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Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry

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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.

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Acknowledgments

The authors are indebted to all the participants, without whom this work would not have been possible. The authors are grateful to the team of technicians from Abbott which helped in the tasks of care and maintenance of animals and in the surgical process that allowed the microdialysis experiments, especially María Luisa Jiménez López. Finally, the authors are grateful to Marti Bergana (Principal Research Scientist, Abbott Nutrition Columbus, Ohio) for his valuable suggestions in the manuscript.

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Authors and Affiliations

  1. Discovery R&D, Abbott Nutrition Research and Development, Abbott Laboratories, Camino de Purchil 68, 18004, Granada, Spain

    A. Santos-Fandila, A. Barranco, R. Rueda & M. Ramírez

  2. Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, 18071, Granada, Spain

    A. Santos-Fandila, A. Zafra-Gómez & A. Navalón

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  1. A. Santos-Fandila
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Correspondence to A. Santos-Fandila.

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Santos-Fandila, A., Zafra-Gómez, A., Barranco, A. et al. Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry. Anal Bioanal Chem 406, 2863–2872 (2014). https://doi.org/10.1007/s00216-014-7694-y

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