3) The applicability of the 50 mM ammonium acetate buffer (pH 9.3) in the preparation of AQC amino acid derivates for direct infusion experiments was evaluated. Derivatized amino acid standard solutions (1 × 10−2 g/L) were infused into the Xevo TQ mass spectrometer. Multiple reaction monitoring (MRM) transitions were determined for 26 amino acids, and the optimal cone voltage and collision energy associated Inhibitors,research,lifescience,medical with each transition were established (Table 1). Unlike previous

direct infusion experiments performed with the borate buffer, signal suppression and source contamination were not observed with this alternative buffer system, after 78 consecutive infusions. AQC amino acid derivatives were stable for more than three weeks when stored at room temperature in the dark, further advocating the effectiveness of this buffer for the derivatization reaction (data not shown). Table 1 MRM transitions, cone voltage (CV) and collision energy (CE) determined for AQC-derivatized standard amino acids

buffered Inhibitors,research,lifescience,medical with ammonium acetate (50 mM, pH 9.3). Experimental conditions: Waters XEVO TQ mass spectrometer; direct infusion at 20 µL/min; … The reproducibility of the derivatization method Inhibitors,research,lifescience,medical with the 50 mM ammonium acetate buffer (pH 9.3) was confirmed by the UPLC-ESI-MS/MS analysis. The peak area of the isotopically labeled amino acids derivatized with AQC in ammonium acetate medium was measured in Inhibitors,research,lifescience,medical nine replicates (final concentration of adducts = 4 × 10−4

g/L) (Table S1). As shown in Table S1, the relatively relative standard deviation (RSD) of the peak area for all isotopically labeled amino acids was below 9%, indicating high reproducibility of the derivatization reaction. The Inhibitors,research,lifescience,medical efficiency of the reaction in the alternative buffer was further studied by evaluating the linearity of the detector response for standard amino acid solutions over the concentration range from 250 μM to 3.05 pM. Figure S1A and Figure S1B (supplementary information) show typical internal calibration curves of phenylalanine obtained by UPLC-ESI-MS/MS analysis under the conditions described in section 3.5. The response factors for these calibration curves were AZD9291 calculated using relative peak areas, in which the area of phenylalanine was divided GSK-3 by the area of the internal standard, 4-hydroxyphenyl-2,6-d2-alanine-2-d1 (present at a constant concentration of 4 × 10−4 g/L after derivatization). Figure S1A displays the internal calibration curve for phenylalanine obtained with the conventional borate buffer, whereas Figure S1B shows the internal calibration curve obtained with the alternative 50 mM ammonium acetate buffer (pH 9.3). Clearly, both internal calibration curves exhibit similar response factors, correlation coefficients and slopes, providing additional evidence for the suitability of the ammonium acetate buffer for AQC derivatization of amino acids.