title: 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.
authors.affiliation: Syngene International Limited, Biocon Park, Bangalore, India.
authors.forename: Parag
authors.initials: P
authors.lastname: Deshpande
authors.forename: Zhang
authors.initials: Z
authors.lastname: Jie
authors.forename: Ramesh
authors.initials: R
authors.lastname: Subbarayan
authors.forename: Vijay Kumar
authors.initials: VK
authors.lastname: Mamidi
authors.forename: Raja Haranadha Babu
authors.initials: RH
authors.lastname: Chunduri
authors.forename: Tapas
authors.initials: T
authors.lastname: Das
authors.forename: Sathyavageeswaran
authors.initials: S
authors.lastname: Shreeram
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D016014:Linear Models; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014631:Valerates / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D014631:Valerates; C004961:beta-hydroxyisovaleric acid