High-Performance Isotope Labeling for Profiling Carboxylic Acid-Containing Metabolites in Biofluids by Mass Spectrometry

Abstract

We have developed a new isotope labeling method, based on the use of isotope-coded p-dimethylaminophenacyl (DmPA) bromide as a reagent, combined with liquid chromatography-mass spectrometry (LC-MS) for high-performance metabolome analysis with a focus on profiling carboxylic acid-containing metabolites. Derivatization is simple, fast (1 h plus 30 min for quenching the reaction), and applicable to a wide range of carboxylic acids with a high yield and little or no side reaction products. This labeling method is demonstrated to be not only effective in introducing an isotope tag for accurate metabolite quantification but also improving the chromatographic retention of the metabolites in reversed-phase (RP) LC, enhancing ESI efficiency by 2-4 orders of magnitude, and facilitating the identification of metabolite peaks in LC-MS. In triplicate experiments of a 1:1 ratio of (13)C-/(12)C-DmPA labeled human urine, we were able to detect 2671, 2546, and 2820 ion pairs from metabolites containing one or more carboxylic acid groups.

Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

“…CIL LC-MS has been shown to provide high accuracy and precision for metabolome profiling [1][2][3][4], which can be attributed to simultaneous measurement of the co-eluted analytes and their isotope counterparts to overcome ion suppression, instrument drift and other technical problems. Furthermore, chemical derivatization provides a useful means of improving the separation of polar or ionic metabolites in reversed phase (RP) LC as well as enhancing the ionization efficiency in electrospray ionization (ESI) by introducing a relatively hydrophobic and easily chargeable ESIactive tag [1,5,6].…”

“…To date, several CIL reagents have been developed to quantify organic amines, carboxylic acids, carbonyl compounds and some other types of metabolites with varying degrees of performance [1,4,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. These research activities are mainly driven by the need of dealing with different classes of metabolites; a universally applicable derivatization reagent for LC-MS is not available at the present.…”

“…On the other hand, chemical labeling can be used as a way of dividing the entire metabolome into several submetabolomes according to their functional groups (e.g., amine submetabolome, acid submetabolome, etc. ), thereby reducing the complexity of a metabolomic sample [5,25]. This divide-andconquer approach requires the use of robust chemical labeling reagents for profiling submetabolomes with different intended purposes (e.g., untargeted quantification, targeted quantification, chemical identification, etc.…”

Chemical Isotope Labeling LC-MS for Monitoring Disease Progression and Treatment in Animal Models: Plasma Metabolomics Study of Osteoarthritis Rat Model

“…In our previous work, we have shown that CIL LC-MS using a rationally designed isotope labeling reagent could increase the metabolite detectability and quantification accuracy significantly for chemical-group-based submetabolome profiling2324. For example, 13 C-dansyl or 12 C-dansyl chloride could be used to differentially label amine- and phenol-containing metabolites for relative quantification of the amine/phenol submetabolomes with high coverage2325.…”

Chemoselective reactions and their applications in metabolite isolation and analysis.

“…CAs are predominantly analyzed using gas chromatography coupled to mass spectrometry (GC-MS) after their chemical derivatization with reagents such as boron trifluoride / butanol [184], hexamethyldisilazane [185], N-methyl-N-tertbutyldimethylsilyltrifluoroacetamide [186] or N-methyl-N-trimethylsilyltrifluoroacetamide [187,188]. Another common technique for analysis of organic CAs is anion exchange chromatography [189] or ion exclusion chromatography [190] coupled to mass spectrometry.…”