HILIC UPLC/ QTof MS Method Development for the Quantification of AGEs Inhibitors - Trouble Shooting Protocol

Objective: The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N--carboxymethyl-L-lysine (CML) in vitro, testing N-ε- carboxy[D2]methyl-L-lysine (d2-CML), and N-ε-carboxy[4,4,5,5-D4]methyl-L-lysine (d4-CML) as internal standards. Methods: During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated. Result: A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented. Conclusion: Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way.