syn-thetic penicillin, a-formamidopenicillanic acid, of low antibiotic activity. Sub-sequently a similar test was described by Cox and Green well (1965) for phene-thecillin. Except for the natural penicillins and some of the synthetic analogsshown to have high biological activity, which may be detected by bioassay, therehave heretofore been no sensitive tests suitable for locating penicillins on paperchromatograms.N-acylated thiazolidines, which include the penicillins, do not react with iodinein neutral solutions at an appreciable rate. However, thiazolidines which arenot N-acylated do consume approximately 9 equivalents of iodine (Scudi andWoodruff, 1949). The test found applicable for the detection of penicillins ondeveloped paper chromatograms begins by treating the paper to which the peni-cillin has been applied with ammonia. Ammonia cleaves the 0-lactam ring ofthe penicillin, producing the a-amide of the corresponding penicilloic acid. Sodiumsalts of penicillin are cleaved quantitatively by ammonia in a few minutes(Mozingo and Folkers, 1949).S CH3 NH3 S CH3RCONHCN—CH C—CH3 RCONHCH CH C—CH3I I I I I ICO—N CHCOO- CONH2 NH CHCOO~The developed paper chromatogram, to which 10-1000 micrograms of penicillinhas been applied, is sprayed with aqueous ammonia (concentrated) and then sus-pended in a chamber over concentrated aqueous ammonia for approximately onehour. After removing the strip and allowing the ammonia to escape, it and acontrol strip, untreated with ammonia but otherwise developed in an identicalmanner, are passed through dilute iodine (5-15 x 10~4 N) in acetone (freshlyprepared). As soon as the acetone has evaporated, the strips are sprayed withaqueous 1% starch - 1% potassium iodide. The penicillin appears as a whitespot or zone on a violet background on the strip treated with ammonia, but noton the control. Easily oxidized impurities also appear in identical positions onboth strips.It is possible to detect IO7 of either the penicillin or its penicilloic acid onWhatman No. 1 paper very readily. Amounts as small as 27 (3 Oxford Units ofpenicillin G) can be detected. Somewhat lower sensitivity is observed on heavierwork was supported in part by a grant from NIH(AI-04326).2Manuscript received October 5, 1966.THE OHIO JOURNAL OF SCIENCE 67(3): 190, May, 1967.No. 3 TEST FOR PENICILLINS 191paper, such as Whatman 3MM. The order of addition of the starch-potassiumiodide to the paper and iodine is not critical.—MARGARET GOLDMAN and RICHARDA. DAY, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221.REFERENCESCox, W. H. and B. E. Greenwell. 1965. Paper Chromatographic Method for DetectingPhenethicillin Contamination in Drugs. J. Pharm. Sci. 54: 1076-1077.Day, R. A. 1957. The Synthesis and Cyclization Studies of New Penicilloic Acids, Ph.D.Thesis, Massachusetts Institute of Technology.Mozingo, R. and K. Folkers. 1949. The Penilloic and Penicilloic Acids and Their Derivativesand Analogs, p. 535-656, In H. Clarke, J. R. Johnson and R. Robinson. The Chemistryof Penicillin, Princeton University Press.Scudi, J. V. and H. B. Woodruff. 1949. Assay of Penicillins p. 1026-1042. In H. Clarke,J. R. Johnson and R. Robinson. The Chemistry of Penicillin, Princeton University Press.

A Test for Penicillins on Chromatograms and in Spot Tests

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A Test for Penicillins on Chromatograms and in Spot Tests

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