Bioanalytical method development and validation of isosorbide mononitrate in human plasma by using LC-MS/MS.

To analyze Isosorbide Mononitrate in human plasma, a highly sensitive and accurate LC–MS/MS bioanalytical method was developed and validated using Isosorbide mononitrate-¹³C₆ as the internal standard. Validate a suitable analytical method for the estimation of unknown drug concentrations in plasma. It can be used for the quantitation of isosorbide mononitrate in K₂EDTA human plasma for bioequivalence and bioavailability studies. The method was found to be specific, sensitive, linear, precise, accurate, and reproducible for the extraction and analysis of isosorbide mononitrate in K₂EDTA human plasma samples over the investigated concentration range of 5.000 ng/mL to 1800.000 ng/mL using a processing volume of 0.100 mL. Chromatographic separation was achieved using a 2 mM ammonium acetate (acidified) mobile phase with acetonitrile containing 0.1% acetic acid in a ratio of 55:45 (v/v) at a flow rate of 0.5 mL/min. Detection was carried out using multiple reaction monitoring with transitions (m/z) of 249.90 → 59.00 for isosorbide mononitrate and 255.90 → 58.90 for the internal standard. The analytes were detected using tandem mass spectrometry (LC–MS/MS). The proposed method was comprehensively validated in terms of linearity, accuracy, precision, specificity, sensitivity, recovery, and stability, with results meeting the acceptance criteria. The developed and validated method was found to be simpler, faster, more specific, precise, and cost-effective compared with previously reported methods.