Development and validation of HPLC method for determination of S-phenylmercapturic acid (S-PMA) in urine

Objective: The aim of this study is to obtain the optimum condition of HPLC method for the determination of benzene metabolite, S-phenyl mercapturic acid (S-PMA), in urine samples. Methods: Liquid-liquid extraction (LLE) was used as sample preparation method for urine sample. HPLC analysis was performed using Hypersil ODS column 125 x 4 mm ID, 5 μm, the column temperature 25°C and the DAD detector was set at wavelength of 205 nm. Results: Optimum separation was accomplished using gradient system with eluent consists of methanol and phosphate buffer pH 3. The result showed linear correlation with a regression coefficient (r) = 0.994. The detection and quantification limit of S-PMA were 0.7832 ±0.081;} 0.0329 μg/mL and 2.6108 ±0.0940 μg/mL, respectively. The recovery of the method was 88.34 - 117.16% with a mean of 99.96% and coefficient variation (CV) = 8.98%. Conclusion: The proposed method has met the acceptance criteria for validation. The method is simple, fast, and has reproducible recoveries. Therefore, this method can be used for routine analysis of S-PMA.