ISSN 1061-9348, Journal of Analytical Chemistry, 2009, Vol. 64, No. 7, pp. 714–720. © Pleiades Publishing, Ltd., 2009. 714 1 Sulfanamides are N1-substituated derivates of p-aminobenzenesulfonamide (sulfanilamide) [1] and commonly used to combat a variety of bacteria [2]. Sul- famethoxazole, a derivate of sulfonamide, has been also widely used to treat the Acquired Immunodefi- ciency Syndrome (AIDS) but it can occasionally cause adverse reactions such as delayed-type hypersensitivity and hepatitis [3]. Metal complexes of aromatic/hetero- cyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared with the uncomplexed sulfonamides [4]. Several low nanomolar mitochondrial isozyme car- bonic anhydrase (CA) V (murine origin) inhibitors were detected such as acylated sulfanilamide, ureido- benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide and aminobenzolamide type of compounds [5]. Sulfan- amides are especially used for vaginal infection since they are rapidly absorbed through the vaginal mucosa; however, the mechanism of absorption is not known yet. Bristol et al. reported that the use of sulfanilamide as vaginal cream in the cervical cancer induced methe- moglobinemia by an unknown mechanism [6]. Kauffman et al. determined herbicide asulam and its antibacterial active metabolite sulfanilamide in honey with liquid chromatography—tandem mass spectrome- try (LC-MS/MS) [7]. The authors explained this situa- tion by that bees occasionally collect nectar from mead- ows treated with asulam. Such honeys were not only contaminated with asulam but also by its degradation 1 The article is published in the original. product sulfanilamide [7]. According to the results of Reybroeck [8], sulfanilamide was detected among 48 imported honey samples available in Belgian mar- kets. In this research, the limits of quantification for sul- fonamides (sulfaguanidine, sulfanilamide, sulfaceta- mide, sulfadiazine, sulfathiazole, sulfadimidine, sul- famethoxazole) were found to be 20 μg/g. It is also necessary to determine the sulfanilamide content in human liquids like urine. The exposure to sulfanilamide by consumption of contamined honey may be a problem for health. To determine the concentrations of sulfanamides and its derivates, various analytical methods have been developed. These include ultraviolet-spectrophotome- try [9], capillary supercritical fluid chromatography— Fourier transform infrared spectroscopy (cSFC-FT-IR) [2], high performance liquid chromatography (HPLC) [1, 10, 11], LC-MS/MS [7], liquid chromatography [12], capillary electrophoresis (CE) [13] and flow injection-spectrophotometric detection [14]. The major disadvantage of HPLC for the determination of the res- idues of sulfanilamides in complex matrices is that an extensive clean-up step is needed prior to analysis. GC methods have been shown to be sensitive and specific but the nonvolatile nature of sulfanamides means that conversion to the N 1 -methyl or N 1 -acyl derivatives is required [2]. Moreover, none of above mentioned methods is a stability-indicating method. The International Confer- ence on Harmonization (ICH) guideline entitled ‘sta- ARTICLES Stability-Indicating High Performance Thin Layer Chromatographic Determination of Sulfanilamide in Human Urine 1 E. Kilinc, B. Gumgum, C. Hamamci, and F. Aydin Department of Chemistry, Faculty of Art and Science, University of Dicle, Diyarbakir, TR-21280 Turkey e-mail: ekilinc@dicle.edu.tr Received December 20, 2007; in final form December 4, 2008 Abstract—A simple, sensitive, selective, precise and stability-indicating high-performance thin-layer chro- matographic (HPTLC) method was developed and applied to human urine for the densitometric determination of sulfanilamide. A mixture of chloroform-ethyl acetate-xylene (2.5 : 4.0 : 1.0, v/v/v) was used as a mobile phase. The system was found to give compact spots for sulfanilamide (retardation factor, R f = 0.21 ± 0.02). The linear regression analysis data for the calibration plots showed good linear relationship with r 2 = 0.9970 ± 0.0003 and r 2 = 0.9947 ± 0.020 within the concentration range of 50–250 ng per spot and 100–1000 ng per spot with respect to peak area, respectively. The limit of detection (LOD) and quantification (LOQ) were 8 and 25 ng per spot, respectively. Sulfanilamide was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery and robustness. The ultraviolet (UV) spectra of the degradation products which had different spectra from sulfanilamide were also recorded. DOI: 10.1134/S1061934809070090