Chromatography

Ethyl acetate

Extraction (chemistry)

Electrospray ionization

Hexane

Selected reaction monitoring

Analytical Chemistry (journal)

Mass spectrometry

Chemistry

Liquid chromatography–mass spectrometry

Electrospray

Tandem mass spectrometry

Journal of Chromatography B

A quantitative assay method by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the simultaneous determination of azelnidipine and its two metabolites, M-1 (aromatized form) and M-2 (hydroxylated form), in human plasma was developed and validated. Plasma samples, each of 1.0 mL, were extracted by a single step liquid–liquid extraction using a mixture of ethyl acetate and hexane (1:1, v/v), and analyzed by the LC/ESI-MS/MS method. Three analytes were separated by isocratic elution on a C18 column, and ionized using a positive ion electrospray ionization source. The ion transitions were monitored in selected reaction monitoring (SRM) mode. The chromatographic run time was 11 min per injection, with retention time of 3.6, 10.2 and 6.8 min for azelnidipine, M-1 and M-2, respectively. The calibration curves for azelnidipine, M-1 and M-2 well fitted to equations by a weighted (1/X2) quadratic regression over the range of 0.5–40.0 ng/mL (r2 > 0.9979). The intra- and inter-assay precisions (coefficient of variation: C.V.), calculated from quality control (QC) samples, were less than 8.7 and 8.4%, 3.8 and 4.7%, and 11.9 and 13.9%, respectively, for azelnidipine, M-1 and M-2. The accuracy was within ±9% for azelnidipine, within ±7% for M-1 and within ±16% for M-2. The overall recoveries for azelnidipine, M-1 and M-2 were 68.8–78.6%, 54.3–62.9% and 80.4–89.7%, respectively. All analytes evaluated demonstrated acceptable short-term, long-term, auto-sampler and stock solution stabilities. Furthermore, the method developed was successfully applied to pharmacokinetic studies on azelnidipine, M-1 and M-2 after an oral dose of 16 mg CALBLOCK® tablets (2 mg × 8 mg tablets) to healthy volunteers.

Simultaneous determination of azelnidipine and two metabolites in human plasma using liquid chromatography-tandem mass spectrometry