A novel LC-MS/MS method for simultaneous estimation of chlordiazepoxide and clidinium in human plasma and its...

A highly sensitive and selective LC-MS/MS assay was developed and validated for the simultaneous quantification of Chlordiazepoxide and Clidinium in human plasma for the first time, employing solid-phase extraction. Chromatographic separation of the analytes and their deuterated internal standards was performed on a reversed-phase Kinetex XB-C18 (150 × 4.6 mm, 5 μm) column with a gradient mobile phase. Mass spectrometric detection was achieved using electrospray ionization in positive ion mode, employing the ion transitions: m/z 300.0 → 227.1 for Chlordiazepoxide, m/z 352.1 → 142.1 for Clidinium, m/z 305.1 → 232.1 for Chlordiazepoxide D5, and m/z 357.2 → 142.2 for Clidinium D5. The assay demonstrated a linear calibration range of 504.0-500,198.3 pg/mL for Chlordiazepoxide and 5.0-3,004.7 pg/mL for Clidinium, ensuring precise pharmacokinetic evaluation. The method was validated with a lower limit of quantification (LLOQ) of 504.0 pg/mL for Chlordiazepoxide and 5.0 pg/mL for Clidinium, precision within 15% RSD, and accuracy within 85-115% of the nominal values. No matrix interference from haemolysed or lipemic plasma was observed, and recovery exceeded 90%. This study presents a novel LC-MS/MS method with significant improvements in sensitivity and specificity, facilitating its direct application in pharmacokinetic and bioequivalence studies.