Determination of free amino acids in plants by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)

Abstract

A robust and sensitive method for identification (quantification and confirmation) of 19 free amino acids in the plant matrix, Stellaria media, based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), with a triple quadrupole analyser, has been developed. Regarding MS optimization, flow injection analysis (FIA) was used in scan and selected reaction monitoring (SRM) mode. The collision energies optimized varied from −12 to −39 eV. The acquisition of three MS/MS transitions for most of the compounds allowed the accurate confirmation of these analytes, which was supported by the accomplishment of ion intensity ratios and retention time as compared with the corresponding standards. The use of a Phenomenex EZ:faast™ Free (Physiological) Amino Acid kit speeds up the sample preparation immeasurably. Nineteen amino acids were separated within 18 minutes on a reverse-phase column under a gradient stepwise programme using 10 mM ammonium formate both in water and methanol. The detection limit (LOD) for free amino acids varied from 0.4 to 9.1 pmol mL⁻¹, except for asparagine amounting to 3000 pmol mL⁻¹. The quantification precision (RSD) of free amino acids for intra- and interday assays was 0.05–19% and 0.2–19%, respectively, but for most of the compounds, it did not exceed 5%. The optimized and validated method was subsequently utilized for free amino acid identification in weed collected from field locations in Poland.