Analysis of 3-Methyl-4-nitrophenol, A Major Metabolite of Fenitrothion, In Mice Urine Using HPLC
Introduction: 3-methyl-4-nitrophenol is a major metabolite of fenitrothion and is considered as a potential risk to public health owning to its genotoxicity and carcinogenicity. Objective: To develop and validate an HPLC method for the quantification of 3-methyl-4-nitrophenol in urine. Methods: A sample of mice urine was collected following the administration of fenitrothion solution through oral gavage and subsequently, 3-methyl-4-nitrophenol was extracted using a simple liquid-liquid extraction method. The newly developed method uses acetonitrile: water 60:40 mobile phase at 270 nm with a flow rate of 1 mL/min HPLC condition. An optimization to a previously used extraction method was made by using ethyl acetate instead of acetonitrile to avoid extraction of water-soluble components of urine other than 3-methyl- 4-nitrophenol. The developed method was validated using the ICH guideline for the validation of analytical procedures. Accordingly, the selectivity, linearity, limit of detection and quantification, accuracy, precision, extraction efficacy, robustness and stability of the method was determined. Results: All of the validation parameters showed that the method is valid for the determination of 3-methyl-4-nitrophenol in urine sample. Using this method it was possible to detect low concentration of 3-methyl-4-nitrophenol in urine samples down to 0.87 μg/mL. Conclusion: The sample was analyzed with this validated method and the respective concentration of the 3-methyl-4-nitrophenol was found to be 31.11 μg/mL. Accordingly, 10.37% of the total fenitrothion administered to mice was found metabolized into 3-methyl-4-nitrophenol. The HPLC retention time for 3-methyl-4-nitrophenol was 2.81 minutes.