Two MS friendly volatile buffers, namely, ammonium formate, and ammonium acetate were studied. For comparison purposes, control experiments using the well established borate selleckchem buffer system (pH 8.8) as the reaction medium were carried out in parallel. An initial judgment on the suitability of the media under evaluation was made based on the physical appearance of their

respective amino acid standard solutions following derivatization with AQC. The use of ammonium formate buffer Inhibitors,research,lifescience,medical (pH 7.6) produced dark-yellowish solutions upon AQC amino acid derivatization, possibly indicating the formation of unwanted byproducts. The ammonium acetate buffer (pH 9.3), on the other hand, yielded Inhibitors,research,lifescience,medical clear colorless solutions similarly to the borate buffer system and was selected for further experiments. The effect of the buffer concentration on the derivatization reaction was investigated

next, while keeping the pH constant at 9.3. Six concentrations of ammonium acetate buffer (10, 20, 50, 100, 200 and 500 Inhibitors,research,lifescience,medical mM) were tested. All six concentrations yielded clear colorless solutions upon AQC amino acid derivatization. Nevertheless, subsequent UPLC-ESI-MS/MS analysis revealed a decrease in ion intensity with the increase in buffer concentration. Evidently, high buffer concentrations led to an increase in salt deposits in the sample cone surface, decreasing the signal intensity. Signal intensity was particularly affected at buffer concentrations 100 mM and higher. The increased LC-MS/MS signal suppression with increasing buffer concentration has been reported Inhibitors,research,lifescience,medical by other authors [48]. Ammonium acetate buffer concentrations equal or less

Inhibitors,research,lifescience,medical than 50 mM did not show significant signal suppression and were found appropriate for AQC amino acid derivatization. Using a constant ammonium acetate buffer concentration of 50 mM, the pH was then adjusted to 9.0, 9.3 and 10.3. Buffered amino acid solutions at pH 9.0 turned slightly yellowish upon AQC derivatization. At pH 9.0, lowering the buffer concentration from 50 mM to 20 mM produced even darker yellowish solutions, further indicating that both the pH and the buffer concentration why affect AQC amino acid derivatization. Ammonium acetate buffer concentrations greater than 50 mM at the pH of 9.0 were not tested based on our previously results, showing a decrease in ion intensity with an increase in buffer concentration. Keeping derivatization conditions at pH = 10.3 also proved suitable for AQC adduct formation, and no differences were observed compared to the results obtained at pH 9.3 (data not shown). All further infusion experiments were performed using the 50 mM ammonium acetate buffer system at pH 9.3. 2.1.2.