On-Line Recognition and Quantitation of Coeluting Hypoxanthine and Guanine in Reversed-Phase High-Performance Liquid Chromatography of Placental Tissue Extracts: Photodiode-Array Detection and Spectral Analysis of Coeluting Peaks

Abstract

In a reversed-phase HPLC method, optimized for resolution and quantitation of nucleosides and nucleobases in human term placental extracts, guanine coeluted with hypoxanthine. Three numerical techniques that treat spectral data generated by photodiode-array detection to yield indices of peak purity were compared for sensitivity of on-line recognition of overlapped guanine and hypoxanthine peaks in chromatograms of 7.0 μM hypoxanthine containing 0.075-0.54 μM guanine. These techniques were purity parameter, the average wavelength of a peak spectrum (λw) peak area ratio, and peak absorbance ratio. Purity parameter of a 7.0 μM hypoxanthine peak containing 1% guanine was significantly different from that of pure hypoxanthine (P < 0.01); peak area and peak absorption ratios were four and seven times, respectively, less sensitive than purity parameter. In chromatograms of human term placental extracts, purity parameter values generated on-line signalled the presence of guanine in hypoxanthine peaks. Quantitation of coelutiaghypoxanthine and guanine was performed on-line from dual-wavelength detection at 249 and 278 nm via calibration curves for both analytes at these wavelengths. Hypoxanthine and guanine concentrations in seven samples from five human term placentas were 41-161 and 3-76 nmol/g, respectively. These findings demonstrate that on-line signalling of hypoxanthine and guanine coelution is achieved by purity parameter, area ratio, and absorbance ratio, that purity parameter is the most sensitive index of coelution, and that quantitation of the coeluting analytes can be performed on-line. These approaches should be applicable to recognition and quantitation of coeluting analytes in chromatograms ofmixtures of nucleosides and nucleobases such as occur in extracts of other tissues.