The development and validation of a high-throughput LC-MS/MS method for the analysis of endogenous β-hydroxy-β-methylbutyrate in human plasma.

A high-throughput, sensitive, and rugged liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for the rapid quantitation of β-hydroxy-β-methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC-MS/MS technique under negative mode electrospray ionization conditions. A 13C-labelled stable isotope internal standard was used to achieve accurate quantitation. Multi-day validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2X), extraction recovery, freeze-thaw sample stability (3 cycles), benchtop sample stability (6 hours and 50 minutes), autosampler stability (27 hours, and frozen storage sample stability (146 days). Linearity was demonstrated between 10 – 500 ng mL-1. Inter-day accuracies and coefficients of variation (CV) were 91.2 – 98.1% and 3.7 – 7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers. This article is protected by copyright. All rights reserved.