Isotopic labeling of multiple functional groups for improving the quantification of urine metebolome

Abstract

A high proportion of metabolites in human urine possess hydroxy and amino groups. Dangly chloride has been widely used in chemical isotope labeling to detect the compounds with phenol and amines by liquid chromatography‐mass spectrometry (LC‐MS), but not for alcohols. Therefore, we carried out the chemical isotope labeling with two kinds of catalysts (DMAP and DIPEA) for labeling both amino group and hydroxy group simultaneously using 12C or 13C‐dansyl chloride. After labeling, the products were extracted with water for three times and collected the organic layer. We dried the extracted sample, separated as two aliquots which were labeled separated with 12C / 13C‐dansyl chloride. The mixtures of 12C / 13C‐labeled metabolites were analyzed by LC‐MS for metabolite identification and quantification. Using penal and cholesterol as standards, this new dansylation method is proved be able to label the compounds with phenol and hydroxyl groups. To optimize the whole work flow, we optimized the variables including reaction solvent ratio, reaction time, reaction volume and compared this technique with other dansylation methods in the peak pairs, retention time and the functional group of labeled metabolites. In summary, this technique improves the detection of many metabolites in LC‐MS analysis, particularly hydroxy group and amino group.