Quantitation of clevidipine in dog blood by liquid chromatography tandem mass spectrometry: Application to a pharmacokinetic study

Clevidipine, a vascular selective calcium channel antagonist of the dihydropyridine class, is rapidly metabolized by ester hydrolysis because of incorporation of an ester linkage into the drug molecule. To characterize its pharmacokinetic profiles in dogs, a simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of clevidipine in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm × 4.6 mm, 5 μm) with a gradient mobile phase consisting of methanol and 5 mM ammonium formate at a flow rate of 0.5 mL/min. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M−H]− → m/z 234.1 for clevidipine and m/z 256.1 [M−H]− → m/z 227.1 for elofesalamide (internal standard) in the negative ion mode with electrospray ionization (ESI) source. This validated LC-MS/MS method showed good linearity over the range 0.5-100 ng/mL with the lower limit of quantitation (LLOQ) of 0.5 ng/mL together with the satisfied intra- and inter-day precision, accuracy, extraction recovery and matrix effect. Stability testing indicated that clevidipine in dog blood with the addition of denaturant methanol was stable on workbench for 1 h, at −80 °C for up to 30 days, and after three freeze-thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler at 4 °C. The validated method has been successfully applied to a pharmacokinetic study of clevidipine injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg/h for 0.5 h.