High performance liquid chromatography coupled to electrospray ionisation mass spectrometry method for the detection of salivary human neutrophil alpha defensins HNP1, HNP2, HNP3 and HNP4

Abstract

Human neutrophil alpha defensins are antimicrobial peptides involved in the first line of defence against invading pathogens. To develop a deeper understanding of the immune responses in relation to airway inflammation and exercise induced epithelial damage it is necessary to have a sensitive method that can detect these peptides in a saliva matrix. A selective and sensitive Liquid Chromatography Mass Spectrometry (LC-MS) method for the detection of four salivary HNP (HNP1, HNP2, HNP3 and HNP4) peptides has been developed and validated. The LC-MS responses of HNPs 1–3 were compared with the response obtained from the traditionally used enzyme-linked immunosorbent assay (ELISA) that measures the combined levels of these three defensins. The peptides were separated on a Phenomenex Kinetex® C8 column (50 × 3.0 mm, 2.6 μm) on an Agilent 1200 series HPLC system using a linear MeOH : H₂O : acetic acid (0.1% v/v) gradient. The HPLC was coupled to a Waters Synapt G1 Electrospray Quadrupole Time of Flight mass spectrometer. A full scan range from 100–2000 m/z in the positive ion mode was used for the acquisition. The LC-MS method was linear for concentrations of HNP2 between 0.05 and 1 ng μL⁻¹ with a LOD of 0.05 ng μL⁻¹ and LOQ of 0.1 ng μL⁻¹. Inter- and intra- assay precisions (% CV) were 0.3 and 14.95%, respectively. HNPs were extracted from saliva by solid-phase extraction (SPE) with a recovery of 80–91%. The cross-validation data revealed no significant quantitative difference between LC-MS and ELISA (R² = 0.96) and confirms that the developed LC-MS method is a reliable method for the detection of these antimicrobial markers. However, superior selectivity in the developed LC-MS method provides a unique opportunity to assess individual alpha defensin levels in the same assay. HNP1, HNP2, HNP3 and HNP4 were evaluated in young athletes before, and up to, 2.5 h after an exercise intervention in order to assess if the developed LC-MS method was sensitive enough to detect rapid changes in their relative levels.