替代标准品校正函数法定量分析不产氧光合细菌螺菌黄质系类胡萝卜素

Quantification of carotenoids of spirilloxanthin series from anoxygenic phototrophic bacteria by substitute reference standard calibration function method

【目的】针对不产氧光合细菌(APB)类胡萝卜素(Car)标准品缺乏的问题,以替代标准品实现螺菌黄质系多种Car组分同步、快速、准确的定量分析。【方法】以沼泽红假单胞菌CQV97为材料,采用吸收光谱、薄层层析和HPLC等方法制备螺菌黄质系Car标准品;以柠檬黄和番茄红素为替代标准品,采用HPLC法,建立了螺菌黄质系Car多组分的定量方法。【结果】制备的6种Car标准品纯度达95%以上。确定了Car的HPLC分析条件,以Car标准品为对照,得到了螺菌黄质系6种Car组分的定性HPLC指纹图谱。在选择的HPLC条件下,测定了6种Car标准品和2种替代标准品的标准曲线,确立了2种替代标准品分别与6种Car标准品之间的定量校正函数关系,并用于实际样品YL28和CQV97菌株的Car定量分析。采用Car标准品法测定的6种Car含量的RSD小于1.5%、回收率在96%-104%,替代标准品法与Car标准品法测定结果吻合,其相对误差小于0.1%。【结论】通过替代标准品校正函数关系,建立了2种准确定量分析螺菌黄质系Car多组分的方法。替代标准品柠檬黄和番茄红素均能准确地传递待测Car的量值关系,实现了螺菌黄质系6种Car的同步、快速、准确的定量分析,弥补了现有Car组分相对定量方法的不足。讨论了替代标准品法校正因子适用范围的局限性,提出了校正函数关系的思路和方法。这为全面实现APB球形烯系、奥氏酮系等其它Car的快速、准确定量分析提供了借鉴和参考。

[ Objective] In this study, we developed a strategy for accurate, rapid and simultaneous quantification of six carotenoids by substitute reference standard. [ Methods] We prepared six carotenoid standards of spirilloxanthin series from Rhodopesudomonnas palustris CQV97 by speetrophotometry, thin layer chromatography and HPLC. The simultaneous quantification method for six earotenoids was established by HPLC using tartrazine and lyeopene as substitute reference standards. [ Results] We established the HPLC fingerprinting of earotenoids of spirilloxanthin series. The quantitative calibration function relationships between two substitute reference standards and six earotenoids were explored. Based on the quantitative calibration function relationships, we quantitatively analyzed carotenoid contents of two samples of CQV97 and YL28 strains. The RSD and recovery of carotenoid contents determined by substitute reference standards method were consistent with quantitative analysis of carotenoid standards method. [ Conclusion] The substitute reference standards were capable of accurately transmitting the quantitative relationship of tested samples. The method could realize the simultaneous quantification of six earotenoids.