Bioanalysis of underivatized amino acids in non-invasive exhaled breath condensate samples using liquid chromatography coupled with tandem mass spectrometry

Exhaled breath condensate (EBC) is receiving increased attention as a novel, entirely non-invasive technique for collecting biomarker samples. This increased attention is due to the fact that EBC is simple, effort independent, rapid, can be repeated frequently, and can be performed on young children and patients suffering from a variety of diseases. By having a subject breathe tidally through a cooling system for 15-20 min, a sufficient amount of condensate is collected for analysis of biomarkers in clinical studies. However, bioanalysis of EBC involves an unavoidable sample preparation step due to the low concentration of its components. Thus, there is a need for a new and more sensitive analytical method of assessing EBC samples. While researchers have considered analyses of single and small quantities of amino acids - for example, those connected with leukemia - no one has previously attempted to simultaneously analyze a panel of 23 amino acids. Moreover, the present study is well-justified, as prior studies focusing on single amino acids and leukemia at the moment of diagnosis and during chemotherapy (33 days of treatment) are inconsistent. In the present study, amino acids were separated using an XBridge Amide column (3 mm × 100 mm, 3.5 μm). The mobile phase consisted of 10 mM of ammonium buffer in water with a pH of 3 (Phase A) and 10 mM ammonium buffer in acetonitrile (Phase B) under gradient program elution. The analytes were detected in electrospray positive ionization mode. Under optimal conditions, the proposed method exhibited limits of quantification (LOQ) in the range of 0.05-0.5 ng/mL, and good linearity, with the determination coefficient (R2) falling between 0.9904 and 0.9998. The accuracy in human exhaled breath condensate samples ranged between 93.3-113.3% for the 23 studied amino acids, with intra- and inter-day coefficient of variation (CVs) of 0.13-9.92% and 0.17-10.53%, respectively. To demonstrate the liquid chromatography with hydrophilic interaction with electrospray source coupled to tandem mass spectrometry (LC-HILIC-ESI-MS/MS) method's applicability for biomedical investigations, it was verified and applied to determine amino acids in pediatric patients with leukemia. These tests confirmed that glutamine, arginine, homoarginine, asparagine, histidine, methionine, proline, hydroxyproline, threonine, tyrosine, and valine were present in significantly higher levels in pediatric leukemia patients than in the healthy control group. The developed assay is an attractive alternative to standard analytical methods, because it allows for the non-invasive, fast, sensitive, and reliable analysis of amino acids without derivatization in EBC.