Simultaneous quantification of fosinopril and its active metabolite fosinoprilat in rat plasma by UFLC-MS/MS: Application of formic acid in the stabilization of an ester-containing drug.

Abstract

Fosinopril is an angiotensin-converting enzyme inhibitor containing a phosphate ester group which undergoes esterase hydrolysis to its active metabolite, fosinoprilat. EDTA was utilized as an anticoagulant to inhibit the hydrolysis of fosinopril in whole blood during blood collection and processing. To prevent the ex vivo conversion to fosinoprilat, formic acid was added to rat plasma to effectively stabilize fosinopril. A sensitive, rapid and robust ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for simultaneous determination of fosinopril and fosinoprilat in rat plasma. Protein precipitation was employed for plasma sample clean-up. Chromatographic separation was achieved on a Welch Ultimate XB-C18 column using gradient elution with a total run time of 5min. Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometric assay. The assay involves quantitation of both analytes in small-volume (50μL) plasma, with the lower limit of quantification of 0.1 and 1ng/mL for fosinopril and fosinoprilat, respectively. The method was fully validated in linear calibration ranges of 0.1-150ng/mL for fosinopril and 1-1500ng/mL for fosinoprilat with acceptable accuracy and precision. Assay recoveries were high (>95% for fosinopril and >91% for fosinoprilat) and matrix effect was negligible. Both analytes were found to be stable in stabilized rat plasma for 6h at room temperature, 30 days at -80°C, and following three freeze-thaw cycles and were also stable in processed samples for 36h at 4°C. The validated method was successfully applied to sample analyses for pharmacokinetic study of fosinopril and can be extended to the measurement of fosinopril in other biological samples.