Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

Abstract

A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of β-hydroxy-β-methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water-acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C(8) (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30-4600 ng/mL (r > 0.998) for HMB. The intra- and inter-day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench-top, autosampler freeze-thaw cycles and long-term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats.

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.…”

Quantification of β-hydroxymethylbutyrate and leucine by ultrahigh performance liquid chromatography tandem mass spectrometry at different situations and stages of a rodent life

“…Since HMB is a molecule with amphipathic chemical character, it was reasonable to avoid protocols that include liquid-liquid partition steps, where polar and non polar solvents are used, and then HMB could be partitioned between both mediums. In fact, Deshpande et al [29] used the liquid-liquid extraction for plasma samples, obtaining very low recovery rates. In the present work, a simple treatment of sample by dilution with a solvent was evaluated to reduce the interferences and to degrade proteins.…”

“…Recently, two analytical methods have been published to improve these inconveniences. First, in 2013 Deshpande et al published a study of the bioavailability of HMB in plasma by LC-MS [29] and second, our research group has recently published a method to measure HMB and Leu by UHPLC-ESI-MS/MS in relatively clean biological fluids, namely cell cultures and brain microdialysates [30].…”

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

“…These were based on gas chromatography−mass spectrometry (GC/MS) after derivatization 15,28,33,34 or high performance liquid chromatography with derivatization 32 or without derivatization, 35 and more recently on liquid chromatography−mass spectrometry (LC/ MS). 30,31 The above-referenced GC/MS-based methods and one LC/MS-based method 31 employed an in-house synthesized stable isotope labeled internal standard.…”