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.
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.
复制标题
胎盘组织提取物反相高效液相色谱中共洗脱次黄嘌呤和鸟嘌呤的在线识别和定量：光电二极管阵列检测和共洗脱峰的光谱分析。
DOI：
10.1006/abio.1993.1583
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发表时间：
1993
影响因子：
2.9
通讯作者：
Maguire,MH
中科院分区：
文献类型：
--
作者：
Szabo,I;Maguire,MH
在反相HPLC方法中，优化了人足月胎盘提取物中核苷和核碱基的分离和定量，鸟嘌呤与次黄嘌呤共洗脱。本文比较了三种处理光电二极管阵列检测器产生的光谱数据以产生峰纯度指数的数值技术，用于在线识别含0.075-0.54 μM鸟嘌呤的7.0 μM次黄嘌呤色谱图中重叠的鸟嘌呤和次黄嘌呤峰的灵敏度。这些技术是纯度参数、峰光谱的平均波长（λw）、峰面积比和峰吸光度比。含1%鸟嘌呤的7.0 μM次黄嘌呤峰的纯度参数与纯次黄嘌呤的纯度参数有显著差异（P < 0.01），峰面积和峰吸收比分别为4倍和7倍，灵敏度低于纯度参数。在人足月胎盘提取物的色谱图中，在线生成的纯度参数值表明次黄嘌呤峰中存在鸟嘌呤。通过在249和278 nm处的双波长检测，通过在这些波长处的两种分析物的校准曲线，在线定量腔上囊次黄嘌呤和鸟嘌呤。从5个人类足月胎盘的7个样本中，次黄嘌呤和鸟嘌呤的浓度分别为41-161和3-76 nmol/g。这些研究结果表明，在线信号的次黄嘌呤和鸟嘌呤共洗脱实现的纯度参数，面积比，吸光度比，纯度参数是最敏感的指标共洗脱，和共洗脱分析物的定量可以进行在线。这些方法应适用于识别和定量的共洗脱分析物在色谱ofmixtures的核苷和核碱基，如发生在其他组织的提取物。
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.