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

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolarconcentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides toEppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is pre-sented to reduce adsorption of these peptides during standard preparation. Within this respect, allprocedural steps from dissolution of the lyophilized powder until the injection of the sample onto thesystem are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N,and a neuromedin N-related neuropeptide, neurotensin, are evaluated.The first part of this study outlines a number of parameters which are known to affect peptide solubility.The main focus of the second part involves the optimization of the sample composition in the UHPLC vialby using design of experiments.Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix com-ponents. They are found important for injection of the peptides into the system, but crucially need tobe excluded from the dilution solvent. Furthermore, the type of surface material, temperature and thepipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results ofthe central composite design reveals that the highest peptide responses are obtained with the injectionsolvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can beidentified as the main contributor to the gain in method sensitivity.Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in aclear and quantifiable signal of the three peptides, optimization of both issues should be considered inthe early stage of method development, in particular when the analysis of low-concentration peptidesolutions is envisaged.

Publication year:2014

Keywords:Neuropeptides, Sensitivity