Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

An Ultrasensitive Nano UHPLC–Esi–MS/MS Method for The Quantification of Three Neuromedin-Like Peptides in Microdialysates

“…The ACN and FA proportion in the loading solvent is much more critical compared to its proportion in the injection solvent. Indeed, we have previously shown [31] that the addition of much higher proportions of these solvents, 13.1% V/V ACN and 4.4% V/V FA, to the sample in the UHPLC vial (injection solvent) are needed to improve sensitivity. This contrast strengthens the necessity of optimizing every single step of the analysis.…”

“…The optimum conditions, solvent compositions and container materials for standard preparation and injection have been previously determined [31] and are applied in this study. Initial stock solutions of NT, NMB and NMN are prepared by dissolving 1 mg of the lyophilized peptide powders in 1 ml of water/ ACN/FA (45/50/5 V/V/V), thus creating standard solutions of 598 μM NT, 883 μM NMB and 1340 μM NMN.…”

“…The solvents applied in our previous study [31] for reducing adsorption of peptides during standard preparation, namely water/FA (95/5 V/V), water/ACN (50/50 V/V), water/ACN/FA (45/50/5 V/V/V), ACN/ FA (95/5 V/V) and ACN are tested as strong wash.…”

“…The dwell time is set at 84 ms. The source and analyzer settings on the Quattro Premier are as previously described [31]. The settings on the XEVO TQ-S instrument are: capillary voltage 4.0 kV; source offset 50 V; source temperature 80°C; extractor voltage 5.00 V; RF lens voltage 0.0 V; entrance and exit voltages of the collision cell 1.0 V. The ion energies are set at 0.5 and 1.0, and the multiplier voltage is 531 V. All measurements on both MS detectors are controlled by MassLynx® version 4.1 operating software (Waters, MA, USA).…”

“…The first challenge has already been extensively investigated in our previous study [31]. Indeed, a significant improvement in sensitivity is observed after reducing aspecific adsorption of peptides during standard preparation (for review, see [32,33]), and by optimizing the injection solvent used for loading the peptides onto the trap column.…”