Quantitative analysis of an anti-viral immune escape compound ML-7 in feline plasma using ultra performance liquid chromatography/electrospray ionization mass spectrometry

An analytical method for the quantitative measurement of ML-7, a product with possible anti-immune escape activity for feline infectious peritonitis virus (FIPV), in feline plasma was developed and validated. The sample preparation consists of a solid-phase extraction step on an MCX cartridge. ML-7 and ML-9, used as the internal standard for the analysis, were separated on an ACQUITY UPLC™ BEH C18 reversed-phase column (1.7 μm, 50 mm × 2.1 mm I.D.), using isocratic elution with acetonitrile and 0.1% formic acid in water as the mobile phase. Both compounds were subsequently quantified in MRM mode on a Micromass® Quattro Premier™ XE triple quadrupole mass spectrometer. The use of a Thermo Scientific® Exactive™ orbitrap mass spectrometer made it possible to confirm the proposed fragmentation pattern of both ML-7 and ML-9. A validation study according to EC requirements was carried out, in which the method showed good performance. Linear behaviour was observed in the 1–2500 ng ml−1 range, which is relevant for real sample analysis. Accuracy and precision were within the criteria requested by the EC requirements throughout this concentration range. Extraction recovery of ML-7 was 72%. Matrix effect for ML-7 was not higher than 8%. The method was successfully used for the monitoring of ML-7 in feline plasma after intravenous, subcutaneous or oral administration of an ML-7 formulation, for the determination of pharmacokinetic parameters, with a limit of quantification of 1 ng ml−1 and a limit of detection of 0.4 ng ml−1. The proposed method also shows good characteristics for the analysis of ML-7 in plasma of other animal species and human plasma.

► An analysis method for ML-7 in feline plasma by UPLC–ESI-MS/MS was optimized. ► Solid-phase extraction clean-up on MCX column was used to minimize matrix effect. ► Validation was performed according to EC guidelines, with an LOQ at 1 ng ml−1. ► Method useful for the analysis of ML-7 in plasma of other animal species or human. ► Method allows for monitoring of ML-7 in treated cats for pharmacokinetic analysis.