The manuscript " Determination of free amino acids in plants by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)" describes the analysis of 19 proteinogenic amino acids by UHPLC-MS/MS using the EZ:faast™ LC-MS Free Amino Acid kit commercially available from Phenomenex. Optimization of the MS as well as MS/MS parameters for confirmation and quantification transitions of each amino acid is presented. Validation of the method including the extraction step was performed with standard solutions and spiked weed plant samples with regard to the linear range, linearity, LODs, LOQs and intra/inter-day precision. Recoveries had been determined by weed samples spiked with amino acid internal standards from the EZ:faast™ LC-MS Free Amino Acid kit. The validated method has been applied to the analysis of Stellaria media weed samples.Application of the EZ:faast™ LC-MS Free Amino Acid kit has been published by several other authors, however the novelty of this paper is its first application to plant samples after the comprehensive validation of the method in order to achieve accurate analysis of free amino acids. Our aim was to validate the method according to strict guidelines -ICH Q2 (R1) and also European SANCO, due to specific criteria concerning LC-MS/MS technique parameters in order to demonstrate that this analytical procedure is suitable for its intended purpose in plant matrices. As the EZ:faast™ kit is known and used widely among scientists, we think that complete validation for all proteinogenic amino acids in plants would be desirable, as we were not able not find any manuscript dealing with these issues, therefore societal impact is justified. Furthermore, many papers were published utilizing this kit, but most of them analyse only few amino acids. They neither present the MS/MS optimization parameters for all amino acids nor validation procedure, so the reliability of those methods is doubtful. Extraction using water when plants amino acids remain in native state is a merit for many scientists dealing with plant stress, when they are interested in non-altered plant composition in order to check how plants react after physiological stress. Moreover, not all amino acids are soluble in organic solvents or their solubility is hindered. As far as we are concerned there has not been any article incorporating water and the EZ:faast™ kit to study free amino acids so we feel it is worth and significant to fill this niche. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ABSTRACT A robust and sensitive method for identification (quantification and confirmation) of 19 free amino acids in plant matrice -Stellaria media, based on liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), with a triple quadrupole analyser, has been developed. Regarding MS optimization, the flow injection analysis (FIA)...