Glavnik Vesna, Vovk Irena, Albreht Alen
Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
J Chromatogr A. 2017 Jan 27;1482:97-108. doi: 10.1016/j.chroma.2016.12.059. Epub 2016 Dec 21.
On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol-formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MS methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6'-O-malonyl)-hexoside).
基于在线洗脱的薄层色谱-质谱联用技术现已成熟，但所获数据的质量有时会受到严重光谱背景或强离子抑制的影响，尤其是当硅胶板与展开溶剂中的酸性改性剂联合使用时。我们通过对硅胶板进行两次预展开，简单而有效地解决了这个问题，第一次用甲醇-甲酸（10:1，v/v），第二次用乙腈-甲醇（2:1，v/v）。这种方法显著提高了高效薄层色谱-质谱联用方法的灵敏度。该方法的适用性在虎杖（Fallopia japonica Houtt.）根茎粗提物中黄烷-3-醇和原花青素的分析中得到了验证。在高效薄层色谱硅胶板和高效薄层色谱硅胶质谱级板上，使用展开溶剂甲苯-丙酮-甲酸（3:3:1、6:6:1、3:6:1，v/v）和二氯甲烷-丙酮-甲酸（1:1:0.1，v/v）进行分离，然后用4-二甲基氨基肉桂醛（DMACA）检测试剂进行色谱后衍生化。对起始点处分析物稳定性的检测证实，在点样标准品和样品测试溶液后应立即展开硅胶板。在展开后的五小时稳定性测试中，我们发现了在280nm处吸收增强的意外现象。然而，基于用DMACA检测试剂进行色谱后衍生化的实验，在高效薄层色谱-质谱联用分析的时间范围内，分析物被证明具有足够的稳定性。这对于开发用于鉴定从单体到十聚体的黄烷-3-醇和B型原花青素的首个高效薄层色谱-质谱联用和高效薄层色谱-质谱方法很重要。基于此研究方法，首次在虎杖根茎中初步鉴定出三聚体、三聚体没食子酸酯、四聚体没食子酸酯、五聚体、五聚体没食子酸酯、六聚体、六聚体没食子酸酯、七聚体、八聚体、九聚体和十聚体。此外，所有开发的高效薄层色谱-质谱联用方法都能够同时鉴定芪类化合物（白藜芦醇、云杉新苷、白藜芦醇苷）和蒽醌类化合物（大黄素、大黄素-O-己糖苷、大黄素-O-（乙酰基）-己糖苷和大黄素-O-（6'-O-丙二酰基）-己糖苷）。
