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.
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.
Abbott Laboratories , 3300 Stelzer Road, Columbus, Ohio 43219, United States.
Stefan
S
Ehling
Abbott Laboratories , 3300 Stelzer Road, Columbus, Ohio 43219, United States.
Todime M
TM
Reddy
D002087:Butyrates / Q000737:chemistry* / Q000378:metabolism; D002208:Caproates / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D006880:Hydroxy Acids / Q000737:chemistry* / Q000378:metabolism; D007651:Keto Acids / Q000737:chemistry* / Q000378:metabolism; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008895:Milk, Human / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure
C521666:2-hydroxy-2-methylbutyric acid; D002087:Butyrates; D002208:Caproates; D006880:Hydroxy Acids; D007651:Keto Acids; C019419:alpha-hydroxyisocaproic acid; C013082:alpha-ketoisocaproic acid; D007930:Leucine
human breast milk; leucine; liquid chromatography−mass spectrometry; α-hydroxyisocaproic acid (HICA); α-ketoisocaproic acid (KIC); β-hydroxy-β-methylbutyric acid (HMB)