title: Use of 'dilute-and-shoot' liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma.
abstract: This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 μL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy. Electronic address: s.esposito@irbm.it.
authors.forename: Simone
authors.initials: S
authors.lastname: Esposito
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Bracacel
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Nibbio
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Speziale
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Veneziano
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Edith
authors.initials: E
authors.lastname: Monteagudo
authors.affiliation: IRBM Science Park, Via Pontina km 30,600, 00040 Pomezia, Roma, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Bonelli
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D010480:Perhexiline / Q000097:blood* / Q000493:pharmacokinetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010480:Perhexiline
keywords: Dilute-and-shoot; LC–HRMS; Orbitrap; Perhexiline; Plasma pharmacokinetics; Preclinical DMPK