Use of ‘dilute-and-shoot’ liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma

• Perhexiline was quantified and metabolites were detected in a single LC–HRMS run.
• Sample preparation is limited to a 100-fold dilution of plasma (“dilute-and-shoot”).
• The amount of information obtained in a single DMPK experiment is increased.
• Times and costs for sample preparation are reduced.
• A low sample volume is needed.

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 (r2 = 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.

Figure optionsDownload as PowerPoint slide