University of Hohenheim, Institute of Food Chemistry, Garbenstrasse 28, 70599 Stuttgart, Germany.
J Chromatogr A. 2013 May 10;1289:105-18. doi: 10.1016/j.chroma.2013.03.005. Epub 2013 Mar 13.
An efficient HPTLC method was developed, which required minimal sample preparation for quantitation of the main anthocyanes in pomace, animal feed as well as various foods. The best separation of 11 anthocyanes was achieved on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate-2-butanone-formic acid-water for anthocyanins and ethyl acetate-toluene-formic acid-water for anthocyanidins. Due to the high flexibility of the HPTLC method, both anthocyane groups could be developed in a combined 2-step method. The second development was only necessary if anthocyanidins were detected in the samples. This normal phase separation was found superior to the best separation achieved on RP-18 phases with a mixture of water-n-propanol-formic acid. Absorbance measurement was performed using the multi-wavelength scan at 505 (or 510), 520, 530 and 555 nm. The correlation coefficients of the calibrations ranged between 0.9993 and 0.9999 for the 11 anthocyanes. LOQs were all ≤90 ng/zone, most even ≤30 ng/zone and for pn-3-glc and pg-3-glc even ≤7 ng/zone. With regard to the analysis of mv-3-glc in grape seed/marc meal and supplemented animal feed samples, the mean repeatabilities were 1.4% (laboratory 1) and 1.8% (laboratory 2). The intermediate precisions within a laboratory over several months were ≤6.7%. The ruggedness of the method was ≤5.5%. The method was transferred to other sample types. Juice and wine samples, which were from the same plant source, showed a comparable anthocyanin pattern, whereas the pattern was characteristically different between plant sources. Unknown anthocyanin sample components were analyzed via HPTLC-ESI-MS by eluting the zones of interest with the TLC-MS Interface, which was helpful for further characterization of unknowns. An interesting tool was demonstrated by effect-directed analysis with regard to radical scavenging properties and general bioactivity based on detection with Vibrio fischeri bacteria.
开发了一种高效的 HPTLC 方法，该方法仅需少量样品制备即可定量测定废渣、动物饲料和各种食品中的主要花色苷。在 HPTLC 板硅胶 60 F254 上，使用乙酸乙酯-2-丁酮-甲酸-水混合物分离 11 种花色苷，使用乙酸乙酯-甲苯-甲酸-水混合物分离花色苷苷元，可实现最佳分离。由于 HPTLC 方法的高度灵活性，可以使用两步法同时开发花色苷和花色苷苷元。如果样品中检测到花色苷苷元，则仅需进行第二次展开。这种正相分离优于使用水-正丙醇-甲酸混合物在 RP-18 相上获得的最佳分离。使用 505（或 510）、520、530 和 555nm 的多波长扫描进行吸光度测量。11 种花色苷的校准曲线相关系数在 0.9993 至 0.9999 之间。LOQs 均≤90ng/zone，大多数甚至≤30ng/zone，对于 pn-3-glc 和 pg-3-glc 甚至≤7ng/zone。关于葡萄种子/果皮粉和补充动物饲料样品中 mv-3-glc 的分析，实验室 1 的平均重复性为 1.4%，实验室 2 的平均重复性为 1.8%。在几个月内，同一实验室的中间精密度≤6.7%。该方法的稳健性≤5.5%。该方法已转移到其他样品类型。来自同一植物来源的果汁和葡萄酒样品显示出相似的花色苷模式，而植物来源之间的模式明显不同。通过使用 TLC-MS 接口洗脱感兴趣的区域，通过 HPTLC-ESI-MS 分析未知花色苷样品成分，这有助于进一步鉴定未知物。基于 Vibrio fischeri 细菌检测的自由基清除特性和一般生物活性的定向分析证明了这是一种有趣的工具。
