Kanemitsu Yoshitomi, Asaji Kei, Matsumoto Yotaro, Tsukamoto Hiroki, Saigusa Daisuke, Mukawa Chikahisa, Tachikawa Tatsuki, Abe Takaaki, Tomioka Yoshihisa
Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Nov 15;1068-1069:1-8. doi: 10.1016/j.jchromb.2017.10.009. Epub 2017 Oct 5.
Column choice is crucial to the development of liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods because analyte selectivity is dependent on the nature of the stationary phase. Recently, mixed-mode chromatography, which employs a combination of two or more stationary phases and solvent systems, has emerged as an alternative to multiple, complementary, single-column systems. This report describes the development and validation of a novel analytical method based on LC-MS/MS employing a reversed-phase/cation-exchange/anion-exchange tri-modal column (Scherzo SS-C18; Imtakt) for the simultaneous quantification of various uremic toxins (UTx), including creatinine, 1-methyladenosine, trimethylamine-N-oxide, indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and 4-ethylphenyl sulfate. Stable isotope-labeled compounds were prepared as internal standards (ISs) for each analyte. Mobile phase optimization and appropriate gradient conditions resulted in satisfactory retention and peak resolution that could not have been attained with a single stationary phase LC system. The essential validation parameters, including intra- and inter-assay precision and accuracy, were adequate. The validated method was applied to measure serum levels of the aforementioned compounds in 19 patients with chronic kidney disease. This is the first report detailing the simultaneous quantification of these analytes using stable isotopes as ISs. Our results suggest that Scherzo SS-C18 columns will be considered breakthrough tools in the development of analytical methods for compounds that are difficult to quantify simultaneously in traditional LC systems.
色谱柱的选择对于液相色谱/串联质谱（LC-MS/MS）方法的开发至关重要，因为分析物的选择性取决于固定相的性质。最近，混合模式色谱法应运而生，它采用两种或更多种固定相和溶剂系统的组合，可替代多个互补的单柱系统。本报告描述了一种基于LC-MS/MS的新型分析方法的开发与验证，该方法采用反相/阳离子交换/阴离子交换三模式色谱柱（Scherzo SS-C18；Imtakt）同时定量多种尿毒症毒素（UTx），包括肌酐、1-甲基腺苷、氧化三甲胺、硫酸吲哚酚、对甲酚硫酸盐、苯硫酸盐和4-乙基苯硫酸盐。为每种分析物制备了稳定同位素标记的化合物作为内标（IS）。流动相优化和适当的梯度条件产生了令人满意的保留和峰分辨率，这是单一固定相LC系统无法实现的。包括批内和批间精密度与准确度在内的关键验证参数均符合要求。经验证的方法应用于测量19例慢性肾脏病患者血清中上述化合物的水平。这是第一份详细描述使用稳定同位素作为内标同时定量这些分析物的报告。我们的结果表明，Scherzo SS-C18色谱柱将被视为开发传统LC系统中难以同时定量的化合物分析方法的突破性工具。
