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Purine Nucleotides
      Fast Separation of XMP and GTP

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Notes:
Among the most important purine nucleotides, XMP and GTP are often analyzed from the lymphocytes of healthy people and HIV-1 seropositive patients at different stages of the disease (ARC-AIDS). Several differences in metabolism of purine nucleotides in the lymphocytes of the AIDS patients, were observed [2]. XMP does not normally appear in free nucleotide cell extracts, however it is the product of the important cell differentiation enzyme IMP dehydrogenase in ribavirin therapies.

[1]. “Aqueous normal phase retention of nucleotides on silica hydridebased columns: Method development strategies for analytes relevant in clinical analysis”, M.T. Matyska, J.J. Pesek, J. Duley, M. Zamzami, S.M. Fisher, J. Sep. Sci. (2010/, 33, 930-938.
[2]. “Analysis of purine nucleotides in lymphocytes from healthy subjects and AIDS patients”, A Tabucchi, F Carlucci, E Ramazzotti, MC Re, F Marinello, M Rubino, R Pagani, Biomedecine & Pharmacotherapy, Volume 46, Issue 1, 1992, Pages 25-29.
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
B: 90% acetonitrile/10% DI water/0.1% ammonium formate
Gradient
Time (min) %B
0.095.0
0.595.0
10.075.0
15.030.0
20.030.0
20.195.0
Post time 5 min.
Temperature 25°C
Flow rate 0.4 mL/min.
Injection 1µL
Samples
  1. XMP: Xanthosine -5'-monophosphate
  2. GTP: Guanosine -5'-triphosphate
Detection UV diode array
using chromatograms at 254 nm and 280 nm, extracted from the UV spectra scanned from 230 to 310 nm for all runs.

Discussion
This fast, simple and easy to use method achieves the separation of nucleotides XMP from GTP. The analysis of these polar compounds is achieved at high concentration of an organic solvent as part of the mobile phase (usually acetonitrile, but acetone may be used as well when MS detection is used) which provides increased sensitivity. Retention times were very reproducible with %RSD approximately 0.4, even when red blood cells extracts were injected in between the standard samples. Using a mass sprectometer this method can be specific and sensitive.




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