Anidulafungin - Challenges in development and validation of an LC-MS/MS bioanalytical method validated for regulated clinical studies

摘要

Anidulafungin is a semi-synthetic echinocandin with antifungal activity, usually administered as an intravenous infusion. In order to determine the pharmacokinetics (PK) of anidulafungin in pediatric patients, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) bioanalytical method (M1) was developed and validated for quantification of anidulafungin in plasma. During analysis of incurred samples (samples collected from patients enrolled in a clinical study) an isobaric chromatographic interference was observed. The source of interferencewas identified as an anidulafungin open-ring form (D1) and its impact on the quantification of anidulafungin was investigated. It was found that accurately quantifying anidulafungin in incurred samples required chromatographic separation of the open-ring form from anidulafungin. The method was redeveloped to achieve the appropriate baseline separation and to avoid experimental conditions that favored opening the anidulafungin ring. The extraction of anidulafungin from plasma by protein precipitation remained unchanged, but the changes in chromatography warranted validation of a new method,M2, 2 years afterM1 was validated. Incurred samples from three studies that were previously analyzed by M1 and were within confirmed long-term frozen stability were then reanalyzed by M2. Although the incurred sample reproducibility tests on those samples passed for each of the two methods, comparison of concentrations from the same samples obtained by M1 and M2 revealed that an overestimation of anidulafungin following the M1 method exceeded acceptance criteria. The new HPLC-MS/MS method (M2) is applicable for quantification of anidulafungin within a nominal range 50-20,000 ng/mL and requires a 50 μL human plasma aliquot. A linear, 1/concentration squared weighted, leastsquares regression algorithm was used to generate the calibration curve and its parameters were used to quantitate the incurred samples. The inter-assay accuracy in heparin human plasma validation ranged from -4.33 to 0.0386 % and precision was ≤7.32 %. The method M2 was validated for use in regulated bioanalysis and is presently used to quantitate anidulafungin in plasma samples from clinical studies.