9810 and 1.000, within the low and high limits of linearity specific for each amino acid, as presented in Table 3. Table 3 Evaluation results for linearity and sensitivity of UPLC-ESI-MS/MS method for the

analysis of AQC-derivatized amino acids. In addition, the overall process efficiency was calculated as PE (%) = 100 (area spiked before extraction/area standard solution) as described in Gu et al. [10]. The area standard solution corresponds to the area of the internal standard in the neat standard solutions used to prepare the calibration curves, and area spiked before extraction corresponds to the Inhibitors,research,lifescience,medical peak area in the sample extract. The overall process efficiencies ranged from 65.0 to 99.4%. As stated by Gu et al. [10], process efficiencies greater than 100% occurs when coeluting species present in the sample matrix contribute to the detected signal of the amino acid. There was no evidence of such contribution according to the results presented Inhibitors,research,lifescience,medical in Table S4. Subsequently, the limits of detection (LOD) were established by using the method of the blank and were calculated as three times the standard deviation of the peak areas observed from the blank signals, divided by the slope of the calibration

curve obtained for the given amino acid. The LOD values obtained (Table 3) ranged Inhibitors,research,lifescience,medical from 1.02 × 10−11 to 1.06 × 10−8 M, suggesting that the analytical method presented in this study is 1 to 5 orders of magnitude more sensitive than other existing LC-MS and LC-MS/MS approaches [14,15,17,18,21,22,36,37,38,39,49,50,51,52,53] for the analysis of native or derivatized amino acids, as showed in Table 4. The LOD values reported by Shimbo et al. [37] Inhibitors,research,lifescience,medical for 20 amino acids derivatized with TAHS are comparable to those obtained in our study, however, our UPLC analysis for AQC-amino acids has higher throughput (38 amino acids Inhibitors,research,lifescience,medical and 15 internal standards separated three times faster).

Table 4 also shows the faster separation time (5 times shorter chromatographic run) and better sensitivity (3 orders of magnitude lower LOD) added to the analysis of AQC-amino acids by the combination of UPLC with PFT�� purchase tandem mass spectrometry operated because in the MRM mode. Table 4 Comparison on the sensitivity of selected LC/MS-based approaches for amino acid analysis. 2.3. Method Application to Screening of Arabidopsis Mutants Currently, metabolomic studies require high-throughput and sensitive targeted analytical platforms for screening of a large number of genetic variants. Thus, after its evaluation, our AccQ•Tag-UPLC-ESI-MS/MS method was used for the quantitative amino acid determination in A. thaliana leaf extracts (9 wild-type samples and 75 mutants; 6 biological replicates each; 504 samples in total) to demonstrate its applicability as a targeted approach for metabolomics analysis (for complete list of A. thaliana mutant stocks used in this study refer to Ref. 7).