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. 2015 Sep 2;63(34):7567-73.
doi: 10.1021/acs.jafc.5b02563. Epub 2015 Aug 20.

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

Stefan Ehling  1 , Todime M Reddy  1
Affiliations

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

Stefan Ehling et al. J Agric Food Chem. .

Abstract

A direct, quantitative, and confirmatory method based on stable isotope dilution liquid chromatography-mass spectrometry was developed and validated for the analysis of leucine and metabolites β-hydroxy-β-methylbutyric acid (HMB), α-ketoisocaproic acid (KIC), and α-hydroxyisocaproic acid (HICA) in human breast milk. Chromatographic resolution was achieved between isobaric leucine and isoleucine. Accuracy and intermediate precision were 89-117% and <10% relative standard deviation (RSD) across three validation runs. Limits of quantitation for HMB, KIC, HICA, and leucine in human breast milk were 20 μg/L, 20 μg/L, 10 μg/L, and 1 mg/L. Measured concentrations of HMB, KIC, HICA, and free leucine in human breast milk from six donors at various stages of lactation were 42-164 μg/L, < 20-1057 μg/L, < 10 μg/L, and 2.1-88.5 mg/L. HMB and KIC were confirmed in human breast milk by orthogonal hydrophilic interaction chromatography (HILIC). This work provides a tool for further study of human breast milk composition and its effect on protein turnover in developing infants.

Keywords: human breast milk; leucine; liquid chromatography−mass spectrometry; α-hydroxyisocaproic acid (HICA); α-ketoisocaproic acid (KIC); β-hydroxy-β-methylbutyric acid (HMB).

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