A method of high performance liquid chromatography-fluorescence detection-mass spectrometry identification (HPLC-FLD-MS) coupled with pre-column derivatization to determine bile acids (BAs) from bio-sample has been developed here. The labeling reagent of 2-(2-(pyren-1-yl)-1H-benzo[d]imidazol-1-yl)-ethyl-4-methyl benzenesulfonate (PBITS) contains six benzene rings and two weak basic nitrogens in the structure, resulting in a higher degree of conjugation and perfect ionization efficiency. The PBITS could easily and quickly label BAs at the optimized derivatization conditions and the labeling yield was close to 100%. Separation of the derivatized BAs exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Hypersil BDS C₁₈ column. Calculated detection limits for HPLC-FLD, at a signal-to-noise ratio of 3, were 10.09–16.33 fmol. Excellent linear responses were observed with correlation coefficients >0.9995. The mean inter-day precision for all standards was <3.63% and the experimental recoveries were 89.9–106.3%. Good compositional data were obtained from the analysis of the extracted BAs from pig bile sample. Therefore, the facile PBITS derivatization coupled with HPLC-FLD-MS analysis allowed the development of a highly sensitive method for the quantization of trace levels of BAs from biological samples.

中文翻译：

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2-（2-（Pyren-1-yl）-1 H-苯并[d]咪唑-1-基）-乙基-4-甲基苯磺酸酯（PBITS）及其在HPLC-FLD-MS测定胆汁酸中的应用†

本文开发了一种高效液相色谱-荧光检测-质谱鉴定（HPLC-FLD-MS）结合柱前衍生化方法从生物样品中测定胆汁酸（BAs）的方法。2-（2-（吡喃-1-基）-1 H-苯并[d]咪唑-1-基）-乙基-4-甲基苯磺酸盐（PBITS）的标记试剂含有6个苯环和2个弱碱性氮结构，导致更高的共轭度和完美的电离效率。PBITS可以在优化的衍生化条件下轻松快速地标记BA，且标记收率接近100％。衍生化BA的分离具有良好的基线分辨率，并在反相Hypersil BDS C ₁₈上进行了梯度洗脱柱子。信噪比为3时，HPLC-FLD的检测限为10.09–16.33 fmol。观察到极好的线性响应，相关系数> 0.9995。所有标准液的平均日间精确度均＜3.63％，实验回收率为89.9–106.3％。从猪胆汁样品中提取的BAs的分析获得了良好的成分数据。因此，简便的PBITS衍生化与HPLC-FLD-MS分析相结合，为开发定量分析生物样品中痕量BA的高度灵敏的方法提供了可能。