Equipe de Chimie Analytique des Molécules Bio-Actives, Université de Strasbourg, Illkirch-Graffenstaden, France.
J Sep Sci. 2012 Aug;35(16):2026-31. doi: 10.1002/jssc.201200071. Epub 2012 Jul 2.
The largely adopted reversed-phase HPLC analysis of the molecular species of isothiocyanates (ITCs) was performed and showed losses during the chromatographic run with eight ITCs. These losses, which obviously impact the accuracy of quantitative determinations, were due to precipitation in the chromatographic system. At 22°C, they ranged from 5.4% for sulforaphane (SFN) to 11.0% for benzyl-ITC when ITCs were injected at 80 μg mL(-1) , but they were up to three times higher at 1 mg mL(-1) reaching 31.9% for benzyl-ITC. The water solubility of the ITCs was a key determinant of the extent of the measured loss. When the column was heated at 60°C, losses in injected ITCs were reduced, in comparison with 22, 40, and 50°C, by two to ten times depending on the ITC considered. A reversed-phase HPLC method based on column heating was suggested and its quantitative performance was determined. It was then applied to the separation of methylene chloride extracts of various cruciferous vegetables. Ally-ITC, SFN, and iberin in cabbage; SFN and iberin in cauliflower; and allyl-ITC and phenylethyl-ITC in horseradish could be identified and quantified. The obtained results cast doubt on quantitative determinations of ITCs that are carried out at room temperature using reversed-phase HPLC.
反相高效液相色谱法已广泛用于异硫氰酸酯（ITC）的分子种类分析，结果显示在八种 ITC 的色谱运行过程中存在损失。这些损失显然会影响定量测定的准确性，是由于色谱系统中出现沉淀造成的。在 22°C 下，当 ITC 以 80 μg mL(-1)的浓度进样时，八种 ITC 的损失范围从 5.4%的萝卜硫素（SFN）到 11.0%的苄基-ITC；但当 ITC 浓度提高至 1 mg mL(-1)时，损失率最高可达 31.9%，这也是苄基-ITC 的损失率。ITC 的水溶性是衡量测量损失程度的关键决定因素。与 22°C、40°C 和 50°C 相比，当柱温加热至 60°C 时，注入的 ITC 损失减少了 2 至 10 倍，具体取决于所考虑的 ITC。建议采用基于柱加热的反相高效液相色谱法，并对其定量性能进行了测定。然后将其应用于各种十字花科蔬菜的二氯甲烷提取物的分离。白菜中的丙烯基-ITC、SFN 和异硫氰酸-3-丁酯；花椰菜中的 SFN 和异硫氰酸-3-丁酯；辣根中的丙烯基-ITC 和苯乙基-ITC 都可以被识别和定量。这些结果表明，在室温下使用反相高效液相色谱法进行 ITC 的定量测定值得怀疑。
