Multifunctional and highly sensitive precolumn reagents for amino acids in liquid chromatography/tandem mass spectrometry.

We have developed novel precolumn derivatization reagent, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS), for sensitive analyses of amino acids using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). TAHS, an activated carbamate, was reacted briefly with the amino group to form a ureide bond under mild condition. The derivatives provided selective cleavage at the binding site between the reagent and the amino acid in the collision cell of the mass spectrometer and produced a characteristic fragment derived from the reagent moiety. Using the precursor ion scan mode of the tandem mass spectrometry, amino acids derivatized with the reagents were simultaneously measured on the chromatogram. Selective cleavage also enabled the straightforward isotope ratio analysis of amino acids by the selected reaction monitoring mode, which was applicable in (13)C metabolic flux analysis. TAHS, which contains a cationic quaternary amine, achieved subfemtomole to attomole levels of amino acids detection by measurement in the selected reaction monitoring mode. We also synthesized trideuteriummethyl-substituted TAHS, TAHS-d(3), and demonstrated that the combination of TAHS and TAHS-d(3) is useful in comparing amino acid concentrations between two different samples using a single LC/MS/MS measurement.

Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), followed by reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI-MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra- and inter-day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra-day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter-day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra-day and inter-day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin.