HPLC Separation of β‑Hydroxy-β-methylbutyric Acid, 2-Hydroxyisocaproic Acid and Leucine on Primesep 100 Column
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Description
High Performance Liquid Chromatography (HPLC) Method for Analysis of β‑Hydroxy-β-methylbutyric Acid (HMB) , D-Leucine , Leucine , 2-Hydroxyisocaproic Acid (HICA)
Both β-hydroxy-β-methylbutyric acid (HMB) and 2-hydroxyisocaproic acid (HICA) are metabolites of the essential dietary branched chain amino acid leucine.
β‑Hydroxy-β-methylbutyric Acid (HMB) , D-Leucine , Leucine , 2-Hydroxyisocaproic Acid (HICA) can be retained and analyzed using the Primesep 100 stationary phase column. The analysis utilizes a gradient method with a simple mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid buffer. Detection is performed using UV.
Method Parameters
| Mobile Phase | MeCN – 5%, 3 min hold, Gradient MeCN 5-30% 12 min |
|---|---|
| Buffer | H2SO4 – 0.05 % 3 min hold, GradientH2SO4 0.05-3% 12 min |
| Flow Rate | 1.0 ml/min |
| Detection | UV, 210 nm |
| Class of Compounds | Acid, Hydrophilic, Ionizable, Normal human metabolite, Dietary supplement, Essential amino acid, Branched-chain amino acid (BCAA) |
| Analyzing Compounds | β‑Hydroxy-β-methylbutyric Acid (HMB),D-Leucine,Leucine,2-Hydroxyisocaproic Acid (HICA) |
HPLC Column Used
Primesep 100, 4.6 x 150 mm, 5 µm, 100 A, dual ended