Enhancing microflow LC-MS/MS analysis of neuromedin U through reduced aspecific adsorption

Quantifying neuropeptides in biological samples from preclinical studies is often challenging due to their low concentrations (low picomolar (pM) to femtomolar (fM) range) in volume-limited samples, even for sensitive hyphenated mass spectrometry (MS) methods. Sensitivity is further limited by aspecific adsorption of the target molecules. Peptides are prone to bind non-specifically to the surfaces of tubes, vials, pipette tips and the tubing of the LC system, which impedes accurate quantification. In this study, a microflow ultra-high performance liquid chromatography system coupled to tandem mass spectrometry (UHPLC-MS/MS) (Waters AQUITY UPLC M-class system with CSH C18 130Å 1.7µm 150µm x 50mm iKey separation device coupled to Xevo TQ-XS) was used in multiple reaction monitoring (MRM) mode to optimize a method to quantify mouse neuromedin U (NmU). NmU is a 2.7 kDa neuropeptide belonging to the neuromedin family that is involved in the stress response. Different approaches to combat aspecific adsorption of NmU at the level of sample preparation and analysis were investigated.
In order to prevent aspecific adsorption, solvents with varying water and organic modifier compositions were used to dissolve the lyophilized NmU standard and to perform further dilution steps. The highest responses were obtained by dissolving NmU in H2O/Acetonitrile/Formic Acid (70:30:0.1 v/v/v) followed by making dilutions with the same solvent composition. The signal could further be increased by adding 10% (v/v) of both acetonitrile and formic acid to the sample in Quanrecovery UHPLC vials before injection. Prior to the analysis of samples, it is crucial that the LC system is passivated by repeatedly injecting a 40 µg/mL bovine serum albumin (BSA) solution, which acts as an adsorption competitor to NmU, saturating all non-specific binding sites within the system.
After the optimisation of the sample preparation, the UHPLC conditions (mobile phase gradient, column temperature and trapping flow rate, temperature and duration) were investigated to further increase sensitivity and limit carryover. A gradient with an initial fraction of organic modifier of 5% while keeping column temperature at 45°C resulted in the best signal. For trapping on the M-class Trap Symmetry C18 100Å 5µm 300µm x 50mm trapping column, a flow rate of 10 µL/min and a trapping volume of 30 µL were selected. Using the optimized method, the limit of detection in water was found in the 100 pM range.
Finally, microdialysis samples were spiked with NmU at different concentrations to compare the sensitivity in water and in microdialysate. In microdialysis samples lower peak areas were consistently found, showing that further developments are required to measure NmU in in vivo samples.