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A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), apalutamide, darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma as per regulatory guidelines. The analytes and the internal standard (I.S.: apalutamide-d 3 ) were extracted from 50 μL mice plasma by simple protein precipitation using acetonitrile, followed by chromatographic separation using an Atlantis C 18 column with an isocratic mobile (0.2% formic acid:acetonitrile; 30:70, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 465 → 209, 451 → 195, 478 → 450, 399 → 178, 397 → 194 and 481 → 453 for enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide, ORM-15341 and the I.S. respectively. The calibration curves were linear from 1.07 to 2000 ng/mL with r 2 ≥0.99 for all the analytes. The intra- and inter-batch accuracy and precision (% CV) across quality controls varied from 88.5-111% and 1.13-13.1, 85.4-106% and 3.15-14.3, respectively for all the analytes. All the analytes were found to be stable under different conditions. The method was applied to an intravenous pharmacokinetic study in mice., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Abiraterone acetate is administered as a prodrug to patients with metastatic, castration-resistant prostate cancer (mCRPC) and is readily metabolized into the potent 17a-hydroxylase/17,20-lyase (CYP17) enzyme inhibitor and androgen receptor inhibitor abiraterone and Δ(4)-abiraterone (D4A), respectively. To investigate pharmacokinetic variability in abiraterone acetate metabolism we developed highly sensitive liquid chromatography/mass spectrometry (LC/MS) assays for the simultaneous quantitation of abiraterone and D4A in human plasma using high-resolution mass spectrometry (HRMS) on an Orbitrap mass spectrometer. This study demonstrates the quantitative performance of HRMS and compares the conventional Parallel Reaction Monitoring (PRM) mode of quantitation with the unconventional Full scan MS mode conducted at high resolution (>70,000 resolution). The use of HRMS for quantitation of abiraterone and D4A yielded assays that were linear over a broad concentration range (0.074-509.6 ng/mL for abiraterone; 0.075-59.93 ng/mL for D4A) in both Full scan MS and PRM modes. The assay precision for abiraterone and D4A was below 5% in PRM mode and 7% in Full scan MS mode. Accuracies fell within 98-107% for abiraterone and 104-112% for D4A in PRM mode, and 96-116% for abiraterone and 96-105% for D4A in Full scan MS mode, each meeting the acceptance criteria of FDA approved guidelines for bioanalytical methods The PRM analysis of abiraterone and D4A provided high specificity and reduced background interference, however the Full scan MS detection at a resolution of 70,000 was advantageous in that it required minimal optimization, was simple to implement, yielded comparable quantitative characteristics to PRM and the data is useful for re-analysis. Use of the assays were demonstrated for quantitation of these metabolites in steady state trough level plasma of seventeen (17) patients with mCRPC, demonstrating the inter-patient variability of up to 10-fold concentration., (Copyright © 2018 Elsevier B.V. All rights reserved.)