High Performance Liquid Chromatography of Plasma Pyrimidines and Purines and Its Application in Cancer Chemotherapy

Abstract

A variety of sensitive high performance liquid chromatography (HPLC) methods have been reported for the separation of pyrimidine and purine compounds^1–4. These methods have involved anion- and cation-exchange and reverse phase chromatography. The use of these common techniques for the analysis of plasma is sometimes hampered by the incomplete fractionation of bases and nucleosides or inadequate separation of these compounds from other plasma components. Also, in most cases elution has required more than one buffer or the use of a gradient. Eksteen et al.⁵ recently demonstrated that bases and nucleosides could be rapidly and efficiently separated by HPLC using an anion-exchange resin and isocratic elution with an alcoholic phosphate buffer. Optimal results were reported achieved using 0.05 M sodium phosphate-0.005 M citric acid, pH 9.25, in 55% ethanol as the eluent and a column temperature of 70°. We initially attempted to use the same method for the analysis of plasma for pyrimidines and purines. However, it was noted that uric acid was not well separated from oxypurines. Moreover, using the conditions described above, we encountered poor column stability and inconsistent flow rates. By decreasing the phosphate and ethanol concentrations in the eluent, it was possible to obtain good column stability.

Supported in part by the Queen Wilhelmina Fund (Project UUKC 77–3).