ORAL PRES.: The application of superchargers to increase nanoUHPLC-MS/MS method sensitivity for the quantification of the neuropeptide neuromedin U.

A sensitive and selective nanoUHPLC-ESI-MS/MS method is developed to quantify neuromedin U in microdialysates. To obtain maximal sensitivity, the duty cycle of the first quadrupole is preferably set to 100%. As a consequence, only one precursor ion can be selected, resulting in a loss of the other charge states in the spray. In this study, we have investigated the possibility to increase signal intensity by maximizing the abundancy of the +4 precursor ion of neuromedin U through the addition of the superchargers m-nitrobenzylalcohol, sulfolane and/or dimethylsulfoxide. Based on several direct infusion screening experiments, an experimental domain is selected in which the optimal concentration and combination of superchargers is identified executing a Box-Behnken optimization design. MS detection is performed in the positive mode using single ion monitoring. Total ion current chromatograms are used for integration. The relative signal of the +4 precursor ion is calculated based on the total peptide signal. The correlation between the obtained average (n= 3) responses and the factors is modeled using a second order polynomial model. An increase to 80% abundancy of the +4 charge state is predicted with a mobile phase containing 0.008% m/v m-nitrobenzylalcohol and 0,5% v/v dimethylsulfoxide, compared to 60% without supercharger. Sulfolane has only a small impact on the abundancy, is moreover difficult to handle and is therefore not included in the final composition. Currently, the practical implications of the addition of these superchargers on sensitivity and method robustness are further investigated.