Relative quantification of carboxylic acid metabolites by liquid chromatography-mass spectrometry using isotopic variants of cholamine

Abstract

Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC-MS conditions, regardless of mobile-phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reversed-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for 10 observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements.

Figure 1

Relative quantification by isotopic labeling. Metabolites containing a certain functional group are derivatized with light- and heavy- isotopic tags prior to mixing the two samples and LC-MS analysis. The ratio of mass spectral peak intensities for each metabolite (e.g. M1_L/M1_H) provides relative quantification between samples A and B. Note that the two adjacent (large and small) peaks for each compound (e.g. M1_L) reflect the natural isotopic distributions of these small molecules.

Figure 2

Extracted ion chromatograms (left) and representative mass spectra (right) of light- and heavy-cholamine labeled fatty acids. For clarity, the EICs are divided between two panels: the upper one shows five more abundant fatty acids and the lower one shows five less abundant fatty acids. Perfect co-elution is observed for the isotopic pairs of peaks (dotted and solid lines for light and heavy labels, respectively). The expected 1:4 intensity ratio and the 9 Da shift are also evident. Note that each compound yields two or three peaks in the mass spectra due to the natural isotopic distribution, but only the tallest (monoisotopic) peak was employed for quantification.

Figure 3

Quantification of fatty acids in hydrolyzed egg lipid extracts from chickens on various diets. (a) The relative quantification results from heavy- and light- isotopic labeling with cholamine are displayed as ratios. (b) This log plot of absolute intensities of labeled fatty acids shows a dynamic range of 3 orders of magnitude.

Scheme 1

Reaction of a carboxylic acid metabolite with cholamine to form a product with a quaternary ammonium group, thereby enhancing mass-spectrometric analysis in positive-ion mode.

Scheme 2

Synthesis of light(d₀)- and heavy(d₉)-cholamine.