title: 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.
abstract: Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system 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 vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified 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 a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Maes
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Joeri
authors.initials: J
authors.lastname: Van Liefferinge
authors.affiliation: Department of Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Viaene
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Jolien
authors.initials: J
authors.lastname: Van Schoors
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Van Wanseele
authors.affiliation: Waters Corporation, Connexion Business Park, Brusselsesteenweg 500, 1731 Zellik, Belgium.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Béchade
authors.affiliation: Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, USA.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Chambers
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Hugo
authors.initials: H
authors.lastname: Morren
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Michotte
authors.affiliation: Department of Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Vander Heyden
authors.affiliation: Waters Corporation, Connexion Business Park, Brusselsesteenweg 500, 1731 Zellik, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Claereboudt
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address: Ilse.Smolders@vub.ac.be.
authors.forename: Ilse
authors.initials: I
authors.lastname: Smolders
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address: aveeckha@vub.ac.be.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Eeckhaut
mesh_terms: D000327:Adsorption; D055598:Chemical Phenomena; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009496:Neurotensin / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D012997:Solvents / Q000737:chemistry; D013499:Surface Properties; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D012997:Solvents; C042090:neuromedin N; D009496:Neurotensin
keywords: Aspecific adsorption; Design of experiments; Nano ultra-high performance liquid chromatography-tandem mass spectrometry; Neuropeptides; Sensitivity; Statistical analysis