Investigation of the Presence of β-Hydroxy-β-methylbutyric Acid and α-Hydroxyisocaproic Acid in Bovine Whole Milk and Fermented Dairy Products by a Validated Liquid Chromatography–Mass Spectrometry Method

Abstract

A simple, rugged, quantitative, and confirmatory method based on liquid chromatography-mass spectrometry was developed and comprehensively validated for the analysis of the leucine metabolites β-hydroxy-β-methylbutyric acid (HMB) and α-hydroxyisocaproic acid (HICA) in bovine whole milk and yogurt. Mean accuracy (90-110% for HMB and 85-115% for HICA) and total precision (<10% RSD in most cases, except for <20% RSD for HMB at the limit of quantitation) at four concentration levels across three validation runs have been determined. Limits of quantitation for HMB and HICA in whole milk were 20 and 5 μg/L, respectively. Measured concentrations of HMB and HICA were <20-29 and 32-37 μg/L, respectively, in bovine whole milk and <5 and 3.0-15.2 mg/L, respectively, in yogurt. These concentrations are insufficient by large margins to deliver any musculoskeletal benefits, and fortification of milk and dairy products with HMB and/or HICA appears to be justified.

Transfer of β-hydroxy-β-methylbutyrate from sows to their offspring and its impact on muscle fiber type transformation and performance in pigs

“…Milk and muscle tissue samples were measured for HMB content by a modification of the plasma and milk analysis method previously described by Deshpande et al [16] and Ehling et al [17]. Briefly, acidified muscle tissue homogenates were extracted with methyl-t-butyl ether for 2 h, and a -hydroxy- a -methylbutyric acid (Sigma H40009; Sigma-Aldrich, Saint Louis, MO, USA) was used as an internal standard.…”

Quantitation of α‐hydroxy acids in complex prebiotic mixtures via liquid chromatography/tandem mass spectrometry

“…Various hydroxy acids, including AHAs, have numerous vital implications for a variety of fields of study, including food science, biomedical materials, metabolic disease screening, and meteorite studies; thus a robust analytical technique for these analytes is of general interest. However, previously reported analytical methods suffer from several innate shortcomings, including the use of time‐consuming gas chromatography coupled to mass spectrometry (GC/MS), the need for complex derivatization procedures with expensive reagents, or, in some cases, using derivatization reagents that are potentially explosive .…”

Direct Analysis of Leucine and Its Metabolites β-Hydroxy-β-methylbutyric Acid, α-Ketoisocaproic Acid, and α-Hydroxyisocaproic Acid in Human Breast Milk by Liquid Chromatography–Mass Spectrometry

“…Along with our previous report, 21 this is the first description of the development and comprehensive validation of a direct, quantitative, and confirmatory method based on LC−MS without derivatization for the analysis of leucine metabolites HMB, KIC, and HICA in a biological matrix. It is also the first report on the direct and quantitative analysis of leucine in a biological matrix by stable isotope dilution mass spectrometry, where the need for the chromatographic resolution of leucine from isobaric isoleucine has been adequately addressed.…”