A sensitive method for determination of nimesulide and its hydroxy metabolite in milk using validated UPLC-MS method

Abstract

Nimesulide is a widely used drug for the treatment of pain in humans as well as in animals. The presence of trace amounts of drugs in milk or other food supplement affects seriously human health. This study reports a method based on LC-MS detection for the determination of nimesulide and its hydroxy metabolite in cow milk samples. Waters ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 µm), with gradient elution using 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL min⁻¹ was used for the chromatographic separation. The ion scans were quantified at m/z 307.0393 for nimesulide, m/z 323.0375 for hydroxy nimesulide metabolite and m/z 434.2198 for IS (valsartan) in negative ESI mode. A double extraction method with protein precipitation by acetonitrile followed by solid-phase extraction was developed to detect nimesulide at 1.30 ng mL⁻¹ and hydroxy nimesulide at 1.03 ng mL⁻¹ in milk. The calibration curve was constructed for nimesulide in the range of 1.30 ng mL⁻¹ to 125.07 ng mL⁻¹ while for hydroxy nimesulide calibration curve was constructed in the range of 1.03 ng mL⁻¹ to 99.70 ng mL⁻¹ in whole cow milk without any treatment. The validation parameters like accuracy, precision, selectivity, stability, matrix effect and dilution integrity were also performed on nimesulide and hydroxy nimesulide. The developed processing and chromatographic method can be used to quantitatively determine nimesulide or its hydroxy nimesulide metabolite in biological matrices, i.e., milk, plasma, faecal, tissue, etc.