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.

呼气凝结水（EBC）作为一种新颖的，完全无创的技术来收集生物标志物样品，受到越来越多的关注。这种关注的增加归因于这样一个事实，即EBC简单，独立，省力，快速，可以经常重复使用，并且可以对年幼的儿童和患有多种疾病的患者进行治疗。通过使受试者在冷却系统中进行潮汐呼吸15-20分钟，可以收集到足够量的冷凝物，以用于临床研究中的生物标志物分析。然而，由于EBC的成分浓度低，因此其生物分析涉及不可避免的样品制备步骤。因此，需要一种评估EBC样品的新的且更灵敏的分析方法。尽管研究人员已经考虑了对单个和少量氨基酸的分析-例如，那些与白血病有关的基因-以前没有人试图同时分析一组23种氨基酸。此外，本研究是合理的，因为先前针对诊断时和化疗期间（治疗33天）的单个氨基酸和白血病的研究不一致。在本研究中，使用XBridge Amide色谱柱（3 mm×100 mm，3.5μm）分离氨基酸。在梯度程序洗脱下，流动相由pH值为3的水中的10 mM铵缓冲液（A相）和乙腈中的10 mM的乙腈缓冲液（B相）组成。以电喷雾正电离模式检测分析物。在最佳条件下，所提出的方法的定量限（LOQ）在0.05-0.5 ng / mL范围内，并且线性良好，测定系数为（R^2个）介于0.9904和0.9998之间。人类呼出的冷凝水样品中23种氨基酸的准确度在93.3–113.3％之间，日内和日间变异系数（CV）分别为0.13–9.92％和0.17–10.53％。为了证明液相色谱法与串联电喷雾质谱联用的电喷雾源具有亲水性相互作用（LC-HILIC-ESI-MS / MS）方法在生物医学研究中的适用性，它已被验证并用于确定儿童白血病患者的氨基酸。这些测试证实，小儿白血病患者的谷氨酰胺，精氨酸，高精氨酸，天冬酰胺，组氨酸，蛋氨酸，脯氨酸，羟脯氨酸，苏氨酸，酪氨酸和缬氨酸的含量明显高于健康对照组。