title: The development and validation of a high-throughput LC-MS/MS method for the analysis of endogenous β-hydroxy-β-methylbutyrate in human plasma.
abstract: 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. A13C-labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze-thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. 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.
authors.affiliation: Syngene International Limited, Bangalore, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: Abbott Laboratories, Columbus, OH, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ehling
authors.affiliation: Abbott Laboratories, Columbus, OH, USA.
authors.forename: Sathyavageeswaran
authors.initials: S
authors.lastname: Shreeram
authors.affiliation: Abbott Laboratories, Columbus, OH, USA.
authors.forename: Todime M
authors.initials: TM
authors.lastname: Reddy
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014631:Valerates / Q000097:blood*
chemical_list: D014631:Valerates; C004961:beta-hydroxyisovaleric acid
keywords: human plasma; liquid chromatography/mass spectrometry (LC/MS); muscle synthesis; β-hydroxy-β-methylbutyrate (HMB)