50 INTERNATIONAL JOURNAL of BIOMEDICAL SCIENCE Spectrophotometric Determination of Diazepam in Pure Form, Tablets and Ampoules W. F. El-Hawary, Y. M. Issa, A. Talat Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt ABSTRACT The interaction of diazepam with picric acid (I), 3,5-dinitrobenzoic acid (II) and 2,4-dinitrobenzoic acid (III) was found to be useful for its spectrophotometric determination. The quantitation was carried out at 475, 500, and 500 nm for the reaction with (I), (II) and (III), respectively. The effect of several variables on the coloring process was studied. The proposed methods have been applied successfully for the determina- tion of diazepam in pure samples and in its pharmaceutical preparations with good accuracy and precision. The results were compared to those obtained by the pharmacopoeial methods. The linear ranges for obedi- ence of Beer’s law are up to 85.6, 180.2, and 128.6 μg/ml, Ringbom ranges are 10.0-79.0, 15.2-177.8, 17.0-83.0 μg/ml, and RSD 0.048, 0.028, and 0.026% for reaction of diazepam with I, II, and III, respectively. Keywords: diazepam; 2,4-dinitrobenzoic acid; 3,5-dinitrobenzoic acid; picric acid; spectrophotometric determination Corresponding author: Y. H. Issa, Chemistry Department, Fac- ulty of Science, Cairo University, Giza, Egypt. Tel: 00202-5676579; Fax: +5728843; E-mail: yousrymi@yahoo.com. Copyright: © 2007 W. F. El-Hawary et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Li- cense (http://creativecommons.org/licenses/by/2.5/), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. IntRoDuctIon Diazepam (C 16 H 13 ClN 2 O) [7-chloro-1,3-dihydro- 1-methyl-5-phenyl-2H-1,4-benzodiazpein], M. Wt. 284.75 [CAS (439-14-5)] is an important compound widely used therapeutically because of its relaxant, sedative, hypnotic and anticonvulsant properties. Several analytical procedures have been adapted for the assay of diazepam. They include non-aqueous titra- timetry (1, 2), ultraviolet spectrophotometry (3-5), vis- ible spectrophotometry (6-10), second order-derivative spectrophotometry (11, 12), fluorimetry (13, 14), high performance liquid chromatography HPLC (15-17), gas chromatography (18, 19), thin layer chromatography (20), polarography (21, 22), potentiometry (23, 24) and infrared assay (25). The official methods involve a non- aqueous titration of diazepam by perchloric acid in acetic anhydride medium using Nile blue as indicator (2) and HPLC (17). Most of the old colorimetric methods involve hydroly- sis of the benzodiazepine moiety to benzophenones, and thus lack specificity since this is the usual degradation pathway of benzodiazepines, and other needs solvent ex- traction before measurements. Therefore, a simple spec- tro-photometric method for determination of diazepam is needed. This is fulfilled in the present investigation by the application of Zimmermann reaction to the active methy- lene group adjacent to a carbonyl group in diazepam to produce highly absorbing s-complexes (26) upon reaction with picric acid (I), 3,5-dinitrobenzoic acid (II) and 2,4-di- nitrobenzoic acid (III), respectively. The present study de- scribes the spectrophotometric determination of diazepam in pure samples and in its pharmaceutical preparations. EXPERIMENTAL Apparatus Perkin Elmer Spectrophotometer model Lambda 1, Hanna instrument coductometer model HI8819N and ORIGINAL ARTICLE