A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE-MS and a method for positively pre-charged and 2H-coded derivatization. Novel derivatizing reagents, N-alkyl-4-aminomethyl-pyridinum iodide (alkyl = butyl, butyl-d9 or hexyl), containing quaternary amine and stable-isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99-1.32% RSD of migration time) and eliminated problems with capillary coating known in CE-MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N-butyl-4-aminomethylpyridinum iodide (BAMP) and N-hexyl-4-aminomethyl-pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE-MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.
- Carboxylic acid derivatives
- Group-specific internal standard technology
ASJC Scopus subject areas
- Analytical Chemistry
- Clinical Biochemistry