Optimization of High Performance Liquid Chromatography Method for Simultaneous Determination of Some Purine and Pyrimidine Bases

Abstract

Nine purine and pyrimidine bases were separated and determined simultaneously using reversed phase (RP) high performance liquid chromatography (HPLC) in some food samples and biological fluids. Chromatographic behavior of these ionizable compounds highly depends on the interactions with the solvent as confirmed experimentally and by calculation of distribution of this species as a function of pH. Chromatograms show the optimal separation of five purine (uric acid, hypoxanthine, xanthine, adenine, and guanosine), and four pyrimidine (cytosine, uracil, cytidine and tymine) bases at pH around four. Accordingly, acetate buffer was selected due to high buffer capacity in this region. By variation of pH, concentration of buffer and volume ratio between buffer and methanol, we found that a mixture of 50 mM acetate buffer of pH 4.0 ± 0.1 with 3% of methanol ensures reproducibility, complete separation in less than 15 minutes and compatibility with UV and MS detection. Developed screening method was validated and applied for the analysis of complex clinical and beverage samples.

Electrochemical Characterization of Iron (III) Doped Zeolite-Graphite Composite Modified Glassy Carbon Electrode and Its Application for AdsASSWV Determination of Uric Acid in Human Urine

“…Thus, development of a simple, rapid, and sensitive method for continual monitoring of its level in human body fluids including urine is vital. A range of techniques including chromatography [7, 8], electrophoresis [9, 10], potentiometry [11], spectroscopy [12], and amperometry [13] have been reported for determination of UA in different samples.…”

The determination of salivary oxypurines before and after exercise by combined liquid chromatography-field asymmetric waveform ion mobility spectrometry-time-of-flight mass spectrometry

“…However, significant inter-day variations in salivary levels of HX and XA (and UA) have been reported by Kochańska et al, which may explain the variations in HX and XA concentrations observed in this study. Markelj et al (Kochanska et al 2000;Markelj et al 2016) reported the analysis of saliva and urine by LC-MS, which showed that concentrations of HX and XA were significantly lower, relative to the concentration of UA, in saliva than in urine, although salivary UA has been shown to have a linear correlation with serum UA concentrations (Soukup et al 2012).…”

“…HX and XA have been detected in plasma, serum or urine using a variety of methods including high performance liquid chromatography (HPLC) and capillary electrophoresis (Guan et al 2004(Guan et al , 2005Markelj et al 2016). These oxypurines have also been detected in saliva (Al-Shehri et al 2013;Guan et al 2004Guan et al , 2005Kochanska et al 2000;Markelj et al 2016), which is known to change in response to a variety of different physiological states (Dame et al 2015). However, saliva is a complex biofluid which can require careful sample preparation and/or extended analysis times to separate HX and XA from co-eluting matrix interferences.…”

Application of Carbon Nanotubes in Chiral and Achiral Separations of Pharmaceuticals, Biologics and Chemicals

“…Carboxylic-MWCNTs have been used in CZE and have shown promising achiral separations of purine and pyrimidine bases. Liquid chromatography has been widely used for the separation of pyrimidine and purine bases due to its high sensitivity, selectivity and reproducibility [ 158 , 159 , 160 ]. Capillary electrophoresis also showed promising separation behaviour for the polar and even not fully ionized compounds including pyrimidine and purine bases [ 161 , 162 ].…”