Nitrate and Nitrite Promote Formation of Tobacco …downloads.hindawi.com/journals/jchem/2017/6135215.pdf · Postcured Storage under Warm Temperature JunWang,1 HuijuanYang,1 HongzhiShi,1

Research ArticleNitrate and Nitrite Promote Formation of Tobacco-SpecificNitrosamines via Nitrogen Oxides Intermediates duringPostcured Storage under Warm Temperature

JunWang1 Huijuan Yang1 Hongzhi Shi1 Jun Zhou2 Ruoshi Bai2

Mengyue Zhang1 and Tong Jin1

1Henan Agricultural University National Tobacco Cultivation amp Physiology amp Biochemistry Research CenterZhengzhou 450002 China2Beijing Cigarette Factory Shanghai Tobacco Group Co Ltd Beijing 100024 China

Correspondence should be addressed to Hongzhi Shi shihongzhi88163com

Received 26 December 2016 Revised 7 February 2017 Accepted 7 March 2017 Published 29 March 2017

Academic Editor Yang Xu

Copyright copy 2017 Jun Wang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Tobacco-specific nitrosamines (TSNAs) are carcinogenic and are present in cured tobacco leaves This study was designed toelucidate the mechanisms of TSNAs formation under warm temperature storage conditions Results showed that nitrogen oxides(NOx) were produced from nitrate and nitrite in a short period of time under 45∘C and then reacted with alkaloids to form TSNAsNitrite was more effective than nitrate in promoting TSNAs formation during 45∘C storage whichmay be due to the fact that nitritecan produce a large amount of NOx in comparison with nitrate Presence of activated carbon effectively inhibited the TSNAsformation because of the adsorption of NOx on the activated carbonThe results indicated that TSNAs are derived from a gassolidphase nitrosation reaction betweenNOx and alkaloids Nitrate and nitrite are major contributors to the formation of TSNAs duringwarm temperature storage of tobacco

1 Introduction

Tobacco-specific nitrosamines (TSNAs) are a group of impor-tant and toxic components of tobacco and tobacco smoke[1 2] TSNAsmainly consist of N-nitrosonornicotine (NNN)N-nitrosoanatabine (NAT) N-nitrosoanabasine (NAB) and4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)NNN and NNK are strong carcinogens [3 4] TSNAs areproduced via nitrosation of tobacco alkaloids during the cur-ing and storage of tobacco leaves (Figure 1) [5ndash8] In Chinaboth air-cured and flue-cured tobacco leaves are generallystored for approximately 18 months in warehouses beforebeing processed for cigarette production in order to reducethe unfavorable smells [9]

Many studies reported about the factors that influenceTSNAs formation during air-curing [7 10ndash12] Cui [10] foundthat TSNAs levels in the leaf lamina and midrib increasedsubstantially during the fourth to seventh weeks of air-curing

stage During this stage nitrate is reduced to nitrite viamicrobial activity and the resulting nitrite in turn is involvedin nitrosation reactions with the naturally existing alkaloidsduring air-curing then leading to the formation of TSNAs(Figure 2) [11]

However to our knowledge there are few reports availableon themechanisms of TSNAs formation during storage stageTSNAs levels may increase several fold in comparison to thelevels in freshly air-cured leaves [13 14] but the mechanismsof TSNAs formation during storage are not clear It has beenreported that as the storage temperature increased TSNAsand nitrite contents increased and the most rapid increase inTSNAs occurred during the warm temperature season [1516] The interactions between temperature and abundantnitrate in cured tobacco leaf could be responsible for TSNAsformation during storage Treatment of the tobaccos withstreptomycin and rifampicin did not inhibit nitrosamine for-mation during storage indicating that TSNAs formation was

HindawiJournal of ChemistryVolume 2017 Article ID 6135215 11 pageshttpsdoiorg10115520176135215

2 Journal of Chemistry

N

N

N

NH

N

NH

N

NH

N

N

O

O

N

N

N

N ON

NN O

N

NN O

AlkaloidDemethylation

Nicotine

Oxidized derivative-PON

Nornicotine

CH3

Anatabine Anabasine

N-nitrosation Activated nitrosating agents NO2minus NO NO2 N2O3

TSNA

NNK NNN NAT NAB

Figure 1 Proposed formation pathways of the major TSNAs found in cured tobacco leaves Nitrosating agents can directly interact withnornicotine anatabine and anabasine to form NNN NAT and NAB respectively Nicotine is less susceptible to nitrosation thus NNK isproduced from pseudo-oxynicotine an oxidized derivative of nicotine

Air-curing process

Nitrosation

NO3minus

NO3minus

NO2minus

NO2minus

Microorganism

Microorganism

In fresh tobacco leaf

TSNA

TSNAPostcured storage

Nitrosation

+

+

Alkaloid

Alkaloid

In cured tobacco leaf

Figure 2The activated nitrosating agents participate in nitrosation of alkaloid to formTSNAs during different processes of air-cured tobaccoproduction Nitrate (NO

3

minus) is available for reduction to nitrite (NO2

minus) via microbial activity and the nitrite in turn becomes involved innitrosation reactions with the alkaloids during air-curing leading to the formation of TSNAs The nitrite is the most important nitrosatingagent during air-curing TSNAs formation was almost not influenced by microorganism during the storage processes

not influenced bymicroorganisms during the storage process(Figure 2) [14] How the nitrate and nitrite trigger the increasein TSNAs during warm temperature storage is still not clear(Figure 2) The objectives of this study were to elucidate themechanisms of TSNAs formation during the storage time ofcured tobacco leaf and to verify the hypothesis that nitrogenoxides produced from nitrate and nitrite in tobacco areresponsible for the formation of TSNAs during storage

2 Experiments

21 PlantMaterials Tobacco sampleswere grown in 2015 andcured locally Leaves from themiddle stalk positionswere col-lected Flue-cured tobacco (N tabacum cv ldquoHongdardquo) was

fromMidu county Yunnan province Leaf samples were pre-pared by removing the stems cut to strips mixed thoroughlysealed in plastic bags and then stored at 4∘C for the testsThemoisture content of tobacco was 12

22 Methods

221 Direct Addition of Nitrate and Nitrite to Tobacco Thetreatments included four levels of NaNO

3(10 20 30 and

40mgmL) andNaNO2(5 10 15 and 20mgmL) whichwere

from 10mL solution each equal to 73 146 219 and 292mg ofNO3

minus and 33 67 100 and 133mg of NO2

minusThe solutions weresprayed onto each of eight 20 g flue-cured tobacco samplesThe samples were then placed to an ambient environment for

Journal of Chemistry 3

air-dryingThemoisture content of tobacco samples after air-drying was 13 Then the samples were stored in an airtightvacuum desiccator (15 cm diameter) at 45∘C with relativehumidity of 70 for 15 d The sample sprayed with the samevolume of deionized water was used as the control Themoisture content of tobacco samples after treatment was118

222 Separating the Tobacco and NitrateNitrite Sources in aClosed System under 45∘C Storage Conditions Avacuumdes-iccatorwith a porcelain platewas used to forma closed systemin which tobacco cuts and added nitrate and nitrite could beseparated during storage Five mL of each aqueous solutionof NH

4NO3 KNO

3 NaNO

3 and NaNO

2at 1molL (equal

to 310mg of NO3

minus and 230mg of NO2

minus) and 5mL volumeof deionized water (control) were sprayed onto the medicalgauze pads (5 cm times 5 cm) After air-drying the sprayedgauzes were placed in the bottom of the vacuum desiccatortogether with each 20 g sample of flue-cured tobacco Thevacuum desiccators were then tightly closed and sealed withpetroleum jelly The containers were then placed into achamber at a temperature of 45∘C and a relative humidity of70 and stored for 15 d The moisture content of tobaccosamples after 45∘C treatments was 111

223 Effect of Indirect Addition of Nitrate andNitrite onNitro-gen Oxides Formation in a Closed System with Flue-CuredTobacco Eight treatments in this experiment were divided totwo groups Group I (1) tobacco (2) tobacco and NaNO

3

(3) tobacco and NaNO3+ 20 g activated carbon (AC) (4)

tobacco and NaNO3+ 100 g AC and Group II (1) tobacco

(2) tobacco and NaNO2 (3) tobacco and NaNO

2+ 20 g AC

(4) tobacco and NaNO2+ 100 g AC The amount of added

NaNO3or NaNO

2was 08 g and 04 g for treatments 2 to 4

(equal to 583mgofNO3

minus and 266mgofNO2

minus) in each groupThe weight ratios of tobacco NO

3

minus and ACwere 68 1 3 and68 1 17 respectively And theweight ratios of tobaccoNO

2

minusand ACwere 150 1 7 and 150 1 37 respectively NaNO

3and

NaNO2were dissolved in 5mL deionized water and then

sprayed onto the gauze pads After air-drying the gauze padswere placed on the bottom of the vacuum desiccator with40 g sample of flue-cured tobacco leaf in each desiccator ACsample was made from wood with the particle size being75120583m produced by Zhengzhou Tianhe Water PurificationMaterial Co Ltd in Henan province The surface area was9017m2g and the total pore volume was 0518 cm3g withthe micropore mesopore and macroporous volume being0372 cm3g 0129 cm3g and 0016 cm3g respectively ACwas activated in 100∘C oven for 30min then was wrappedin a medical gauze and suspended under the porcelain plateof the vacuum desiccator for the relevant treatments All vac-uum desiccators were tightly closed sealed and placed into achamber at a temperature of 45∘C and a relative humidity of70 for 24 h

TheACused in the above experiments were taken out andthen were placed in the new airtight desiccator respectivelyTwo grams of AC was put into another container as controlAll airtight desiccators were placed into a chamber at a

temperature of 60∘C and a relative humidity of 70 for 15minand 90min

224 Effects of Activated Carbon (AC) on TSNAs Formationin Flue-Cured Tobacco in Response to Nitrate and NitriteAddition Twelve treatments in this experiment were dividedinto two groups Group I (1) tobacco (4∘C) (2) tobacco(warm temperature control 45∘C) (3) tobacco and NaNO

3

separately (4) tobacco and NaNO3+ 10 g AC separately (5)

tobacco and NaNO3+ 50 g AC separately and (6) tobacco

and NaNO3+ 100 g AC separately and Group II (1) tobacco

(4∘C) (2) tobacco (warm temperature control 45∘C) (3)tobacco and NaNO

2separately (4) tobacco and NaNO

2

+ 10 g AC separately (5) tobacco and NaNO2+ 50 g

AC separately and (6) tobacco and NaNO2+ 100 g AC

separately The amount of NaNO3or NaNO

2added to each

sample of treatments 3 to 6was 03 g (equal to 219mg ofNO3

minus

and 200mg of NO2

minus) The weight ratios of tobacco NO3

minusandACwere 91 1 4 91 1 22 and 91 1 45 respectively Andthe weight ratios of tobacco NO

2

minus and AC were 100 1 5100 1 25 and 100 1 50 respectively NaNO

3NaNO

2were

dissolved in 5mL deionized water and then were sprayedonto the gauze pads After air-curing the gauzes were placedon the bottom of the vacuum desiccators with 20 g sampleof flue-cured tobacco leaf in each desiccator All treatmentswere performed in the vacuum desiccator as describedabove The activated AC also was used as described aboveThe vacuum desiccators were put into a chamber with aconstant temperature of 45∘C and a relative humidity of 70respectively The moisture content of cured tobacco samplesafter treatment was 116

23 Chemical Analyses

231 TSNAs Measurements In each experiment tobaccosamples were lyophilized ground to powder sieved through a025mm screen and then measured for the content of NNNNNK NAT and NAB TSNAs contents were determined atthe BeijingCigarette Factory according to themethod of SPE-LC-MSMS [12 17 18]

232 Nitrate Nitrite and Alkaloid Measurements NO3-N

and NO2-N were quantified according to the method of

Crutchfield and Grove [19] The individual alkaloids wereanalyzed with a gas chromatograph as described by Jack andBush [20] Methyl tert-butyl ether was used as the extractionsolvent with N-hexadecane as the internal standard

233 Nitrogen Oxides Analysis The first step was dilutingthe standard gas with a dynamic gas calibrator (Model 146 iThermo Scientific USA EPA) to give a concentration withinthe operational range of the instrument The high puritynitric oxide (NO) and nitrogen dioxide (NO

2) standards

in N2(component content 698 ppm gas sample number

L120911099 National Institute of MetrologyNational Stan-dard Material Research Center Beijing China) were config-ured into nitrogen oxides gas of a low concentration (1 ppm)by 146i calibrator It was necessary to modify the original


EDE CD C

B

NNN A

Addition of NO3minus (mg)

Stored at 45∘C for 15d

Beforestorage

0 73 146 219 292

00

01

02

03

04

05

06

NN

N (120583

gg)

(a)

F

E DC

B

NAT A



Beforestorage

0 73 146 219 292

000025050075100125150175200

NAT

(120583g

g)

(b)

B

B

B

A A

NAB

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NA

B (120583

gg)

(c)

DC C

BA

NNK

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NN

K (120583

gg)

(d)

FE D

C

B

TSNAsA



Beforestorage

0 73 146 219 292

00

05

10

15

20

25

30

TSN

As (

120583g

g)

(e)

Figure 3 Effects of added nitrate on TSNAs formation in flue-cured tobacco stored at 45∘C for 15 d Note For all experiments each valuerepresents the mean of three independent samples Uppercase letters indicate significant differences between the treatments at 119875 lt 005

procedure by configuring the standard gas again if the levelsof NOx exceeded the limit of detection The NO and NOx inthe air were defaulted to the zero point by the instrument

After experiment the airtight vacuum desiccators weretaken out and then connected with a vacuum pump (DOA-P504-BNGASTManufacturing AUnit of IdexCorporationMI USA) to extract the gas into a gas collecting bag After25 s the gas bag was pulled out and connected with theNO-NO

2-NOX analyzer (Model 42i Thermo Scientific USA

EPA ReferencemethodRFNA-1289-074) forNOx analysis bychemiluminescence detection

24 Statistical Analyses Analysis of variance (ANOVA) andleast significant difference (LSD) of TSNAs and NOx valueswere performed at the 005 level of significance Data werestatistically analyzed with SPSS 200 Figures were drawnwith Origin 85 All treatments were randomly designed intriplicate

3 Results and Discussion

31 Effect of Nitrate Concentration on TSNAs Formation inFlue-Cured Tobacco The mean contents of nicotine nor-nicotine anabasine and anatabine of tobacco used in this

experiment were 198 12 03 and 15mgg respectivelyand the NO

3-N and NO

2-N content were correspondingly

119 120583gg and 10 120583gg With the increasing amount of NO3

minus

added both individual and total TSNAs contents increasedsignificantly (Figure 3) After the addition of 04 g NaNO

3

(equal to 292mg of NO3

minus) the NAT and total TSNAs con-tents increased by 340and 311 respectively in comparisonwith the control

No significant change of NNN was observed in thetobacco samples when less than 02 g of NaNO

3(146mg

of NO3

minus) was added However when the amount of NO3

minus

added increased to 219mg (03 g NaNO3) the NNN content

was approximately double that of 02 g NaNO3addition

Although the NAB content was the lowest of the four indi-vidual TSNAs a significant increase inNAB content occurredas a result of the addition of NaNO

3reached to 02 g The

NNK content also increased significantly as nitrate additionincreased

32 Effect of Nitrite Concentration on TSNAs Formation inFlue-Cured Tobacco Table 1 shows that as the concentrationof added NO

2

minus increased the individual and total TSNAscontents showed rapid and dramatic increases and the rela-tive increments were greater for the samples with greater


Con

trol

CBBB

NNN A


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

10

20

30

40

50

60

NN

N (120583

gg)

(a)

BBBB

NATA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0005101520

180

200

220

240

NAT

(120583g

g)

(b)

B BB B

NABA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

02

04

06

08

10

NA

B (120583

gg)

(c)

BBBB

NNKA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

01

02

03

04

05

NN

K (120583

gg)

(d)

CB B B

ATSNA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0

2

4

20

25

30

TSN

As (

120583g

g)

(e)

Figure 4 Effects of indirect addition of three nitrate compounds and sodium nitrite on TSNAs formation in flue-cured tobacco during warmtemperature storage Note The 1molL of nitrate nitrite compounds (310mg of NO

3


minus resp) and tobacco were addedseparately in the closed vessel which was then stored at 45∘C for 15 d Uppercase letters indicate significant differences between the treatmentsat 119875 lt 005

amounts of addedNO2

minusWhen tobacco samples were treatedwith 133mg of NO

2

minus (02 g of NaNO2) total TSNAs content

was 117 120583gg at a 207 increment compared with the control(056 120583gg) For the individual TSNA levels of NNN NNKNAT andNAB showed 257- 38- 203- and 110-fold increasesrespectivelyThe results showed that NO

2

minus wasmore effectivein promoting TSNAs formation in the cured leaf than NO

3

minus

during warm temperature storageThe higher TSNAs levels in burley tobacco are partly

due to the relatively higher levels of TSNAs precursorssuch as alkaloids and oxide of nitrogen that are present inthe leaf tissue [21ndash23] It is reported that nitrite which isderived from the bacterially mediated reduction of nitrateis considered to be the limiting factor in TSNAs forma-tion in air-cured tobacco during air-curing [6 24] In thisresearch the addition of nitrate and nitrite in flue-curedtobacco to the levels which are equivalent to those in burleytobacco can increase the TSNAs concentration comparable

to burley tobacco especially coupled with warm temperatureResults indicated that the great amount of nitrosating speciesavailable is a major contributor to the formation of TSNAsobserved in cured tobacco during storage

33 Effects of Indirect Addition of NitrateNitrite on TSNAFormation in Flue-Cured Tobacco during Warm TemperatureStorage TSNAs contents increased as the storage temper-ature increased [15 16] and the abundance of nitrite andnitrate could be a major contributor To clarify how nitrate ornitrite affects the formation of TSNAs under warm temper-ature indirect addition of nitrate or nitrite experiments wascarried out

After tobacco leaf treated with 1molL NaNO2 total

TSNAs content increased almost by 54 times compared withthat in the control sample (Figure 4) When tobacco leaf isplaced separately with gauze pad containing 310mg nitrateobvious increases were observed both in individual and


Table 1 Effects of added nitrite on TSNAs formation in flue-cured tobacco stored at 45∘C for 15 d

Storage conditions Addition of NO2

minus (mg) NNN (120583gg) NAT (120583gg) NAB (120583gg) NNK (120583gg) Total TSNAs (120583gg)Before storage 0 008 plusmn 001E 011 plusmn 001E 0004 plusmn 000E 003 plusmn 000D 022 plusmn 000E

45∘C for 15 d

0 013 plusmn 001E 039 plusmn 002E 002 plusmn 000E 003 plusmn 001D 056 plusmn 003E

33 585 plusmn 028D 1897 plusmn 031D 031 plusmn 003D 009 plusmn 001CD 2522 plusmn 007D

67 1285 plusmn 088C 4173 plusmn 195C 077 plusmn 006C 023 plusmn 003C 5557 plusmn 274C

100 1922 plusmn 113B 5196 plusmn 154B 115 plusmn 015B 066 plusmn 009B 7299 plusmn 244B

133 3366 plusmn 218A 7968 plusmn 096A 225 plusmn 023A 121 plusmn 016A 11680 plusmn 353A

Note Each value represents the mean of three independent samples and uppercase letters indicate significant differences between the treatments at 119875 lt 005

D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)

2 431Stored at 45∘C for 24h

(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)

Figure 5 Effect of indirect addition of nitrate and activated carbon (AC) on nitrogen oxides formation in a closed system with flue-curedtobacco Note (1) tobacco (2) tobacco + NaNO

3 (3) tobacco + NaNO

3+ AC 20 g and (4) tobacco + NaNO

3+ AC 100 g 40 g of flue-cured

tobacco and 08 g NaNO3(583mg of NO

3

minus) The NO3

minus AC and tobacco were added separately in the vacuum desiccators stored at 45∘C for24 h Uppercase letters indicate significant differences between the treatments at 119875 lt 005

in total TSNA content compared with the control Datapresented here suggested that even though the tobaccosample is placed separately with nitrate or nitrite TSNAcontent in tobacco also increased The formation of TSNAsin storage triggered by nitrate and nitrite is likely a gas phasereaction

34 Effect of Indirect Addition of NitrateNitrite on NOxFormation in a Closed Systemwith Flue-Cured Tobacco Flue-cured tobacco leaves could generate trace concentrations ofNOx under 45∘C after 24 h treatment (Figures 5 and 6) Asa main component NO accounted for more than 95 ofthe NOx produced from the tobacco sample As 08 g of


000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)

Figure 6 Effect of indirect addition of nitrite and activated carbon (AC) on nitrogen oxides formation in a closed system with flue-curedtobacco Note (1) tobacco (2) tobacco + NaNO





2

minus) The NO2

minus AC and tobacco were added separately in vacuum desiccators stored at 45∘C for24 h Uppercase letters indicate significant differences between the treatments at 119875 lt 005

NaNO3(equal to 583mg of nitrate) was added in the vacuum

desiccator the concentration of NO and NOx increasedsignificantly (119875 lt 005) NOx reached 06 ppm (Figure 5(c))When 04 g of NaNO

2(equal to 266mg of nitrite) was added

NOx concentrations in the desiccator dramatically increasedto 83 ppm which was 246-fold greater than the control(Figure 6(c))

It is interesting that when 2 g of AC was added to thesystem (the weight ratio of tobacco NO

3

minus and AC is 68 1 3the weight ratio of tobacco NO

2

minus and AC is 150 1 7) a 97decrease of NOx level was observed in comparison to theadded nitrate treatment (Figure 5(c)) and almost 79 ppm ofNOx were adsorbed by AC (Figure 6(c)) Meanwhile similaradsorption effects were observed for NO The concentrationof NO and NOx decreased significantly (119875 lt 005) as theaddition of AC increased indicating strong adsorption forNOx generated from nitrate and nitrite by AC

Table 2 presents the desorption rate of NOx from the ACsamples which were used for the adsorption experiments andwere significantly higher than those from control group Thedesorption rates of NOx and NO were obviously increasedwith the prolonging of treating time and the increase of NOxadsorption observed in Figures 5 and 6 Results indicatedthat NOx were indeed adsorbed by AC As the weight ratioof tobacco NO

2

minus and AC is 150 1 7 NOx decreased by79 ppm (Figure 6(c)) at the same time 0632 ppmofNOxwasdesorbed by the AC after treatment at 60∘C for 90min Sincethe desorption rate of NOx in AC has close relationship withthe temperature and time [25] further research is needed todetermine the effect of desorbing temperature and time onthe desorption rate of NOx adsorbed by AC

During flue-curing process direct-fired systems allowcombustion products specifically NOx to mix with the airand expose the green tobacco leaves to these gases [26] The


Table 2 The desorption rate of NOx at 60∘C for different time

Treatment NO (ppm) NOx (ppm)15min 90min 15min 90min

AC (Control) 00094C 00249C 00203B 00486B

AC used in Figure 5 Treatment 3 (2 g AC) 00200B 00466B 00220B 00505B

Treatment 4 (10 g AC) 00216B 00471B 00244B 00511B

AC used in Figure 6 Treatment 3 (2 g AC) 00732A 06200A 00864A 06320A

Treatment 4 (10 g AC) 00740A 06302A 00887A 06435A


Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)

Figure 7 Effect of activated carbon (AC) on TSNAs formation in flue-cured tobacco in response to nitrate additionNote (1) tobacco (beforestorage 4∘C) (2) tobacco (3) tobacco +NaNO

3 (4) tobacco +NaNO

3+AC 10 g (5) tobacco +NaNO

3+AC 50 g and (6) tobacco +NaNO

3

+ AC 100 g 20 g of flue-cured tobacco and 03 g NaNO3(219mg of NO

3

minus)The NO3

minus AC and tobacco were added separately in the vacuumdesiccators stored at 45∘C for 15 d Uppercase letters indicate significant differences between the treatments at 119875 lt 005

previous research showed that TSNAs may be formed bynitrosation via the elevated levels of NOx in the air surround-ing the leaves during the curing process [27] However therewere few reports about the relationship between NOx andTSNAs during storage process Data in Figure 4 suggestedthat TSNAs in storage is likely a gas phase reactionThis resultshowed that NOx can be generated from NO

2-N and NO

3-N

under 45∘C after 24 h Nitrite was more effective than nitratein the production of NOx which in turn would probablypromote the formation of TSNAs

35 Effect of AC on TSNAs Formation in Flue-Cured Tobaccoin Response to Nitrate and Nitrite Added Having a veryporous structure and special surface properties AC has beenused to trap TSNAs in tobacco solution [28 29] Lin et al[30] reported that AC made from coconut shells exhibited ahigh affinity for TSNAs The impregnated sorbent ZnAC canremove 73of the TSNAs in solution offering a cost-effectivecandidate for industrial applications [29]

As shown in Figures 7(e) and 8(e) total TSNAs contentin tobacco incubated separately with NO

2

minus (treatment 3) at


C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)

Figure 8 Effect of activated carbon (AC) on TSNAs formation in flue-cured tobacco in response to nitrite additionNote (1) tobacco (beforestorage 4∘C) (2) tobacco (3) tobacco +NaNO

2 (4) tobacco +NaNO



2

+AC 100 g 20 g of flue-cured tobacco and 03 g NaNO2(200mg of NO

2

minus)TheNO2


45∘C increased markedly to 25 120583gg a 59-fold increase overthe control (treatment 2) By treating tobacco with NO

3

minushowever total TSNAs increased to 1 120583gg also significantlyhigher than the control The huge difference of TSNAscontent between tobacco treated with NO

2

minus and NO3

minus couldbe attributed to the high concentration of NOx which wereemitted from NO

2

minus under the same conditionHowever after adding 1 g of AC to the separating system

of tobacco and NO2

minus the weight ratio of tobacco NO2

minusand AC was 100 1 5 both individual and total TSNAscontents of tobacco decreased markedly with total TSNAscontent decreasing to 13120583gg 48 lower than in treatment3 and NNN NAT NAB and NNK decreasing by 479476 673 and 41 respectively When the addition of ACincreased to 5 g the weight ratio of tobacco NO

2

minus and ACreached to 100 1 25 total TSNAs levels further decreased to12 120583gg indicating a crucial role of AC in inhibiting TSNAsformation by adsorbing the NOx generated fromNO

2-N and

NO3-N The results also showed no significant differences in

reducing TSNAs levels by increasing AC usage greater than5 g (Figures 7 and 8)Themaximum inhibitory effect ofAConTSNAs formation occurred at an ACNO

2

minus ratio of 251 Theabove results indicated that AC may have the potential to be

used as adsorbent agent to reduce TSNAs formation duringtobacco leaf storage Our results strongly support the theorythat TSNAs formation in storage is a gas phase reaction andthe NOx which is produced from nitrate and nitrite couldintermediate the TSNAs formation duringwarm temperaturepostcured storage of tobacco Since NOx can be producedfrom nitrate and nitrite under warm temperature in a shortperiod it can readily react with alkaloids to formTSNAsThisresult also explained why the most rapid increase in TSNAsoccurred during the warm temperature season [16]

Decreasing NOx level by AC adsorption significantlyreduced TSNAs formation of tobacco which indicated thatthe removal of NOx from storage environment could be aneffective way to inhibit TSNAs formation in storing tobaccoleaf Therefore controlling the storage environment andscavenging gaseous nitrosation agents would be crucial toreduce or inhibit TSNAs formation during leaf storage

4 Conclusion

The results proved that TSNAs are derived from a gassolidphase nitrosation reaction betweenNOx and alkaloids during


storage Nitrogen oxides produced from nitrate and nitriteare responsible for the formation of TSNAs during storageunder warm temperature Presence of activated carbon in thetobacco storage containers effectively inhibited the TSNAsformation due to the adsorption of NOx on the activatedcarbon

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

JunWang and Huijuan Yang contributed equally to this workand should be considered co-first authors

References

[1] J D Adams S J Lee N Vinchkoski A Castonguay and DHoffmann ldquoOn the formation of the tobacco-specific car-cinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone dur-ing smokingrdquo Cancer Letters vol 17 no 3 pp 339ndash346 1983

[2] D Hoffmann M Dong and S S Hecht ldquoOrigin in tobaccosmoke of N1015840-nitrosonornicotine a tobacco-specific carcinogenbrief communicationrdquo Journal of the National Cancer Institutevol 58 no 6 pp 1841ndash1844 1977

[3] P C Gupta P R Murti and R B Bhonsle ldquoEpidemiology ofcancer by tobacco products and the significance of TSNArdquoCritical Reviews in Toxicology vol 26 no 2 pp 183ndash198 1996

[4] S S Hecht ldquoBiochemistry biology and carcinogenicity oftobacco-specific N- nitrosaminesrdquo Chemical Research in Toxi-cology vol 11 no 6 pp 559ndash603 1998

[5] H Shi and J Zhang ldquoThe significant of alkaloidsrdquo in TobaccoAlkaloids vol 1st p 14 China Agriculture Press Beijing China2004

[6] L P Bush M Cui H Shi et al ldquoFormation of tobacco specificnitrosamines in air-cured tobaccordquo Recent Advances in TobaccoScience vol 27 pp 23ndash46 2001

[7] M V Djordjevic S L Gay L P Bush and J F ChaplinldquoTobacco-specific nitrosamine accumulation and distributionin flue-cured tobacco alkaloid isolinesrdquo Journal of Agriculturaland Food Chemistry vol 37 no 3 pp 752ndash758 1989

[8] I Stepanov A Knezevich L Zhang C H Watson D KHatsukami and S S Hecht ldquoCarcinogenic tobacco-specific N-nitrosamines in US cigarettes three decades of remarkableneglect by the tobacco industryrdquo Tobacco Control vol 21 no1 pp 44ndash48 2011

[9] S-C Shen K-C Tseng and J S-BWu ldquoAn analysis ofMaillardreaction products in ethanolic glucose-glycine solutionrdquo FoodChemistry vol 102 no 1 pp 281ndash287 2007

[10] M Cui The source and the regulation of nitrogen oxide produc-tion for tobacco-specific nitrosamine formation during air-curingtobacco [PhD thesis] University of Kentucky Lexington KyUSA 1998

[11] H R Burton N K Dye and L P Bush ldquoRelationship betweenTSNA and nitrite from different air-cured tobacco varietiesrdquoJournal of Agricultural and Food Chemistry vol 42 pp 2007ndash2011 1994

[12] X Wei X Deng D Cai et al ldquoDecreased tobacco-specificnitrosamines by microbial treatment with Bacillus amylolique-faciens DA9 during the air-curing process of burley tobaccordquoJournal of Agricultural and Food Chemistry vol 62 no 52 pp12701ndash12706 2014

[13] J L Verrier AWiernik M Staaf J l Cadilhac M Onillon andB Vidal ldquoThe influence of post-curing of burley tobacco anddark air-cured tobacco on TSNA and nitrite levelsrdquo in Pro-ceedings of the CORESTACongress Shanghai China November2008

[14] R M Jackisch and J H Rovedder ldquoBurley tobacco post-curing management and its effect in the nitrosamine amountrdquoin Proceedings of the CORESTA Joint Study Group MeetingKrakow PolandOctober 2007 httpswwwcorestaorgabstractsburley-tobacco-post-curing-management-and-its-effect-nitro-samine-amount-1525html

[15] H Saito M Miyazaki and J Miki ldquoRole of nitrogen oxidesin tobacco-specific nitrosamine formation in burley tobaccordquoin Proceedings of the 2006 CORESTA Congress Paris FranceOctober 2006 httpswwwcorestaorgabstractsrole-nitrogen-oxides-tobacco-specific-nitrosamine-formation-burley-tobacco-2254html

[16] H Shi R Wang L P Bush et al ldquoChanges in TSNA contentsduring tobacco storage and the effect of temperature and nitratelevel on TSNA formationrdquo Journal of Agricultural and FoodChemistry vol 61 no 47 pp 11588ndash11594 2013

[17] W Morgan J Reece C Risner et al ldquoA collaborative study forthe determination of tobacco specific nitrosamines in tobaccordquoBeitrage zur Tabakforschung International vol 21 no 3 pp 192ndash203 2014

[18] J Zhou R Bai and Y Zhu ldquoDetermination of four tobacco-specific nitrosamines in mainstream cigarette smoke by gaschromatographyion trap mass spectrometryrdquo Rapid Commu-nications in Mass Spectrometry vol 21 no 24 pp 4086ndash40922007

[19] J D Crutchfield and J H Grove ldquoA new cadmium reductiondevice for the microplate determination of nitrate in water soilplant tissue and physiological fluidsrdquo Journal of AOAC Interna-tional vol 94 no 6 pp 1896ndash1905 2011

[20] A Jack and L Bush The lsquoLCrsquo Protocol-Appendix 3 LaboratoryProcedures University of Kentucky Lexington Ky USA 2007httpwwwukyeduAgTobaccoPdfLC-ProtocolPdf

[21] L P Bush M W Cui and H Z Shi ldquoFormation of tobacco-specific nitrosamines in air-cured tobaccordquo in Proceedings of the55th Tobacco Science Research Conference Greensboro NCUSA September 2001

[22] H Shi N E Kalengamaliro M R KraussW P Hempfling andF Gadani ldquoStimulation of nicotine demethylation by NaHCO

3

treatment using greenhouse-grown burley tobaccordquo Journal ofAgricultural and Food Chemistry vol 51 no 26 pp 7679ndash76832003

[23] H Shi R Wang L P Bush H Yang and F F Fannin ldquoTherelationships between TSNAs and their precursors in burleytobacco from different regions and varietiesrdquo Journal of FoodAgriculture and Environment vol 10 no 3-4 pp 1048ndash10522012

[24] R S Lewis R G Parker D A Danehower et al ldquoImpact ofalleles at the Yellow Burley (Yb) loci and nitrogen fertilizationrate on nitrogen utilization efficiency and tobacco-specificnitrosamine (TSNA) formation in air-cured tobaccordquo Journal ofAgricultural and Food Chemistry vol 60 no 25 pp 6454ndash64612012


[25] W J Zhang S Rabiei A Bagreev M S Zhuang and FRasouli ldquoStudy ofNOadsorption on activated carbonsrdquoAppliedCatalysis B Environmental vol 83 no 1-2 pp 63ndash71 2008

[26] D M Peele M G Riddick and M E Edwards ldquoFormation oftobacco specific nitrosamines in flue-cured tobaccordquo RecentAdvances in Tobacco Science vol 27 pp 3ndash12 2001

[27] G H Ellington andMD Boyette ldquoInvestigation into the corre-lation among nitrogen oxides and tobacco-specific nitrosaminein flue-cured tobaccordquo Tobacco Science vol 50 pp 11ndash18 2013

[28] S Tatsuoka ldquoProcess for producing regenerated tobacco mate-rialrdquo EP Patent1782702 A1 2005 httpwwwfreepatentsonlinecomEP1782702html

[29] X D Sun W G Lin L-J Wang et al ldquoLiquid adsorption oftobacco specific N-nitrosamines by zeolite and activated car-bonrdquoMicroporous andMesoporous Materials vol 200 pp 260ndash268 2014

[30] W G Lin B C Huang B Zhou et al ldquoTrapping tobacco spe-cific N-nitrosamines in Chinese-Virginia type tobacco extract-ing solution by porous materialrdquo Journal of Porous Materialsvol 21 no 3 pp 311ndash320 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal ofInternational Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


N

N

N

NH

N

NH

N

NH

N

N

O

O

N

N

N

N ON

NN O

N

NN O

AlkaloidDemethylation

Nicotine

Oxidized derivative-PON

Nornicotine

CH3

Anatabine Anabasine

N-nitrosation Activated nitrosating agents NO2minus NO NO2 N2O3

TSNA

NNK NNN NAT NAB

Figure 1 Proposed formation pathways of the major TSNAs found in cured tobacco leaves Nitrosating agents can directly interact withnornicotine anatabine and anabasine to form NNN NAT and NAB respectively Nicotine is less susceptible to nitrosation thus NNK isproduced from pseudo-oxynicotine an oxidized derivative of nicotine

Air-curing process

Nitrosation

NO3minus

NO3minus

NO2minus

NO2minus

Microorganism

Microorganism

In fresh tobacco leaf

TSNA

TSNAPostcured storage

Nitrosation

+

+

Alkaloid

Alkaloid

In cured tobacco leaf

Figure 2The activated nitrosating agents participate in nitrosation of alkaloid to formTSNAs during different processes of air-cured tobaccoproduction Nitrate (NO

3

minus) is available for reduction to nitrite (NO2

minus) via microbial activity and the nitrite in turn becomes involved innitrosation reactions with the alkaloids during air-curing leading to the formation of TSNAs The nitrite is the most important nitrosatingagent during air-curing TSNAs formation was almost not influenced by microorganism during the storage processes

not influenced bymicroorganisms during the storage process(Figure 2) [14] How the nitrate and nitrite trigger the increasein TSNAs during warm temperature storage is still not clear(Figure 2) The objectives of this study were to elucidate themechanisms of TSNAs formation during the storage time ofcured tobacco leaf and to verify the hypothesis that nitrogenoxides produced from nitrate and nitrite in tobacco areresponsible for the formation of TSNAs during storage

2 Experiments

21 PlantMaterials Tobacco sampleswere grown in 2015 andcured locally Leaves from themiddle stalk positionswere col-lected Flue-cured tobacco (N tabacum cv ldquoHongdardquo) was

fromMidu county Yunnan province Leaf samples were pre-pared by removing the stems cut to strips mixed thoroughlysealed in plastic bags and then stored at 4∘C for the testsThemoisture content of tobacco was 12

22 Methods

221 Direct Addition of Nitrate and Nitrite to Tobacco Thetreatments included four levels of NaNO

3(10 20 30 and

40mgmL) andNaNO2(5 10 15 and 20mgmL) whichwere

from 10mL solution each equal to 73 146 219 and 292mg ofNO3

minus and 33 67 100 and 133mg of NO2

minusThe solutions weresprayed onto each of eight 20 g flue-cured tobacco samplesThe samples were then placed to an ambient environment for




4NO3 KNO

3 NaNO

3 and NaNO

2at 1molL (equal

to 310mg of NO3




3




2+ 20 g AC


NaNO3or NaNO





3


2


3and






3






2




2added to each


minus

and 200mg of NO2



2


3NaNO

2were








2) standards




EDE CD C

B

NNN A



Beforestorage

0 73 146 219 292

00

01

02

03

04

05

06

NN

N (120583

gg)

(a)

F

E DC

B

NAT A



Beforestorage

0 73 146 219 292

000025050075100125150175200

NAT

(120583g

g)

(b)

B

B

B

A A

NAB

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NA

B (120583

gg)

(c)

DC C

BA

NNK

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NN

K (120583

gg)

(d)

FE D

C

B

TSNAsA



Beforestorage

0 73 146 219 292

00

05

10

15

20

25

30

TSN

As (

120583g

g)

(e)










3-N and NO



minus


3




3(146mg

of NO3


minus







2



Con

trol

CBBB

NNN A


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

10

20

30

40

50

60

NN

N (120583

gg)

(a)

BBBB

NATA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0005101520

180

200

220

240

NAT

(120583g

g)

(b)

B BB B

NABA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

02

04

06

08

10

NA

B (120583

gg)

(c)

BBBB

NNKA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

01

02

03

04

05

NN

K (120583

gg)

(d)

CB B B

ATSNA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0

2

4

20

25

30

TSN

As (

120583g

g)

(e)


3



amounts of addedNO2


2



2


3

minus











45∘C for 15 d







D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)


(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)






3

minus) The NO3





000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




4NO3 KNO

3 NaNO

3 and NaNO

2at 1molL (equal

to 310mg of NO3




3




2+ 20 g AC


NaNO3or NaNO





3


2


3and






3






2




2added to each


minus

and 200mg of NO2



2


3NaNO

2were








2) standards




EDE CD C

B

NNN A



Beforestorage

0 73 146 219 292

00

01

02

03

04

05

06

NN

N (120583

gg)

(a)

F

E DC

B

NAT A



Beforestorage

0 73 146 219 292

000025050075100125150175200

NAT

(120583g

g)

(b)

B

B

B

A A

NAB

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NA

B (120583

gg)

(c)

DC C

BA

NNK

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NN

K (120583

gg)

(d)

FE D

C

B

TSNAsA



Beforestorage

0 73 146 219 292

00

05

10

15

20

25

30

TSN

As (

120583g

g)

(e)










3-N and NO



minus


3




3(146mg

of NO3


minus







2



Con

trol

CBBB

NNN A


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

10

20

30

40

50

60

NN

N (120583

gg)

(a)

BBBB

NATA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0005101520

180

200

220

240

NAT

(120583g

g)

(b)

B BB B

NABA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

02

04

06

08

10

NA

B (120583

gg)

(c)

BBBB

NNKA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

01

02

03

04

05

NN

K (120583

gg)

(d)

CB B B

ATSNA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0

2

4

20

25

30

TSN

As (

120583g

g)

(e)


3



amounts of addedNO2


2



2


3

minus











45∘C for 15 d







D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)


(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)






3

minus) The NO3





000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


EDE CD C

B

NNN A



Beforestorage

0 73 146 219 292

00

01

02

03

04

05

06

NN

N (120583

gg)

(a)

F

E DC

B

NAT A



Beforestorage

0 73 146 219 292

000025050075100125150175200

NAT

(120583g

g)

(b)

B

B

B

A A

NAB

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NA

B (120583

gg)

(c)

DC C

BA

NNK

A



Beforestorage

0 73 146 219 292

000

001

002

003

004

005

NN

K (120583

gg)

(d)

FE D

C

B

TSNAsA



Beforestorage

0 73 146 219 292

00

05

10

15

20

25

30

TSN

As (

120583g

g)

(e)










3-N and NO



minus


3




3(146mg

of NO3


minus







2



Con

trol

CBBB

NNN A


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

10

20

30

40

50

60

NN

N (120583

gg)

(a)

BBBB

NATA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0005101520

180

200

220

240

NAT

(120583g

g)

(b)

B BB B

NABA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

02

04

06

08

10

NA

B (120583

gg)

(c)

BBBB

NNKA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

01

02

03

04

05

NN

K (120583

gg)

(d)

CB B B

ATSNA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0

2

4

20

25

30

TSN

As (

120583g

g)

(e)


3



amounts of addedNO2


2



2


3

minus











45∘C for 15 d







D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)


(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)






3

minus) The NO3





000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Con

trol

CBBB

NNN A


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

10

20

30

40

50

60

NN

N (120583

gg)

(a)

BBBB

NATA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0005101520

180

200

220

240

NAT

(120583g

g)

(b)

B BB B

NABA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

02

04

06

08

10

NA

B (120583

gg)

(c)

BBBB

NNKA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

00

01

02

03

04

05

NN

K (120583

gg)

(d)

CB B B

ATSNA

Con

trol


NH

4N

O3

KNO

3

NaN

O3

NaN

O2

0

2

4

20

25

30

TSN

As (

120583g

g)

(e)


3



amounts of addedNO2


2



2


3

minus











45∘C for 15 d







D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)


(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)






3

minus) The NO3





000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





45∘C for 15 d







D

C

A

B

000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

(ppm

)


(a)

A

AAA


Con

cent

ratio

n of

NO

2(p

pm)

0000

0005

0010

0015

0020

(b)

B

A

D

C


000

002

004

006

020

040

060

080

Con

cent

ratio

n of

NO

x ( p

pm)

(c)






3

minus) The NO3





000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


000

010

020

030

700

800

900

1000

C

D

A

Con

cent

ratio

n of

NO

(ppm

)

B


(a)

000

010

020

030

040

050

BB

A

B

Con

cent

ratio

n of

NO

2(p

pm)


(b)

000

020

040

060

700

800

900

1000

A

B

C

B


Con

cent

ratio

n of

NO

x(p

pm)

(c)






2

minus) The NO2







3


2



2






AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




AC (Control) 00094C 00249C 00203B 00486B






Beforestorage

000

010

020

030

040

050

060

C

NNNA

B

C

ED

NN

N (120583

gg)


1 2 3 4 5 6

(a)

000

010

020

030

040

050

060NAT A

B

C CD

E

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

000

001

002

003

004

005

006

BC

NAB

A

BC

DC

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

000001002003004005006007008

BCBC

NNK A

B

CBC

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

000

020

040

060

080

100

120TSNAs A

B

C CD

E

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


3 (4) tobacco +NaNO



3


3

minus)The NO3



2-N and NO

3-N




2



C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


C C C C

B

NNN A

00

10

20

30

40

50

60

NN

N (120583

gg)

Beforestorage


1 2 3 4 5 6

(a)

C C

C C

B

NAT A

00051080

120

160

200

240

NAT

(120583g

g)

Beforestorage


1 2 3 4 5 6

(b)

C C C C

B

NAB A

000102030405060708

NA

B (120583

gg)

Beforestorage


1 2 3 4 5 6

(c)

C C C C

B

NNK

A

00

01

02

03

04

05

06

NN

K (120583

gg)

Beforestorage


1 2 3 4 5 6

(d)

C CC C

B

TSNAsA

00102030

100150200250300350

TSN

As (

120583g

g)

Beforestorage


1 2 3 4 5 6

(e)


2 (4) tobacco +NaNO



2


2

minus)TheNO2



3


2

minus and NO3


2


of tobacco and NO2



2


2-N and



2




4 Conclusion








References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







References























3




















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Nitrate and Nitrite Promote Formation of Tobacco …downloads.hindawi.com/journals/jchem/2017/6135215.pdf · Postcured Storage under Warm Temperature JunWang,1 HuijuanYang,1 HongzhiShi,1