Validation of high-performance liquid chromatography–tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4′‑des-methyl and cephalotaxine metabolites in human plasma and urine

• These assays were fully validated according to the latest FDA and EMA guidelines.
• Deuterated isotopes were used as internal standards.
• The assays were considered very suitable to support clinical pharmacologic studies of omacetaxine mepesuccinate.

Omacetaxine mepesuccinate (hereafter called omacetaxine) is a modified cephalotaxine and is registered (Synribo®) for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). To evaluate the pharmacokinetics of omacetaxine, sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays for the quantification of omacetaxine and its inactive 4′-des-methyl (4′-DMHHT) and cephalotaxine metabolites in human plasma and urine were developed and validated.Since omacetaxine is mainly metabolised by esterases, the plasma samples were immediately stabilised after collection with an esterase inhibitor and stored at a nominal temperature of −80 °C. Urine samples were stored at −80 °C immediately after collection. Protein precipitation was applied as the sample pretreatment method for the plasma samples, and urine samples were processed using solid-phase extraction (SPE). For both assays, the dried and reconstituted extracts were injected on a XBridge BEH Phenyl column for analysis of all analytes. Gradient elution was applied with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionised using a turbospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer.The validated plasma assay quantifies all analytes in the concentration range of 0.1–100 ng/mL and the urine assay in the range of 0.1–50 ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.