Dansylhydrazine Isotope Labeling LC-MS for Comprehensive Carboxylic Acid Submetabolome Profiling

High-performance chemical isotope labeling (CIL) LC-MS is an important tool for profiling chemical-group-based submetabolomes using different labeling chemistries for quantitative metabolomics. Metabolites containing carboxylic acid groups are a class of molecules playing diverse and significant roles in many metabolic pathways. We report a relatively simple and convenient method for analyzing the carboxyl submetabolome with high coverage. Dansylhydrazine (DnsHz) labeling of the carboxylic acid group in metabolites is used to improve both separation and ionization in LC-MS. Using differential ¹²C- and ¹³C-DnsHz reagents for labeling individual samples and a reference sample (e.g., a pooled sample), accurate relative quantification of individual metabolites among comparative samples can be achieved. DnsHz labeling of carboxylic acids could be carried out at room temperature in water-containing solution in 2 h. A labeled-carboxyl-standard library consisting of 193 endogenous human metabolites was constructed for metabolite identification, which covers more than 55 pathways. To demonstrate the performance of this method, analysis of triplicate human urine samples with duplicate injections ( n = 6) was carried out. A total of 2266 peak pairs or metabolites were commonly detected in all six runs; among them, 81 peak pairs were positively identified and 517 and 1445 peak pairs were matched in the zero- and one-reaction MyCompoundID metabolome libraries, respectively, on the basis of accurate mass search. The possibility of using this method for comprehensive profiling of the carboxyl submetabolome in real world samples was demonstrated in metabolome comparison of 30 urine samples of female and male individuals as well as in absolute quantification of two urinary acids.