Development of a method for quantification of phosphatidylcholine in exhaled breath

Abstract

Non-invasive and quantitative sampling of particles in exhaled air may provide valuable information on the status of the peripheral airways. Studies of particles have confirmed the presence of pulmonary surfactant, including both important phospholipids and surfactant proteins. The aim of this study was to develop a reliable method for the quantification of the major phosphatidylcholines (PCs) in exhaled particles. Methods: Exhaled particles were counted and collected on membranes using the PExA method. The particles were extracted with methanol:chloroform prior to flow injection into an electrospray ionization tandem mass spectrometer. A quantitative method was developed for dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoylphosphatidylcholine (POPC) using selected reaction monitoring. The method was validated by investigating within- and between-batch precision, accuracy, carry-over, stock stability, short-term and long-term stability, freeze-thaw stability and processed sample integrity. The validated method was used to investigate the inter-individual variation in exhaled particles from 6 healthy volunteers. Results: The method had a linear response for DPPC and POPC with a limit of quantification of 0.005 µM. The DPPC and POPC concentration, expressed as weight % in exhaled particles, were 13.9 (median, range 8.8-16%) and 4.1 (median, range 2.4-4.9%), respectively, of the total particle sample. Conclusion: We have developed a reliable method for the quantification of surfactant PCs in exhaled particles using a simple sample extraction and mass spectrometry. The method can readily be applied in clinical studies to investigate the role of PCs in different lung related diseases.