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. 2023 Mar 30;18(3):e0283783.
doi: 10.1371/journal.pone.0283783. eCollection 2023.

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

Nicholas King  1 , Soledad Garcia-Martinez  1 , Eider Alcaraz  1 , Alba Grisalena  1 , Rubin Lubomirov  2 , Raquel Altares  2 , Carlos Fernandez-Teruel  2 , Andrés M Francesch  3 , Pablo M Avilés  3 , Salvador Fudio  2
Affiliations

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

Nicholas King et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Raquel Altares, Rubin Lubomirov, Andrés M. Francesch, Pablo Manuel Avilés and Salvador Fudio are employed by and/or have ownership interests (including patents and/or shareholding) in PharmaMar (Colmenar Viejo, Spain). Carlos Fernández-Teruel was an employee of PharmaMar at the time of the study.

Figures

Fig 1
Fig 1. The chemical structures of lurbinectedin, lurbinectedin metabolites and their stable isotope–labeled internal standards.
Fig 2
Fig 2. Chromatogram of Lurbinectedin Blank (upper), LLOQ (middle), and a Treated Patient (lower) Plasma Samples (with a lurbinectedin concentration of 177 ng/mL), With Internal Standard (right).
ES+, positive mode electrospray ionization; LLOQ, lower limit of quantitation; MRM, multiple reaction monitoring.
Fig 3
Fig 3. Chromatogram of Lurbinectedin Blank (upper), LLOQ (middle), and a Treated Patient Plasma:PBS Samples, With Internal Standard (right).
LLOQ, lower limit of quantitation; MRM, multiple reaction monitoring; PBS, phosphate-buffered saline.
Fig 4
Fig 4. Chromatogram of M4 Blank (upper), LLOQ (middle), and a Treated Patient (lower) Plasma Samples, With Internal Standard (right).
Retention time of M4 is ~1.05 minutes. LLOQ, lower limit of quantitation; MRM, multiple reaction monitoring.
Fig 5
Fig 5. Chromatogram of M6 Blank (upper), LLOQ (middle), and a Treated Patient (lower) Plasma Samples, With Internal Standard (right).
LLOQ, lower limit of quantitation; MRM, multiple reaction monitoring.
Fig 6
Fig 6. Lurbinectedin plasma protein binding in artificial plasma with varying alpha-1-acid glycoprotein levels.
Fig 7
Fig 7. Total and unbound lurbinectedin and M4 and M6 plasma concentration-time profiles of an individual patient treated in study A-019.
See this image and copyright information in PMC

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