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Separation of ATP, UDP-Glucose UDP-Galactose
Separation of ATP, UDP-Glucose UDP-Galactose
Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-7.5P |
| Dimensions |
4.6 x 75 mm |
| Solvents |
A: DI water/0.1% ammonium acetate
B: 90% acetonitrile/10% DI water/0.1% ammonium acetate |
| Gradient |
| Time (min) | %B | Time (min) | %B |
| 0.0 | 100.0 | 10.0 | 80.0 |
| 1.0 | 95.0 | 10.5 | 50.0 |
| 8.0 | 95.0 | 14.0 | 50.0 |
| 9.0 | 80.0 | 14.0 | 100.0 |
|
| Post time |
5 min. |
| Flow rate |
0.4 mL/min. |
| Injection |
1mL |
| Samples |
- UDP-GLU:
UDP-glucose: uridine 5'-diphosphateglucose, 1 mg/mL
- UDP-GAL:
UDP-galactose: uridine 5'-diphosphategalactose, 1 mg/mL
- ATP:
Adenosine 5'-triphosphate, 1 mM
|
| Detection |
UV:254 nm |
Discussion
Figure A shows a sample (spiked with UDP-Galactose) of a red blood cell extract from a patient
for monitoring. Figure B shows separation of standards using the same method. Retention times
were very reproducible with %RSD around 0.4. The desired separation between UDP-Glu and UDP-Gal
was achieved, all other nucleotides eluted later and did not interfere with the analysis. ATP
represents the most abundant nucleotide in the red blood cell extracts and in this example it
eluted after both analytes.
Notes: Even with UV detection it is possible to detect individual nucleotides in the presence of
a biological matrix. The retention time can be compared to the standard (Figure B) showing that
the values of the real sample and the standard are very close. UDP-glucose, UDP-galactose and
galactose 1-phosphate determination can be used for diagnosis of galactosemia in newborn babies
[1-2].
1. Ji-Seon Jeong, Hye-Ran Yoon, Seon-Pyo Hong, Development of a new diagnostic method for
galactosemia by high-performance anion-exchange chromatography with pulsed amperometric
detection, J. Chromatography A, 1140 (2007) 157-162.
2. M.T. Matyska, J.J. Pesek, J. Duley, M. Zamzami, S.M. Fischer, Aqueous normal phase
retention of nucleotides on silica hydride-based column: Method development strategies for
analytes relevant in clinical analysis, J. Sep. Sci. 33 (2010) 930-938.
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