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Phosphorylated Sugars by LCMS
UDP, ADP, TDP and CDP
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printable Application Sheet
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Notes: Please note the addition of small amount of ammonia to the sample matrix. The alkaline environment of the
sample matrix assured efficient and symmetrical peaks for all analytes.
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Discussion:
UDP hexanolamine was used as the internal standard in the analysis of nucleotide sugars in this rapid analysis method. The mobile
phase was designed to maximize the detector response in LC-MS for maximum efficiency. The simple “inverse gradient” which produces
ANP (aqueous normal phase) HPLC method was required for the results shown therefore a Cogent Diamond Hydride column was chosen. This
method can be used in measuring metabolite concentration.
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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% ammonium formate pH 7.2
B: 90%acetonitrile + 10% DI water + 0.1% ammonium formate pH 6 |
| Mobile Phase |
Gradient:
| Time |
%B |
Time |
%B |
| 0.0 |
95 |
12.1 |
95 |
| 10.0 |
75 |
15.0 |
95 |
| 12.0 |
75 |
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| Flow rate |
0.3 µL/min. |
| Peaks |
- ADP – glucose, RT = 3.68 min, monitored MRM
transitions were m/z 588 to m/z 346
- Proprietary sugar nucleotide, RT = 6.03 min,
monitored MRM transitions were m/z 563 to m/z 321
- Proprietary sugar nucleotide, RT = 6.20 min,
monitored MRM transitions were m/z 606 to m/z 385
- CDP – glucose, RT = 7.13 min, monitored MRM
transitions were m/z 564 to m/z 322
- UDP hexanolamine (internal standard), RT = 8.40 min,
monitored MRM transitions were m/z 502 to m/z 258
MRM – multiple reaction monitoring in LC/MS/MS
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| Sample Preparation |
400 µL DI water + 400 µL acetonitrile + 20 µL of stock
solution of each compound + 5 mL of 12% ammonia |
| Detection |
ESI – neg - Agilent 6210 MSD TOF mass spectrometer |
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