Development of a rapid and sensitive LC–MS/MS assay for the determination of combretastatin A4 phosphate, combretastatin A4 and combretastatin A4 glucuronide in beagle dog plasma and its application to a pharmacokinetic study

This study details the development and validation of a simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the quantification of combretastatin A-4 3-O-phosphate (CA4P), combretastatin A4 (CA4) and its main metabolite, combretastatin A4 glucuronide (CA4G), in beagle dog plasma. Sample pretreatment includes simple protein precipitation by adding methanol to the plasma sample containing an internal standard (colchicine). LC separation was successfully accomplished on a Waters RP8 Symmetryshield™ column (3.0 mm×150 mm, i.d., 5 μm) with a gradient mobile phase of methanol (0.1% formic acid, v/v) and water (20 mM ammonium acetate) at a flow rate 0.8 mL min−1. The three analytes were detected in the positive/negative ion alternate mode, negative ion mode for CA4G and positive ion mode for CA4P and CA4. Multiple reaction monitoring (MRM) was used for determination of three analytes. Calibration curves were linear in the concentration range of 5–5000 ng mL−1 for CA4P (r ≥ 0.999), 5–3000 ng mL−1 for CA4 (r ≥ 0.999) and 5–5000 ng mL−1 for CA4G (r ≥ 0.999). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was reliable and has been successfully applied to a pharmacokinetic study of CA4P in beagle dogs via intravenous drop infusion at dose rates of 1, 3 and 9 mg kg−1. After daily intravenous drop infusions at 1 mg kg−1 for 7 consecutive days, the accumulation ratio was approximately 1.0, indicating no accumulation from multiple doses in beagles.