Reliable and easy-to-use Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) assay for quantification of Olorofim (F901318) - a novel antifungal drug - in human plasma and serum [Analytical Procedures]

Background A fast and easy-to-use liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of a novel antifungal drug, Olorofim (formerly F901318), a member of the novel class of orotomides, in human plasma and serum was developed and validated.

Methods Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. An isotope labeled analogue of F901318 was employed as internal standard. Chromatographic separation was achieved using a 50 mm x 2.1 mm, 1.9μm, polar Hypersil Gold C18 column and isocratic mobile phase consisting of 0.1 % formic acid/acetonitrile (60%/40%, v/v) at a flow rate of 330 μl/min. The analyte was detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI⁺) within a single runtime of t = 2.00 min.

Results The present LC-MS/MS-method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the US Food and Drug Administration (FDA). Linearity of F901318 concentration ranges was verified by the Mandel-test. Calibration curve was tested linear across the range and fitted using least squares regression with a weighting factor of the reciprocal concentration. Limit of detection was: LOD = 0.0011 mg/l, lower limit of quantitation was: LLOQ = 0.0033 mg/l. Intra-day and inter-day precisions ranged from 1.17% to 3.23% for F901318, and intra-day and inter-day accuracies (%bias) ranged from 0.75% to 5.01%.

Conclusion The method was established for the rapid quantitation of F901318 concentrations in serum and plasma samples in patient trials, and optimizes TDM in applying an easy-to-use single method.

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