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1 Supplemetary Iformatio Quatitative detectio of itric oxide i exhaled huma breath by extractive electrospray ioizatio mass spectrometry Susu Pa a, Yog Tia b, Mig Li c, Jiuya Zhao d, Lala Zhu d, Wei Zhag d, Haiwei Gu a*, Haidog Wag a, Jiabo Shi b, Xiag Fag c, Peghui Li a, Huawe he a a Jiagxi Key Laboratory for Mass Spectrometry ad Istrumetatio, East hia Istitute of Techology, Nachag, Jiagxi Provice , P. R. hia b State Key Laboratory of Evirometal hemistry ad Ecotoxicology, Research eter for Eco-Evirometal Scieces, hiese Academy of Scieces, Beijig , P. R. hia c Natioal Istitute of Metrology, Beijig , P. R. hia d Departmet of Respiratory Medicie, The First Affiliated Hospital of Nachag Uiversity, Nachag, Jiagxi Provice , P. R. hia : These authors cotributed equally to this work. *: orrespodig author: Dr. Haiwei Gu guhaiwei2004@gmail.com; Tel/fax:
2 Experimetal Details 1. alculatio procedure for the mass cocetratio of the stock solutio of ( ) After reactio with a NO gas stadard, the cocetratio of surplus O ca be calculated accordig to the calibratio curve of O. Oe mole of NO is oxidized by O to yield oe mole of NO 2 ad oe mole of. Hece, (1) O where is the umber of moles ( mol ) of produced; O is the umber of moles of O cosumed. Eterig the mass cocetratio of stock solutio of O (1.0 mg L -1 ) to eq. (1), the O ca be calculated as below: O gL O VO w (2) m Eterig eq. (2) ito eq. (1), the umber of moles of produced ca be calculated usig the equatio below: O gl O VO m w (3) where 1 is the mass cocetratio ( gl ) of remat O after reactio with a O NO gas stadard, O shall be calculated accordig to the calibratio curve of O, V O is the volume of O solutio, ad m w is the molar mass of O. 2
3 Give VO 0.01L; mw g mol 1 ad eterig these ito eq. (3), thus g L O mol (4) 1 So the mass cocetratio ( gl ) of the stock solutio of after reactio with a NO gas stadard ca be calculated as below: m V (5) where V is the volume of the mixture solutio after reactio. Actually, m is the molar mass of : m V V L. Eterig m, g mol, ad O 0.01 V, ad eq. (4) ito eq. (5), the g mol 0.01L L g mol g L O mol g L O (6) So the mass cocetratio of the stock solutio of ( accordig to eq. (6). ) ca be calculated 2. alculatio procedure of eno For exhaled NO samples, the umber of moles of NO also equals to accordig 3
4 to the proportio relatio of the reactio: NO (7) From eq. (5), ca be calculated as below: V m ' (8) where 1 is the mass cocetratio ( gl ) of produced after reactio with ' NO i breath. Eterig eq. (8) ito (7), the NO V m ' (9) Give V 0.01 L; m g mol 1 ad eterig these ito eq. (9), thus NO ' 0.01L 217.3g mol ' mol (10) VNO NO Vm (11) Accordig to the lapeyro equatio, at a temperature of 25 ad pressure of Pa, the volume of 1 molar NO ca be approximately calculated as below: 4
5 V m RT P 1 1 1mol J K mol 298K Pa L m 3 3 Eterig Vm Lmol ad eq. (10) ito eq. (11), the V V NO NO m 24.5Lmol Lmol mol ' 3 ' NO L (12) Because FeNO V NO V (13) where V is the total volume (15 s for each exhalatio, 10 times) of exhaled breath bubblig through the O solutio, ad the cotrolled flow rate is 0.8 L mi -1, the V 15 mi L mi 60 2 L 1 Eterig V 2L ad eq. (12) ito eq. (13), the V FeNO V NO ' 2 L L 5 ' ppbv 1ppbv (14) 5
6 osequetly, eno was calculated usig eq. (14). The eq. (14) also shows that eno is proportioal to '. 3. Optimizatio of experimetal parameters To achieve better sesitivity for NO detectio, the sigal itesities of [+H] + (m/z 218) ad its characteristic fragmet (m/z 144) were optimized by adjustig the ESI voltage, ESI solvet compositio, ESI solvet ijectio rate, sample ijectio rate, io-trasport capillary temperature, ad sheath gas (N 2 ) pressure. The data obtaied usig either O or showed the same optimal coditios; thus the discussio i this sectio is focused o the optimizatio of O sigal, ad the similar data obtaied usig are ot show. ESI voltage. The effect of electrospray voltage o the sigal itesity of characteristic fragmet (m/z 84) of O is show i Figure S-7a. The data idicated that the optimal ESI voltage to detect O was i the rage kv. The highest sigal itesity for the m/z 84 fragmet was obtaied at 3 kv. Whe a voltage higher tha 3.5 kv was used, a coroa discharge betwee the two chaels of the EESI source was occasioally observed. This resulted i lower efficiecy of ioizatio ad the decreased sigal itesity of O sigal. ESI solvet compositio. For optimizig the primary ESI solvet compositio, differet proportios of methaol/water were tested. The highest itesity was obtaied whe 100% methaol was applied (Figure S-7b). This ca be due to the 6
7 higher desolvatio efficiecy of methaol tha water. ESI solvet ijectio rate. The sigal itesity level of characteristic fragmet m/z 84 was affected by the ESI solvet ijectio rate, ad the result is show i Figure S-7c. For low flow rates (1 5 L mi -1 ), the sigal itesity icreased with the icrease of the ESI solvet ijectio rate. However, the sigal itesity started to decrease whe the ijectio rate exceeded 5 L mi -1, probably because the ebulizatio efficiecy was affected by the higher flow rates. Sample ijectio rate. The sigal itesity of characteristic fragmet (m/z 84) of O was also affected by the sample ijectio rate (eutral chael i EESI), ad the result is show i Figure S-7d. Whe the sample ijectio rate was raised from 1 L mi -1 to 6 L mi -1, the sigal itesity was icreased. However, the sigal was dropped whe the sample ijectio rate exceeded 6 L mi -1. Because higher ijectio rates ca cause chemical cotamiatio to the ilet of mass spectrometer, the sample ijectio rate i this work was fially adjusted to 6 L mi -1. Io-trasport capillary temperature. The desolvatio process of charged droplets ca be facilitated by elevated temperature of the io-trasport capillary, resultig i a better efficiecy of producig gaseous species. Whe the temperature of io-trasport capillary of the LTQ istrumet was icreased from 100 to 300, the sigal itesity of m/z 84 was rapidly icreased, possibly due to the better desolvatio effects (Figure S-7e). However, the sigal itesity decreased slightly whe the io-trasport capillary temperature was higher tha 300, which ca be due to the thermal 7
8 dissociatio of O/ ios iside the io-trasport capillary. Sheath gas pressure. The pressure of sheath gas affected the sigal itesity of characteristic fragmet m/z 84. The result is show i Figure S-7f. Below the optimal pressure of 1.4 MPa, sample ebulizatio was poor, leadig to a lower sigal itesity of m/z 84. The sigal decrease at higher pressures ca be due to the decreased efficiecy of olie liquid-liquid extractio/ioizatio i the EESI plume, sice faster droplets have lower residece time i the extractio area. 4. MS/MS aalysis of [O+2H] + (m/z 235) Upo ID, the ios [O+2H] + (m/z 235) geerated fragmets at m/z 201, 153, 105, 98, 84 ad 83 (Figure S-8). The largest fragmet (m/z 201) was produced by the loss of H 2 O 2 from the precursor ios. The MS 3 spectrum of the ios at m/z 201 (iset of Figure S-8) showed that the precursor ios (m/z 201) decomposed ito the fragmet ios at m/z 144 ad m/z 98 through the loss of 3 H 7 N ad 6 H 5 N, respectively. The low abudat ios at m/z 104 which were observed i the MS 3 spectrum of precursor ios (m/z 201) were attributed to the fragmetatio of the ios at m/z 144. The other fragmets (m/z 153, 105, 84, ad m/z 83) observed i MS/MS spectrum of the ios [O+2H] + were produced by the loss of 6 H 10, 6 H 14 N 2 O, 7 H 7 N 2 O 2, ad 7 H 8 N 2 O 2, respectively. The MS 2 spectrum of m/z 235 showed product io sigals at m/z 105 ad m/z 84, which correspoded to bezoyl ios ad 6 H 12 + ios, respectively. 8
9 5. MS/MS aalysis of [+2H] + (m/z 219) I the MS 2 spectrum of [+2H] +, precursor ios m/z 219 geerated product ios of m/z 145, 137, 119, 98, ad 83, as show i Figure S-3. The ios of m/z 137 were obtaied by rig-opeig of precursor ios m/z 219 through loss of 6 H 10. Sigals at m/z 120, 119, ad 104 i MS 3 spectrum were observed by loss of OH, H 2 O, ad NH 2 OH, respectively. O is a five membered itroge cotaiig heterocyclic compoud, ad the ios m/z 98 observed i MS 2 spectrum were probably geerated by loss of 6 H 5 H 2 NO from m/z 219 through cleavage of =N bod followed by rearragemet. Rig-opeig ad reductio of =N bod i imidazoles could happe with assistace of special catalyst. Therefore the abudace of m/z 98 was relatively low as the efficiecy of rig-opeig is very low. The product ios of m/z 145 ad m/z 83 i MS 2 spectrum of ios at m/z 219 were geerated by loss of 3 H 8 NO ad 7 H 8 N 2 O, respectively. 9
10 Table S-1. The results of spikig experimet usig the breath samples from from 5 subjects. Base value (ppbv) Spiked (ppbv) Total (ppbv) Recovery (%) Subject ± ± Subject ± ± Subject ± ± Subject ± ± Subject ± ±
11 Figures Figure S-1. EESI-MS spectra of O. a) Full sca EESI-MS mass spectrum of O (0.5 mg L -1 ); b) MS 2 spectrum of protoated O (m/z 234), ad the iset shows the MS 3 spectrum of m/z
12 Figure S-2. Proposed structure for the product ios from protoated i positive io detectio mode ([+H] +, m/z 218). 12
13 Figure S-3. MS 2 spectrum of [+2H] + (m/z 219). The iset shows the MS 3 spectrum of ios of m/z 219. See MS/MS aalysis of [+2H] + (m/z 219) i Sectio 5. 13
14 Figure S-4. Detectio of, the resultat of O, ad exhaled NO, by EESI-MS. a) sigal (m/z 218); b) MS 2 spectrum of m/z 218, ad the iset shows the MS 3 spectrum of m/z
15 Figure S-5. alibratio curve of O obtaied usig EESI-LTQ-MS. 15
16 Figure S-6. alibratio curve for the detectio of NO cocetratio obtaied usig EESI-LTQ-MS aalysis of product for the reactio betwee NO ad O. 16
17 Figure S-7. Optimizatio of experimetal parameters for EESI. a) ESI voltage, b) compositio of ESI solvet, c) ESI solvet ijectio rate, d) sample ijectio rate, e) io-trasport capillary temperature, ad f) ebulizig gas (N 2 ) pressure. 17
18 Figure S-8. MS 2 spectrum of [O+2H] + (m/z 235). The iset shows the MS 3 spectrum of ios of m/z 235. See MS/MS aalysis of [O+2H] + (m/z 235) i Sectio 4. 18
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