Determination of doxazosin and verapamil in human serum by fast LC–MS/MS: Application to document non-compliance of patients

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d3 as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid–liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3 → 165.2 and 150.2 for verapamil, m/z 452.2 → 344.4 and 247.4 for doxazosin, m/z 298.2 → 103.1 for trimipramine-d3. Linearity was achieved between 1 and 500 ng/mL (R2 ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.