title: Quantitative analysis of an anti-viral immune escape compound ML-7 in feline plasma using ultra performance liquid chromatography/electrospray ionization mass spectrometry.
abstract: 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 C(18) 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.
authors.affiliation: Ghent University, Faculty of Veterinary Medicine, Department of Pharmacology, Toxicology and Biochemistry, Merelbeke, Belgium. Marc.Cherlet@UGent.be
authors.forename: Marc
authors.initials: M
authors.lastname: Cherlet
authors.forename: Sabine
authors.initials: S
authors.lastname: Gleich
authors.forename: Hannah
authors.initials: H
authors.lastname: Dewerchin
authors.forename: Hans
authors.initials: H
authors.lastname: Nauwynck
authors.forename: Sylvie
authors.initials: S
authors.lastname: Daminet
authors.forename: Patrick
authors.initials: P
authors.lastname: De Backer
authors.forename: Siska
authors.initials: S
authors.lastname: Croubels
mesh_terms: D000818:Animals; D001381:Azepines / Q000097:blood* / Q000276:immunology; D002415:Cats; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005374:Filtration / Q000295:instrumentation; D006801:Humans; D008567:Membranes, Artificial; D009281:Naphthalenes / Q000097:blood* / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001381:Azepines; D008567:Membranes, Artificial; D009281:Naphthalenes; C070571:ML 7