Abstract
Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry. Ultra high sensitivity FA analysis usually is done with gas chromatography of pentafluorobenzyl esters coupled to an electron-capture detector. With the popularity of electrospray ionization (ESI) mass spectrometers coupled to liquid chromatography, it would be convenient to develop a method for ultra high sensitivity FA detection using this equipment. Although FAs can be analyzed by ESI in negative ion mode, this method is not very sensitive. In this study, we demonstrate a new method of FA analysis based on conversion of the carboxylic acid to an amide bearing a permanent positive charge, N-(4-aminomethylphenyl)pyridinium (AMPP) combined with analysis on a reverse-phase liquid chromatography column coupled to an ESI mass spectrometer operating in positive ion mode. This leads to an ∼60,000-fold increase in sensitivity compared with the same method carried out with underivatized FAs. The new method is about 10-fold more sensitive than the existing method of gas chromatography/electron-capture mass spectrometry of FA pentafluorobenzyl esters. Furthermore, significant fragmentation of the precursor ions in the nontag portion improves analytical specificity. We show that a large number of FA molecular species can be analyzed with this method in complex biological samples such as mouse serum.
Highlights
Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry
Amide results in an analyte with a permanent positive charge, which can be analyzed by LC-electrospray ionization (ESI)-MS/MS in positive ion mode
This is more sensitive than negative ion mode detection of the underivatized caboxylate anion because ionization of the latter is greatly suppressed by the protonation resulting from the addition of weak organic acid such as acetic or formic acid, which is necessary for optimal LC on reverse-phase columns
Summary
Low abundant FAs such as AA are usually not present as a contaminant in glassware and reagents; abundant FAs such as oleic, palmitic, and stearic acids are present as common contaminants. It has not been possible to remove these contaminants to a level below the FA detection limit for the method described in this paper. FA contamination to a level usually below the amounts to be detected in the sample of interest. All glassware used for extraction and pre-LC-ESI-MS/MS work-up was baked overnight in a high temperature oven at 450°C to remove any residual FA contamination. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (TCI America, catalog #D1601), 1-hydroxy-7-azabenzotriazole (HOAt) (Sigma, catalog #44545-2), and AMPP [32] were triturated with distilled isooctane to remove any residual FA contamination
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
