Development and validation of an LC–MS/MS method for quantification of paracetamol and camylofin in human serum

Abstract Background The present work aims to develop and validate an LC–MS/MS method for the bioanalysis of a fixed-dose combination containing 325 mg of paracetamol and 50 mg of camylofin in human serum. Results A selective, sensitive and fast LC–MS/MS method was developed and validated for quantitative bioanalysis of the analytes from human serum, using protein precipitation technique and diphenylamine as an internal standard (IS). Chromatographic separation was performed on Agilent Zorbax SB C18 column (50 mm × 2.1 mm, 5 μm), using a mixture of 0.1% formic acid and methanol (50: 50 v/v) as mobile phase at a flow rate of 0.5 mL/min, with a run time of 6 min. Tandem mass spectrometry (MS/MS) was employed for the analysis, utilising positive ionisation mode and a multiple reaction monitoring (MRM) scan type. The method was established with a linear range of 100–20,000 ng/mL for paracetamol and 0.25–200 ng/mL for camylofin using 200 μL of human serum. Conclusion The developed bioanalytical method was validated as per the Bioanalytical Method Validation Guidance for Industry of the United States Food and Drug Administration (US-FDA)-CDER. The developed method is reliable and easy to use and was applied successfully to a clinical pharmacokinetic study involving twelve healthy Indian subjects under fasting and fed conditions.