Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic.
Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 3, 166 28 Prague, Czech Republic.
Int J Mol Sci. 2022 May 20;23(10):5743. doi: 10.3390/ijms23105743.
Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), H, and C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.
磺化反应是自然界中的一种重要反应，而磺酸化酚类化合物作为哺乳动物 II 相代谢物或前药的标准物质备受关注。这些标准物质可以使用芳基磺基转移酶的化学酶法来制备。本工作的目的是获得大量的磺酸化酚类、酚酸类、类黄酮类和黄酮木脂素类化合物，并对其进行 HPLC（高效液相色谱）分析的优化。本文制备了 2,3,4-三羟基苯甲酸、儿茶酚、4-甲基儿茶酚和根皮素的四个新硫酸盐，并使用 MS（质谱）、H 和 C NMR 对其进行了充分表征。使用 HPLC 与 PDA（光电二极管阵列）检测对分离进行了研究，并对 38 种酚类及其硫酸盐标准物质进行了分离。比较了不同的固定相（整体式 C18、C18 极性、五氟苯基、ZICpHILIC）和有无乙酸铵缓冲液的流动相。分离结果强烈依赖于流动相的 pH 值和缓冲能力。所开发的稳健的 HPLC 方法适用于类黄酮、黄酮木脂素、2,3-脱氢黄酮木脂素、酚酸和酚类的酶促磺化反应混合物的分离，并用 PDA 检测。此外，由于低流速以及流动相中没有磷酸盐缓冲液和/或离子对试剂，该方法可直接与质量检测联用。
