Ultrahigh Flow Regulated Discharge Coupling with Targeted Mass Spectrometry Analysis: Real Time Capturing Biomolecules in Exhaled Breath

Online breath analysis is an attractive approach for noninvasive diagnosis of disease and monitoring of human metabolic processes. To date, however, most compounds observed in exhaled breath are volatiles that are not directly associated with blood test results. Here, we proposed an ultrahigh flow regulated discharge (UFD) ionization method that enables online detection of various bioactive breath metabolites and some macromolecules by coupling with mass spectrometry, some of them were commonly observed in blood and body fluids. Through the usage of ultrahigh flow, the corona discharge is transformed into glow discharge in atmospheric pressure, and the mechanism similar to sonic spray may also be involved simultaneously to form a composite ionization manner. Compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), UFD exhibited wide coverage and high sample throughput and ionization efficiency for compounds from small volatiles to proteins in different molecular weights (MW). Based on this method, 81 biomolecules can be detected in exhaled breath, including all the 20 common amino acids, nucleosides, tricarboxylic acids, parts of nonprotein amino acids, nucleotides, coenzymes, polyamines, and related derivatives/intermediates. Notably, a total of 63 components were observed for the first time. Moreover, macromolecules also can be detected in exhalation via this method, though these components could not be unambiguously identified due to the limited selectivity. For method validation, the breath homocysteine from five subjects was measured and presented similar trends with blood test results. The online measurement of these biomolecules in human breath showed the possibility of breath tests as an alternative for some blood test items in clinical diagnosis and offers a new approach for real time investigating the metabolic process of nonvolatile bioactive components. Besides, the UFD also provides a new interface for mass spectrometry with a large mass range, wide polarity, high sensitivity, and high throughput.