Quantitative determination of lurbinectedin, its unbound fraction and its metabolites in human plasma utilizing ultra-performance LC-MS/MS.

AIMS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods to quantify total lurbinectedin, its plasma protein binding to derive the unbound fraction and its main metabolites 1',3'-dihydroxy-lurbinectedin (M4) and N-desmethyl-lurbinectedin (M6) in human plasma, were developed and validated. MATERIALS & METHODS: For lurbinectedin, sample extraction was performed using supported liquid extraction. For metabolites, liquid-liquid extraction with stable isotope-labeled analogue internal standards was used. Plasma protein binding was evaluated using rapid equilibrium dialysis. In vitro investigations at different plasma protein concentrations were carried out to estimate dissociation rate constants to albumin and alpha-1-acid glycoprotein (AAG). RESULTS: Calibration curves displayed good linearity over 0.1 to 50 ng/mL for lurbinectedin and 0.5 to 20 ng/mL for the metabolites. Methods were validated in accordance with established guidance. The inter-day precision and accuracy ranged from 5.1% to 10.7%, and from -5% to 6% (lurbinectedin in plasma); from 3.1% to 6.6%, and from 4% to 6% (lurbinectedin in plasma:PBS); from 4.5% to 12.9%, and from 4% to 9% (M4); and from 7.5% to 10.5%, and from 6% to 12% (M6). All methods displayed good linearity (r2 >0.99). Recovery was evaluated for lurbinectedin in plasma:PBS (66.4% to 86.6%), M4 (7.82% to 13.4%) and M6 (22.2% to 34.3%). The method for lurbinectedin in plasma has been applied in most clinical studies, while the plasma:PBS and metabolites methods were used to evaluate the impact of special conditions on lurbinectedin PK. Lurbinectedin plasma protein binding was 99.6% and highly affected by AAG concentration. CONCLUSIONS: These UPLC-MS/MS methods enable the rapid and sensitive quantification of lurbinectedin and its main metabolites in clinical samples.

Bionalytical validation study for the determination of unbound ambrisentan in human plasma using rapid equilibrium dialysis followed by ultra performance liquid chromatography coupled to mass spectrometry.

Ambrisentan is a highly selective endothelin-1 type A receptor antagonist indicated for use in the treatment of pulmonary hypertension. In this study an assay was developed and validated for the quantification of total and unbound (free) concentrations of ambrisentan in human plasma. Plasma samples were dialysed against phosphate buffered saline in a rapid equilibrium dialysis device to obtain dialysate and plasma for unbound and total ambrisentan, respectively. Subsequently, ambrisentan and deuterated ambrisentan (internal standard) were extracted from plasma or plasma dialysate by solid-phase extraction and separated by ultra performance liquid chromatography using on a reversed-phase C18 column. Detection was conducted with a tandem mass spectrometer with an electrospray ionization source and analysed in positive ion mode with multiple reaction monitoring. Calibration curves were generated over a linear concentration range of 0.1-200 ng/mL in plasma and 0.1-10 ng/mL in plasma ultrafiltrate; with a recovery for ambrisentan of 69.4% and 77.5%, respectively. This assay has been shown to be reproducible and sensitive. The lower limit of quantification in both cases was 0.1 ng/mL; reaching a sensitivity not previously described in the literature. The inter- and intra-batch precision and accuracy were in both cases ≤±15%. The procedure was applied to assess total and free plasma concentrations of ambrisentan in healthy volunteers. Plasma protein binding of ambrisentan was approximately 99%.