Sensitive analysis of N-blocked amino acids using high-performance liquid chromatography with paired ion electrospray ionization mass spectrometry.

Abstract

In this study, a paired ion electrospray ionization (PIESI) mass spectrometry method was developed for sensitive detection of 9-fluorenylmethyl chloroformate (Fmoc)-derivatized amino acids. The structure-optimized ion-pairing reagent was introduced post column to form positively charged complexes which can be detected in the positive ion mode. These complexes are more surface-active than the original analytes, and meanwhile, the intensity of sodium adducts was significantly reduced. The limit of detection of the amino acids obtained with the optimal ion-pairing reagent was 0.5 to 20pg which was 5-100 times lower than the negative mode. In addition, two mass spectrometry platforms-linear ion trap and triple quadrupole-were used to compare the PIESI improvements. Eventually, the method was applied to successfully detect the level of amino acids in human urine samples with high accuracy and the added benefit of minimizing matrix effects. Graphical abstract A HPLC-ESI-MS/MS method by using ion-pairing reagents for sensitive detection of Fmoc amino acids.