Cogent Diamond Hydride™ 4µm, 100Å. 70000-15P-2 2.1 x150 mm A: DI water + 0.1% acetic acid B: acetonitrile + 0.1% acetic acid Mobile phase: Gradient: 0 min 85% B 0–2 min 85.0% B 2–3 min to 80% B 3–5 min 80%B 5–6 min to 75%B 6–7 min 75% B
Post time: Flow rate: Sample:
7–8 min to 70% B 8 to 9 min 70% B 9–10 min to 50% B 10–11 min 50% B 11–11.1 min to 20% B 11.1–14 min hold 20% B
5 min 0.4 mL/min. 1. D,L - Leucine 132.1025 m/z (M + H)+ 2. D,L - Isoleucine 132.1025 m/z (M + H)+ 100 ng/mL of each prepared in 50%A/50%B and diluted 1:10 before analysis.
Chromatogram presented is adapted from: J.J.Pesek, M.T. Matyska, S. M. Fisher, T. R. Sana, Journal of Chromatography A, 1204 (2008) p50.
Notes: D,L - Leucine and D,L - Isoleucine are considered essential amino acids because human beings cannot survive unless these amino acids are present in the diet.
Cat. No. 70000-15P-2
Detection:
ESI – pos - Agilent 6210 MSD TOF mass spectrometer.
Discussion The chromatogram presented was done using a Cogent Diamond column and an ANP gradient. The addition of weak acetic acid to the mobile phase allows the small difference in the pKas of the two compounds to be used to achieve the resolution for this pair of analytes which have identical m/z values. A few microliters of serum suffices to estimate the leucine and isoleucine present. The two branched-chain amino acids are separated and can be easily quantified. The method is especially applicable to the frequent monitoring of serum in treating patients with maple-syrup disease, and may also be used for rapid diagnosis of the disease in suspected infants.
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