Coumarin-based derivatization reagent for LC-MS analysis of amino acids

Abstract

In this paper, we report about the application of a sensitive fluorescent derivatization reagent Coumarin151-N-Hydroxysuccinimidyl Carbamate (Cou151DSC) for amino compounds using high-performance liquid chromatography (HPLC) compatible with ultraviolet (UV), fluorescence detector (FLD) and electrospray ionization - tandem mass spectrometry (ESI-MS/MS)―positive mode. We optimized derivatization procedure and validated an analytical method to determine 24 amino acids in Kvass drink using Norvaline as an internal standard. Compared to 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate (6-AQC), the derivatization with Cou151 DSC is faster and milder, for 5 min at 40°C instead of 15 min at 55°C. The limit of quantitation (LOQ, pmol on column) for 21 amino acids in this work is lower 1.1–30.0 times than values obtained with 6-AQC. The derivatives have excitation wavelength at 355 nm and emission wavelength at 486 nm. Their MS/MS fragmentation behaviors were examined together with 23 other amino compounds. We found three possibilities to lose a neutral group which can be Coumarin 151 isocyanate Cou151NCO (255 Da), amine Coumarin 151 (229 Da) or urea Cou151CONH2 (272 Da). The accuracy of the proposed method was within 83–107% with good relative standard deviations (RSDs) of equal or less than 6%. The recoveries were from 82 to 120% in four spiked concentrations, repeatability was between 0 and 14%. The intra- and inter-day precision are less than 13% and 18%, respectively.