Development and validation of bioanalytical methods to support investigations of AZD9496 in the clinic

Aim: AZD9496 is an oral nonsteroidal, potent and selective antagonist and degrader of ER-α. Two major active metabolites (M3 and M5 as diastereomers) were identified in humans. Methodology/results: Multianalyte, sensitive LC–MS/MS method in human plasma was developed and validated that overcame the challenges encountered. The method demonstrated acceptable precision, accuracy and selectivity for AZD9496 and two major metabolites. Incurred sample reanalysis was acceptable from evaluation in clinical studies, indicating adequate reproducibility. In addition, a urine method for AZD9496 was also developed and validated. Conclusion: Robust and sensitive LC–MS/MS assays for the quantitation of AZD9496 and two diastereomeric metabolites in human plasma and AZD9496 in human urine have been validated and successfully applied to clinical studies.

Download Full-text

Development and Validation of an RP-HPLC Method for Determination of Atorvastatin and its Hydroxyl Metabolites in Human Plasma

Current Pharmaceutical Analysis ◽

10.2174/1573412914666180912110154 ◽

2020 ◽

Vol 16 (3) ◽

pp. 238-245

Author(s):

Dagmara Sowińska ◽

Alicja Pogorzelska ◽

Marlena Rakicka ◽

Justyna Sznura ◽

Justyna Janowska ◽

...

Keyword(s):

Human Plasma ◽

Internal Standard ◽

Hplc Method ◽

Active Metabolites ◽

Peak Separation ◽

Rp Hplc ◽

Increased Risk ◽

Relative Standard ◽

Development And Validation

Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.

Download Full-text