Development of an Improved HPLC-PDA Method for the Determination of Tolfenamic Acid in Meat and Milk

Abstract

Tolfenamic acid (TA) is one of the non-steroidal anti-inflammatory drugs (NSAIDs) which are widely used as analgesic, anti-inflammatory and antipyretic drugs. In this study, we developed an improved detection method focusing on the validation of the TA residue using high performance liquid chromatography (HPLC) and a photodiode array (PDA) detector. TA in bovine, swine muscles and milk were analyzed using a C18 column (5 μm, 250 mm × 4.6 mm i.d.) and the mobile phase consisted of water with 0.1% phosphoric acid (eluent A) and 0.1% phosphoric acid in acetonitrile (ACN) (eluent B). Additionally, to optimize TA detection, liquid-liquid extraction (LLE) and additional purification steps were established to minimize the endogenous peaks and interferences. The developed method was validated through the limit of detection (LOD), the limit of quantification (LOQ), linearity, accuracy, and precision testing. TA in food samples were successfully detected after 20 min in chromatography. The LOQ was evaluated at 0.01 mg/kg and coefficient of determination of the linear regression (r2) was well over 0.99. The recovery rates ranged from 81.9-90.8%, 74.2-92.8% and 74.5-80.7% with relative standard deviations lower than 20% for bovine, swine muscles and milk, respectively, which corresponds to the CODEX International Food Standards guideline. Finally, the developed method has been applied for monitoring samples collected from the markets in major cities and proven great potential to be used as the confirmatory method to analyze and monitor TA residue in animal-based food products.