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Branched-chain amino acids (BCAAs)
Leucine, Isoleucine
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printable Application Sheet
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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.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 0.1% acetic acid
B: acetonitrile + 0.1% acetic acid |
| Mobile Phase |
Gradient:
| 0 min 85% B | 7–8 min to 70% B |
| 0–2 min 85.0% B | 8 to 9 min 70% B |
| 2–3 min to 80% B | 9–10 min to 50% B |
| 3–5 min 80% B | 10–11 min 50% B |
| 5–6 min to 75% B | 11–11.1 min to 20% B |
| 6–7 min 75% B | 11.1–14 min hold 20% B |
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| Post time |
5 min. |
| Flow rate |
0.4 mL/min. |
| Sample |
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.
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| 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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