Skip to Content
Merck
  • 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.

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.

Journal of chromatography. A (2014-08-26)
Katrien Maes, Joeri Van Liefferinge, Johan Viaene, Jolien Van Schoors, Yannick Van Wanseele, Guillaume Béchade, Erin E Chambers, Hugo Morren, Yvette Michotte, Yvan Vander Heyden, Jan Claereboudt, Ilse Smolders, Ann Van Eeckhaut
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Supelco
Potassium Chloride, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Potassium chloride, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.0%
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
Potassium chloride, for molecular biology, ≥99.0%
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium chloride, tablet
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Potassium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Sodium chloride, tested according to Ph. Eur.
Sigma-Aldrich
Potassium chloride, tested according to Ph. Eur.
Supelco
Potassium chloride solution, for Ag/AgCl electrodes, ~3 M KCl, saturated with silver chloride
Supelco
Sodium chloride, reference material for titrimetry, certified by BAM, >99.5%
Sigma-Aldrich
Potassium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Supelco
Potassium chloride solution, conductance standard C acc. to ISO 7888, 0.001 M KCl
Supelco
Potassium chloride solution, BioUltra, ~3 M in H2O
Supelco
Potassium chloride solution, conductance standard B acc. to ISO 7888, 0.01 M KCl
Supelco
Potassium chloride solution, conductance standard A acc. to ISO 7888, 0.1 M KCl
Sigma-Aldrich
Potassium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Potassium chloride, ≥99.99% trace metals basis
Sigma-Aldrich
Potassium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Sodium chloride, random crystals, optical grade, 99.9% trace metals basis