Abstract
We developed a robust analytical method for quantification of malondialdehyde (MDA) in exhaled breath condensate (EBC) via derivatization with 2,4-dinitrophenylhydrazine (DNPH). The target MDA-DNPH hydrazone was separated by ultra-performance liquid chromatography using two reversed-phase analytical columns (C(18) and phenyl-hexyl) inter-connected via a two-position, six-port switching valve to a single-quadrupole mass spectrometer. The target derivative was analyzed under positive electrospray ionization using single ion monitoring mode (m/z = 235 for the target derivative, and m/z = 237 for its labeled isotopic analog). This pseudo two-dimensional chromatographic separation provided optimum separation conditions for the target derivative resulting in the limit of detection of 0.58 nM in EBC sample (or 36.2 pmol on-column amount), which is comparable to those reported previously using different techniques, including tandem mass spectrometry. Based on the calibration solutions, the method had a linear quantification range of 1.0-200 nM (r2 = 0.998). The method showed good relative recoveries (92.2-102.0%) and acceptable precisions (3.6-12.2% for inter-day precision, and 4.3-12.4% for intra-day precision for two quality control levels, prepared from 5 nM and 25 nM solutions). The derivative was found to be stable at room temperature for 48 h or during analysis. The method was used to analyze 205 exhaled breath condensate samples collected from individuals from a healthy population of student athletes. MDA was detected in approximately 95% of these samples, with concentrations ranging from 1.16 to 149.63 nM. The median concentration was 6.82 nM, (IQR 4.08-9.88). These data demonstrate that our method can be successfully used to measure MDA in population studies.