title: Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry.
abstract: The main objective of the present work was to develop a method to determine β-hydroxymethylbutyrate (HMB) and leucine (Leu) in culture media and brain microdialysates. An accurate, selective, and cost-effective method, based on the use of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed for the identification and quantification of both compounds. The method consisted of sample dilution, direct injection onto the chromatographic equipment, and quantification with a triple quadrupole mass spectrometer using an electrospray ionization interface in positive mode. The procedure and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Hilic column using acetonitrile-water gradient with formic acid as additive was employed. The total run time was 4 min. The limits of detection (LODs) obtained ranged from 0.01 to 0.04 μg mL(-1), and the limits of quantification (LOQs) ranged from 0.04 to 0.12 μg mL(-1). Precision (expressed as relative standard deviation) was lower than 15 %, and the determination coefficient (R (2)) was higher than 99.0 % with a residual deviation for each calibration point lower than ±25 %. Mean recoveries were between 85 and 115 %. The method was successfully applied to the analysis of both compounds, HMB and Leu, in samples obtained from an experiment of blood-brain barrier (BBB) passage in vitro and to an experiment of brain microdialysis in rats in vivo after an oral challenge with HMB to detect its appearance in the brain.
authors.affiliation: Discovery R&D, Abbott Nutrition Research and Development, Abbott Laboratories, Camino de Purchil 68, 18004, Granada, Spain, angela.santos@abbott.com.
authors.forename: A
authors.initials: A
authors.lastname: Santos-Fandila
authors.forename: A
authors.initials: A
authors.lastname: Zafra-Gómez
authors.forename: A
authors.initials: A
authors.lastname: Barranco
authors.forename: A
authors.initials: A
authors.lastname: Navalón
authors.forename: R
authors.initials: R
authors.lastname: Rueda
authors.forename: M
authors.initials: M
authors.lastname: Ramírez
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000737:chemistry* / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D007930:Leucine / Q000032:analysis* / Q000378:metabolism; D017551:Microdialysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014631:Valerates / Q000032:analysis* / Q000378:metabolism
chemical_list: D003470:Culture Media; D014631:Valerates; C004961:beta-hydroxyisovaleric acid; D007930:Leucine