Biochemistry, Department of
Date of this Version
2008
Citation
Electrophoresis. 2008 November ; 29(22): 4549–4560
Abstract
A new approach for the selective comparative metabolite profiling of carboxylic acids in rat urine was established using capillary electrophoresis-mass spectrometry (CE-MS) and a method for positively pre-charged and 2H-coded derivatization. Novel derivatizing reagents, N-alkyl-4-
aminomethylpyridinum 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-aminomethylpyridinum iodide (HAMP), 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 BAMP-d9, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and BAMP-d9. The metabolite profiling variation between these two samples was clearly visualized.
Comments
Open Access. Used by permission.