Real-time Breath Analysis By Extractive Electrospray Ionization Mass Spectrometry

Breath analysis has elicited considerable research efforts because it is non-invasive and carries metabolic information released during blood–air exchange.The traditional techniques for analyzing breath are gas chromatography mass spectrometry(GC-MS),select ion flow tube mass spectrometry(SIFT-MS)and proton transfer reaction mass spectrometry(PTR-MS).However,the above-mentioned techniques are difficult to meet the needs of the detection of both volatile and non-volatile metabolite in exhaled breath,while achieving real-time online breath analysis.The ambient electrospray ionization mass spectrometry(EESI-MS)developed in recent years enables rapid and direct analysis of volatile and non-volatile components in liquid,solid,aerosol and other forms.With its unique design features,it plays an irreplaceable role in in-vivo,real-time and online analysis.In this paper,the EESI-MS method is developed to improve the sensitivity of real-time online breath analysis,and the application range of real-time online breath analysis is further extended to the field of sports science,rather than only limited to clinical medicine.The main research contents of this thesis are:(1)A new EESI-MS method for real-time online breath analysis using supercharged proteins as primary reagent ions was established.An aqueous solution containing 5 ?M protein,5%(v/v)1,2-butenyl carbonate and 0.5%(v/v)acetic acid was injected into the electrospray channel of the EESI source to generate supercharged protein primary ions.Then the supercharged protein primary ions collided with the exhaled gas sample molecules,causing more protons to transfer from the supercharged protein ions to the metabolites in the exhaled breath.This resulted in the enhanced ionization efficiency for the breath analytes even with relatively low gas phase basicity.Moreover,the total number of metabolites detected in breath increased by about 260% in the mass range of 200-500 Da,owing to the substantial signal enhancement for breath metabolites,providing a new technique for physiological/pathologic studies.(2)Taking the exhaled breath as samples,a new EESI-MS method for real-time online monitoring of exercise fatigue was established.EESI-MS was used to analyze the breath of athlete in different degrees of exercise fatigue and normal state and thus the breath fingerprint data was obtained.The breath data was then processed by orthogonal partial least squares-discriminant analysis(OPLS-DA)as well as partial least squares-discriminant analysis(PLS-DA)and the signal peaks with VIP > 6 were screened out.The results show that the established OPLS-DA model of mild exercise fatigue and normal state,OPLS-DA model of moderate exercise fatigue and normal state,OPLS-DA model of excessive exercise fatigue and normal state and PLS-DA model of mild fatigue,moderate fatigue and excessive fatigue were stable and no over-fitting.It has also been found that urea,a differential metabolite in breath,can be used as a biomarker for monitoring exercise fatigue.This method is simple,fast and non-invasive.It is a new method that is completely different from traditional blood tests and urine tests by real-time monitoring of exercise fatigue to finely regulate exercise load and prevent exercise injury caused by long-term excessive fatigue.