Leucine and iso Leucine Separated with LCMS - AppNote
May 10, 2012
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Leucine and Isoleucine, Isobaric Compounds Separated for LCMS Detection
This Application Note presents a Method for the Analysis of two Branch Chain Amino Acids (BCAA) with nearly identical m/z values.
Addition of weak Acetic Acid to the Mobile Phase allows the small difference in the pKas of the two Isobaric Compounds to achieve Resolution. A few microliters of Serum suffices as the Sample to estimate the Leucine and Isoleucine present. The two compounds are Separated and can be easily Quantified.
Column: Cogent Diamond Hydride™, 4 μm, 100 Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150 mm
Mobile Phase:
-- A: DI Water / 0.1% Acetic Acid
-- B: Acetonitrile / 0.1% Acetic Acid
Gradient:
Post Time: 5 minutes
Flow rate: 0.4 mL / minute
Detection: ESI – pos - Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: 100 ng / mL of each Compound prepared in 50:50 Solvent A / Solvent B and diluted 1:10.
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. The Method is especially applicable to the frequent monitoring of Serum while treating patients with Maple-Syrup Disease, and may also be used for rapid diagnosis of the disease in suspected infants.
[1] Chromatogram is adapted from: J.J.Pesek, M.T. Matyska, S. M. Fisher, T. R. Sana, Journal of Chromatography A, 1204 (2008) p50.
Attachment No 84 Leucine & iso Leucine Separated with LCMS pdf 0.1 Mb Download File
This Application Note presents a Method for the Analysis of two Branch Chain Amino Acids (BCAA) with nearly identical m/z values.
Addition of weak Acetic Acid to the Mobile Phase allows the small difference in the pKas of the two Isobaric Compounds to achieve Resolution. A few microliters of Serum suffices as the Sample to estimate the Leucine and Isoleucine present. The two compounds are Separated and can be easily Quantified.
Peaks:
1. D,L - Leucine
132.1025 m/z (M+H)+,
2. D,L - Isoleucine
132.1025 m/z (M+H)+
Column: Cogent Diamond Hydride™, 4 μm, 100 Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150 mm
Mobile Phase:
-- A: DI Water / 0.1% Acetic Acid
-- B: Acetonitrile / 0.1% Acetic Acid
Gradient:
| Time (minutes) | %B |
|---|---|
| 0-2 | 85 |
| 2-3 | 80 |
| 3-5 | 80 |
| 5-6 | 75 |
| 6-7 | 75 |
| 7-8 | 70 |
| 8-9 | 70 |
| 9-10 | 50 |
| 10-11 | 50 |
| 11-11.1 | 20 |
| 11.1-14 | 20 |
Flow rate: 0.4 mL / minute
Detection: ESI – pos - Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: 100 ng / mL of each Compound prepared in 50:50 Solvent A / Solvent B and diluted 1:10.
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. The Method is especially applicable to the frequent monitoring of Serum while treating patients with Maple-Syrup Disease, and may also be used for rapid diagnosis of the disease in suspected infants.
[1] Chromatogram is adapted from: J.J.Pesek, M.T. Matyska, S. M. Fisher, T. R. Sana, Journal of Chromatography A, 1204 (2008) p50.
Attachment No 84 Leucine & iso Leucine Separated with LCMS pdf 0.1 Mb Download File