STABILITY INDICATING ASSAY FOR DILTIAZEM AND ITS METABOLITES IN HUMAN PLASMA BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY FOR PHARMACOKINETIC APPLICATION

A simple and sensitive Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry method was developed to perform a stability study of diltiazem and its metabolites in human plasma using various buffer reagents at a different strength. The method was applied for the quantification of diltiazem and its two major metabolites N-desmethyl diltiazem and desacetyl diltiazem in human plasma. The analytes were separated using a binary solvent delivery mode on a reversed-phase column and analyzed by Mass Spectrometry in the multiple reaction monitoring mode using the respective (M+H)⁺ ions, m/z 415.05/178.03 for diltiazem, m/z 401.09/150.04 for N-desmethyl diltiazem m/z 373.21/108.85 for desacetyl diltiazem, m/z 419.22/314.0 for diltiazem-D4 (internal standard). The linearity was 0.93 to 250.10 ng/mL for diltiazem, 0.24 to 64.00 ng/mL for N-desmethyl diltiazem and 0.15 to 40.69 ng/mL for desacetyl diltiazem in human plasma. The lower limit of quantification was 0.93 ng/mL, 0.24 ng/mL and 0.15 ng/mL for diltiazem, N-desmethyl diltiazem, and desacetyl diltiazem, respectively. The plasma samples buffered with 1% of 0.1 M NaF solution was able to limit the degradation of diltiazem to desacetyl diltiazem for longer storage periods at -70 °C.