Analysis of TSNAs in nicotine used as a component in e-liquids

S. C. Moldoveanu*, J. Zhu**, N. Qian***R.J. Reynolds Tobacco Co.**Eurofins Lancaster Labs.

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Background• Nicotine used in the liquids from electronic smoking devices is

obtained from tobacco (Nicotiana tabacum, Nicotiana rustica). • USP grade nicotine manufactured by several suppliers for use in

electronic smoking devices contains, besides nicotine, low levels and traces of a number of compounds present in tobacco such as minor alkaloids, neophitadiene, and possibly tobacco specific nitrosamines (TSNAs, with four of interest: nitrosonornicotine or NNN, nitrosoanatabine or NAT, nitrosoanabasine or NAB, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or NNK).

• When present in the starting nicotine, TSNAs will be present in the e-liquid and in the particulate phase generated by the electronic smoking devices.

• Analysis of TSNAs in nicotine used for preparing e-liquids is important, and because the potential levels are expected to be very low, a very sensitive method for this analysis is necessary.

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Chromatogram of a commercial USP grade nicotine sample

6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

5000000

1e+07

1.5e+07

2e+07

2.5e+07

3e+07

Time-->

Abundance

7.35 9.55 12.41 13.78

14.23

15.7217.27

17.53

18.09

18.67

20.81 22.67

24.36

25.83 27.28

28.0

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Peak identification for the compounds in the nicotine chromatogram

Compound Ret. time min CAS# Area

(to nicot.)

3-Vinylpyridine 9.55 121-55-7 3.11E-03Pyridincarboxaldehyde 12.41 500-22-1 1.34E-031-(3-Pyridinyl)ethanone 13.78 350-03-8 2.60E-043-(1-Methylpyrrolidinyl)-pyridine (Nicotine) 14.22 54-11-5 1.00E+00Unknown 15.42 - 6.72E-041,2,3,6-Tetrahydro-2,3'-bipyridine (Anatabine) 15.72 581-49-7 6.16E-03(1'S,2'S)-Nicotine-N'-oxide 17.27 29419-55-4 4.04E-033-(3,4-Dihydro-2H-pyrrol-5-yl)-pyridine (Myosmine) 17.53 532-12-7 3.06E-023-Pyridineacetonitrile 18.09 6443-85-2 4.84E-03Unknown 18.32 - 1.20E-03β-Nicotyrine 18.67 487-19-4 3.03E-025-Phenyl-1,4-dimethyl-imidazole ? 19.39 62576-11-8 1.18E-03Unknown 20.81 - 9.17E-04N-Methyl-3-pyridincarboxamide 22.67 114-33-0 4.12E-04Cotinine 24.36 486-56-6 2.72E-023-Pyrrol-2-yl-pyridine ? 25.83 494-98-4 4.08E-04N-Formylnornicotine 27.28 3000-81-5 1.38E-03

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Summary of initially utilized TSNAs analytical method

• The nicotine samples were made into 3% solutions in 100 mM aqueous ammonium acetate, containing deuterated TSNAs: NNN-d4, NAT-d4, NAB-d4 and NNK-d4 as internal standards.

• The solutions were analyzed without clean-up on an Agilent 1290 HPLC system coupled with an AB Sciex 6500 triple-quadrupole mass spectrometer with positive ESI source.

• The sample separation was performed on a Phenomenex Kinetex EVO 1.7 µm column using gradient, Solution A: 95 % 10mM ammonium acetate in DI water at pH 6.75 with 5% acetonitrile, Solution B: 0.1% acetic acid in acetonitrile .

• Injections were made at 3µL. • The detection was done in MRM mode for transitions

corresponding to the four TSNAs and their deuterated forms.

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Extracted ion chromatogram for a solution containing1 ng/mL NNN-d4, NNK-d4, NAT-d4 and 0.835 ng/mL NAB-d4

N

N

N O

D

D

D DN

D

D

D D

N

NO

N

D

D

D D

N

NO

N

D

D

D D

O

NN

CH3

O

NNN-d4

182.1→152.1

NAT-d4

194.1→164.1

NAB-d4

196.1→166.1NNK-d4

212.1→126.1

Inte

nsity

, cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Time, min

0.0

5000

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e45.7e4

1.99

2.50

2.19

2.42

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Extracted ion chromatogram for a solution containing 3% nicotine and 1 ng/mL NNN-d4, NNK-d4, NAT-d4 and 0.835 ng/mL NAB-d4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time, min

0

400

800

1200

1600

2000

2400

2800

3200

Inte

nsity

, cps

1.99

2.19

2.42

2.50

N

D

D

D D

O

NN

CH3

O

N

D

D

D D

N

NO

N

N

N O

D

D

D D

N

D

D

D D

N

NO

NNK-d4

212.1→126.1

NNN-d4

182.1→152.1NAT-d4

194.1→164.1

NAB-d4

196.1→166.1?

??

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Requirements for a chromatographic separation of nicotine and TSNAs

• Assure good separation of nicotine and TSNAs. • Accommodate large amount of sample matrix (3% nicotine). • Use a column with very high efficiency (large theoretical plate

number).• Produce separation within a short chromatographic run time.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 2 3 4 5

Sign

al

Ret. time

Noise 1 (N1)

Signal 2 (S2)

Signal 1 (S1)

Noise 2 (N2)

Average baseline

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Sample preparation for the new TSNAs analysis

• Extracting solution: 100 mM ammonium acetate in water and four internal standards: NAB-d4 at 835 pg/mL, NAT-d4, NNK-d4, and NNN-d4 at 1000 pg/mL.

• For nicotine samples: the nicotine samples were made into 3% in the extracting solution.

• For the e-liquids: about 1 g e-liquid was precisely measured and dissolved in 10 mL extracting solution.

• For particulate phase: e-cigarettes were smoked (puffed) on a Cerulean SM 450 smoking machine. The collection of particulate phase was performed on Cambridge pads using typical puffing conditions, with 55 mL puff volume, 3 s puff, and 30 s puff interval. 100 puffs were collected, and the pads were extracted with 15 mL extracting solution.

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Summary of the new analytical method

• The solutions were analyzed without clean-up on an Agilent 1290 HPLC system coupled with an AB Sciex 6500 triple-quadrupole mass spectrometer with positive ESI source.

• The sample separation was performed on a Phenomenex Luna Omega 1.6 µm C18 100 A, 100 x 2.1 mm column using gradient, Solution A: 95 % 10mM ammonium formate in DI water at pH 5.0 (brought with formic acid) with 5% acetonitrile, Solution B: 0.2% formic acid in acetonitrile .

• Injections were made at 3µL. • The detection was done in Scheduled MRM mode for each

TSNA and deuterated TSNA.

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Extracted ion chromatogram for a solution containing 3% nicotine and 1 ng/mL NNN-d4, NNK-d4, NAT-d4 and 0.835 ng/mL NAB-d4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

Inte

nsity

, cps

3.17

3.80

3.47

3.70

NNN-d4

NNK-d4

NAT-d4

NAB-d4

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TSNAs standards in pg/mL used for calibration in the presence of 3%

nicotine

Standard NAB NAT NNK NNNStd. 1 12.2 49.1 50.4 50.8

Std. 2 24.5 98.2 100.7 101.7

Std. 3 98.0 392.9 402.9 406.8

Std. 4 245.0 982.1 1007.2 1016.9

Std. 5 979.8 3928.5 4028.8 4067.7

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Extracted ion chromatogram for a solution containing3% (3∙10+10 pg/mL) nicotine and 98.2 pg/mL NAT, 24.5 pg/mL

NAB, 100.7 pg/mL NNK and 101.7 pg/mL NNN

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0.0

4000.0

8000.0

1.2e4

1.6e4

2.0e4

2.4e4

2.8e4

3.2e4

3.6e4

4.0e4

Inte

nsity

, cps

3.48

3.82

3.183.71

NNNNNK

NAT

NAB

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Linearity of calibration in the presence of 3% nicotine (Validation step)

Y = aX + b, Y is the amount of TSNA in ng/mL and X is the ratio (peak area of analyte)/(peak area of internal standard)

Compound I.S. a b R2

NAB NAB-d4 9.1258 e-1 -1.1868 e-2 0.99981

NAT NAT-d4 1.0421 -3.5315 e-2 0.99982

NNK NNK-d4 9.9914e-1 -8.0306 e-2 0.99865

NNN NNN-d4 1.0073 -1.4665 e-1 0.99937

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Recovery evaluation of the procedure exemplified for one nicotine sample (values in pg/mL) (Validation step)

Analyte Initiallymeasured

Add 1/5 Std. 3 Measured %

Recov.Add 1/5

Std. 5 Measured % Recov.

NAB 222.9 19.6 235.2 97.00% 196 393.8 94.00%

NAT 151.8 78.6 214.3 93.00% 785.7 946.9 101.00%

NNK 1,771.2 80.6 1,833.3 99.00% 805.6 2,525.3 98.00%

NNN 53.1 81.4 138.5 103.00% 813.5 840.6 97.00%

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Values for LOD and LOQ obtained in pg/mL from the SD values of the lowest

calibration standard (Validation step)(LOD = 3 SD, LOQ = 10 SD.)

NAB NAT NNK NNN

LOD 1.9 7.5 15.4 4.8

LOQ 6.2 25.2 51.3 16.1

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Results for TSNAs level in pg/g in four USP grade nicotine samples from

different sources (triplicate measurements)

* Red indicates level below LOQ

Sample NAB RSD% NAT RSD% NNK RSD% NNN RSD%

TRC nicotine 0.015* - 0.018* - N.D.** - 0.002* -

Spl. Nicotine 1 6.306 16.2 4.360 21.7 31.127 8.9 0.415* 22.7

Spl. Nicotine 2 7.247 4.1 19.047 6.2 50.757 1.0 2.084 17.8

Spl. Nicotine 3 5.821 19.9 21.852 9.6 34.678 2.9 1.710 17.1

Spl. Nicotine 4 3.340 2.5 3.927 8.2 31.292 7.4 1.067 18.1

** Not Detected

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Results for TSNAs level in pg/g in the e-liquid* separated from four e-cigarettes

(triplicate measurements for Spl. B, C1, and C2)

Sample NNN RSD%NNN NAT RSD%

NAT NAB RSD%NAB NNK RSD%

NNK

E-Cig. A 7,886 - 47,609 - 3,980 - 3,858 -

E-Cig. B 887 17.7 535 6.0 24 56.7 614 22.4

E-Cig C1 1,555 17.8 525 6.9 167 52.6 2,739 18.3

E-Cig C2 1,374 18.1 524 10.2 137 68.1 2,749 24.7

Red indicates level below LOQ

*Note: Tobacco cigarettes: NNN: ~2,500,000 pg/cig, NAT: ~2,300,000 pg/cig, NAB: 500,000 pg/cig, NNK: 900,000 pg/g (Stepanov et al. Nicotine & Tobacco Research Vol. 8, No. 2 (2006) 309–313)

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Results for TSNAs level in pg/g in the e-liquid separated from e-tanks

(triplicate measurements)

N.D. = Not Detected

Sample NNN RSD%NNN NAT RSD%

NAT NAB RSD%NAB NNK RSD%

NNKE-Tank 1 189 2.1 305 1.0 10 82.2 N.D. -E-Tank 2 215 5.1 311 4.2 11 27.4 N.D. -E-Tank 3 211 4.0 304 1.7 15 25.8 41 79.2

E-Tank 4 264 5.9 690 8.6 19 63.0 116 17.9

E-Tank 5 1,621 4.8 9,845 2.9 225 3.3 1,100 13.0

E-Tank 6 823 2.7 2,704 5.5 233 3.8 1,159 9.0

E-Tank 7 1,567 1.5 29,308 3.3 2,779 2.3 708 21.2

E-Tank 8 278 2.7 996 2.9 72 4.7 1,695 2.7

E-Tank 9 4,083 2.5 13,023 1.4 1,165 3.9 1,380 6.8

E-Tank 10 220 8.8 1,371 1.6 97 45.3 2,075 3.1

E-Tank 11 3,129 3.1 6,601 2.8 562 5.2 91 43.3

E-Tank 12 238 16.7 648 7.5 44 38.0 2 86.6

Red indicates level below LOQ

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Results for TSNAs level in the particulate phase* from e-cigarettes expressed in pg/20 puffs

(averages of three replicates).

Sample NNN RSD%NNN NAT RSD%

NAT NAB RSD%NAB NNK RSD%

NNK

E-Cig. A 933 14.8 3,422 24.0 311 24.7 403 19.2

E-Cig. B 170 6.5 102 6.0 3 24.4 56 16.6

E-Cig C1 225 36.0 113 11.0 39 25.7 43 5.1

E-Cig C2 143 22.3 101 7.7 13 41.5 22 32.4

*Note: 2R4F cigarette smoked in ISO conditions generates:NNN: 133,110 pg/cig, NAT: 119,020 pg/cig, NAB: 16,280 pg/cig, NNK: 115,610 pg/cig (Chen, Moldoveanu, Beitr. Tabak. Int. 20 (2003), 448-458.

Red indicates level below LOQ

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Results for TSNAs level in pg/g collected aerosols (triplicate measurements for Spl. B, C1, and C2)

Red indicates level below LOQ

Sample Weight

g/100 puffsNNN NAT NAB NNK

E-Cig. A 0.328 14,243 52,239 4,748 6,152

E-Cig. B 0.282 3,019 1,811 53 994

E-Cig. C (1) 0.243 4,622 2,321 801 883

E-Cig. C (2) 0.241 2,967 2,095 270 456

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Results for TSNAs level in the particulate phase from e-tanks expressed in pg/20 puffs (averages of

three replicates).

* Red indicates level below LOQ

Sample NNN RSD%NNN NAT RSD%

NAT NAB RSD%NAB NNK RSD%

NNKTank 1 0.140 29.0 0.048* 5.5 0.002 65.6 0.010 83.2Tank 2 Menthol 0.113 18.2 0.047 3.2 0.003 65.1 0.001 44.9Tank 3 0.108 24.9 0.055 12.1 0.002 39.1 0.057 14.8Tank 4 0.142 - 0.078 - 0.003 - 0.035 -Tank 5 0.107 - 0.047 - 0.002 - 0.015 -

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Results for TSNAs level in the particulate phase from e-tanks expressed in pg/g collected aerosol

(averages of three replicates).

* Red indicates level below LOQ

Sample Weightg/200 puffs

NNN NAT NAB NNK

Tank 1 0.791 1.770 0.607 0.025 0.126

Tank 2 Menthol 0.561 2.013 0.837 0.053 0.018

Tank 3 0.227 4.749 2.419 0.088 2.507

Tank 4 0.757 1.876 1.030 0.040 0.462

Tank 5 0.634 1.688 0.742 0.032 0.237

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Conclusions• A very sensitive method for the analysis of TSNAs in nicotine

and e-liquids has been developed.• The method is of the type “dilute and inject” and does not

require elaborate sample preparation as compared to other methods reported in the literature for the analysis of TSNAs in e-liquids (e.g. H.-J. Kim, H.-S. Shin, J. Chromatography A, 1291 (2013) 48-55).

• Good linearity, , recovery, and excellent LOD and LOQ were demonstrated for the method (as validation steps).

• Nicotine samples and several e-liquids used in electronic smoking devices were successfully analyzed.

• Although at very low levels, the TSNAs from the starting nicotine makes their way to the e-liquids and e-cigarettes aerosols.

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