Feasibility Study Of Mass Spectrometry Breath Analysis As A Non-invasive Technique For Influenza

Influenza is one of the most common viral infectious diseases worldwide,which causes considerable damages to the public health and economics every year.Rapid,accurate and non-invasive diagnosis technique for influenza infectioni is of great significance for timely and effective prevention and control of the spread of influenza virus as well as antiviral treatment.Virus infection can lead to changes in metabolites in host cells that are released into the bloodstream,and some of these metabolites further enter into the exhaled breath air through the exchange of blood into the alveoli gas.Therefore,screening out the specific metabolites in the exhalation during influenza virus infection and revealing their sources are key fro the application of breath analysis to the noninvasive detection of the course and treatment of influenza infection.Therefore,in this study we have firstly explored the use of secondary electrospray ionization high resolution mass spectrometry(SESI-HRMS)for noninvasively monitor the process of influenza A(H1N1)virus(IAV)infection through breath analysis in a mouse model.We confirmed that SESI-HRMS can effectively capture more than one thousand ions formed by trace volatile metabolites in mouse breath.Furthermore,we screened out 423specific ions in the infected group,which not only effectively distinguish the infected group from the control group,but also accurately predict the infection on the first day after influenza virus infection.Metabo Ananlyst R analysis showed that the 1770 features identified in the exhalation of infected mice were related to 40 metabolic pathways.The top five most relevant pathways induced by IAV infection includes:1)glyoxylate and dicarboxylate metabolism,2)alanine aspartate and glutamate metabolism,3)nitrogen metabolism,4)purine metabolism and 5)arginine biosynthesis.This study provides a theoretical basis for the development of real-time and non-invasive diagnosis tool for influenza breath.In addition,this method may also be applied to the diagnosis of other infectious diseases(such as COVID-19).In order to further explore the biological evidence for the diagnosis of influenza virus infection by exhalation at cellular level,a quantitative method was developed for twenty two typical aldehyde and ketone metabolites in the headspace of cells.Standard compounds were transformed into stable DNPH-carbonyl adducts using 2,4-dinitrophenylhydrazine(DNPH)impregnated cartridge.These adducts were further eluted and analyzed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry.The standard curves of 22 DNPH-carbonyl adducts showed linearity at high(40-1000μg/L),medium(8-400μg/L)and low(0.08-8μg/L)levels,respectively.The coefficient of correlation(R~2)was≥0.9984 and the detection limits were in the range of 0.003-0.09μg/L.The method demonstrated good precision of≤3.91%(intra-day RSD)and≤5.29%(inter-day RSD)as well as satisfactory recovery of 95.10%-109.58%(RSD≤5.83%).19 aldehydes and ketones were successfully detected in the headspace of A549 cells and A549 cells infected with H1N1 influenza virus(IAV)over a concentration range of 1-400μg/L.Eleven potential carbonyl biomarkers for viral infection were determined:heptaldehyde,nonanal,octanal,hexaldehyde,propionaldehyde,decanal,acetone,benzaldehyde,cyclohexanone,fomaldehyde and acetaldehyde.