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Variations of TSNA Levels in Tobaccos Upon Heating at Moderate Temperatures

Serban C. Moldoveanu
Serban C. Moldoveanu
, 
Marlene Adams
Marlene Adams
 and 
Frank K. St.Charles
Frank K. St.Charles
   | Sep 25, 2020
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Published Online: Sep 25, 2020
Page range: 84 - 96
Received: Mar 19, 2020
Accepted: Jul 28, 2020
DOI: https://doi.org/10.2478/cttr-2020-0009
Keywords
© 2020 Serban C. Moldoveanu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Figure 1

Sealed tubes containing powdered tobacco (Burley lower stalk) before heating.
Sealed tubes containing powdered tobacco (Burley lower stalk) before heating.

Figure 2

Temperature variation inside a tobacco sample placed in a glass tube as a function of heating time of the tube. Heating was stopped at 5 min and the samples were exposed to room temperature.
Temperature variation inside a tobacco sample placed in a glass tube as a function of heating time of the tube. Heating was stopped at 5 min and the samples were exposed to room temperature.

Figure 3

Extracted ion chromatograms for NNN, NAT, NAB and NNK and their d4-labeled compounds measured for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min.
Extracted ion chromatograms for NNN, NAT, NAB and NNK and their d4-labeled compounds measured for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min.

Figure 4

Variation of NNN in FC-Up sample with 2 min heating at different oven temperatures.
Variation of NNN in FC-Up sample with 2 min heating at different oven temperatures.

Figure 5

Variation of NNN in FC-Up sample with 5 min heating at different oven temperatures.
Variation of NNN in FC-Up sample with 5 min heating at different oven temperatures.

Figure 6

Initial levels of TSNAs (data taken from Table 7).
Initial levels of TSNAs (data taken from Table 7).

Figure 7

Levels of TSNAs after heating for 2 min at 200 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 2 min at 200 °C (data taken from Tables 8 to 10).

Figure 8

Levels of TSNAs after heating for 5 min at 200 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 5 min at 200 °C (data taken from Tables 8 to 10).

Figure 9

Levels of TSNAs after heating for 2 min at 250 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 2 min at 250 °C (data taken from Tables 8 to 10).

Figure 10

Levels of TSNAs after heating for 5 min at 250 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 5 min at 250 °C (data taken from Tables 8 to 10).

Concentrations of deuterated TSNAs in the samples containing internal standard solution.

NNN-d4 (ng/mL) NAT-d4 (ng/mL) NAB-d4 (ng/mL) NNK-d4 (ng/mL)
40.663 41.009 10.377 40.275

Concentrations of TSNAs in the standard solutions.

Standard NNN (ng/mL) NAT (ng/mL) NAB (ng/mL) NNK (ng/mL)
1 162.65 164.04 41.51 161.10
2 81.33 82.02 20.75 80.55
3 40.66 41.01 10.38 40.28
4 20.33 20.50 5.19 20.14
5 10.17 10.25 2.59 10.07
6 5.08 5.13 1.30 5.03
7 2.54 2.56 0.65 2.52

Solvent gradient employed for the HPLC-MS/MS analysis of TSNAs.

Time % A % B Flow rate (mL/min)
0.0 100.0 0.0 0.6
1.7 100.0 0.0 0.6
3.5 70.0 30.0 0.6
4.5 60.0 40.0 0.6
8.0 50.0 50.0 0.6
10.0 100.0 0.0 0.6

Variation of TSNAs in flue-cured tobaccos upon heating (in ng/g).

Temperature (°C) Flue-cured lower stalk, FC-Lo Flue-cured upper stalk, FC-Up
2 min 5 min 2 min 5 min
NNN (ng/g)
23 372.5 ± 2.1 372.5 ± 2.1 358.3 ± 3.9 358.3 ± 3.9
23 357.9 ± 0.5 357.9 ± 0.5 342.9 ± 1.7 342.9 ± 1.7
23 — — 361.0 ± 2.9 361.0 ± 2.9
100 397.9 ± 6.9 400.8 ± 0.2 378.9 ± 9.1 389.5 ± 5.3
100 — — 388.8 ± 2.6 380.5 ± 3.5
150 420.5 ± 1.7 465.3 ± 1.4 392.7 ± 10.4 422.6 ± 4.7
150 — — 391.7 ± 14.3 —
200 479.8 ± 1.7 697.4 ± 5.2 457.5 ± 10.2 540.5 ± 9.3
200 — — 461.3 ± 4.7 546.0 ± 3.3
250 496.7 ± 9.2 109.1 ± 0.4 595.8 ± 42.4 102.4 ± 0.6
NAT (ng/g)
23 566.7 ± 7.2 566.7 ± 7.2 469.1 ± 4.7 469.1 ± 4.7
23 566.4 ± 4.8 566.4 ± 4.8 447.9 ± 7.4 447.9 ± 7.4
23 — — 457.9 ± 2.1 457.9 ± 2.1
100 602.8 ± 18.0 645.0 ± 4.6 520.0 ± 12.7 492.6 ± 1.0
100 — — 517.5 ± 22.5 483.6 ± 3.1
150 693.2 ± 3.2 836.3 ± 10.9 529.3 ± 8.4 618.3 ± 1.6
150 — — 520.9 ± 4.1 —
200 909.8 ± 5.0 1366.4 ± 5.6 649.3 ± 11.7 871.2 ± 1.7
200 — — 657.5 ± 1.4 885.8 ± 6.6
250 945.6 ± 5.8 17.1 ± 0.9 1039.2 ± 30.6 10.9 ± 0.2
NAB (ng/g)
23 31.9 ± 0.1 31.9 ± 0.1 34.8 ± 3.1 34.8 ± 3.1
23 34.5 ± 0.2 34.5 ± 0.2 30.4 ± 0.2 30.4 ± 0.2
23 — — 32.7 ± 0.2 32.7 ± 0.2
100 40.3 ± 0.5 38.0 ± 0.8 36.5 ± 0.5 36.9 ± 0.4
100 — — 38.4 ± 0.4 37.1 ± 0.5
150 39.6 ± 0.2 46.4 ± 1.2 37.0 ± 0.2 43.8 ± 0.2
150 — — 38.2 ± 0.8 —
200 51.1 ± 0.5 101.0 ± 0.3 45.7 ± 2.4 72.3 ± 0.6
200 — — 48.6 ± 0.7 74.2 ± 0.6
250 72.4 ± 0.6 41.1 ± 0.2 99.7 ± 5.7 43.5 ± 1.3
NNK (ng/g)
23 270.1 ± 1.5 270.1 ± 1.5 225.3 ± 4.9 225.3 ± 4.9
23 296.1 ± 2.4 296.1 ± 2.4 218.0 ± 1.7 218.0 ± 1.7
23 — — 239.0 ± 1.4 239.0 ± 1.4
100 318.5 ± 7.4 314.5 ± 1.9 242.8 ± 5.7 235.0 ± 1.5
100 — — 236.2 ± 8.1 225.6 ± 3.3
150 341.0 ± 1.33 343.9 ± 3.9 240.6 ± 5.5 225.4 ± 0.1
150 — — 231.4 ± 0.1 —
200 352.7 ± 4.3 658.7 ± 9.4 249.1 ± 4.9 349.1 ± 6.8
200 — — 259.0 ± 0.2 340.3 ± 0.4
250 640.0 ± 13.1 269.7 ± 0.5 430.6 ± 32.1 216.4 ± 2.7

Variation of TSNAs in lower stalk Burley tobaccos upon heating (in ng/g).

Temperature (°C) Burley lower stalk, Bu-Lo Burley upper stalk, Bu-Up
2 min 5 min 2 min 5 min
NNN (ng/g)
23 4678.4 ± 262.0 4678.4 ± 262.0 4666.7 ± 65.8 4666.7 ± 65.8
23 4698.2 ± 40.4 4698.2 ± 40.4 4893.9 ± 15.2 4893.9 ± 15.2
100 4715.8 ± 98.1 4701.7 ± 44.7 5458.9 ± 21.3 5753.9 ± 51.8
150 4903.5 ± 219.2 5968.8 ± 69.2 6288.3 ± 4.4 7508.4 ± 7.5
200 5930.8 ± 215.3 8541.1 ± 160.6 6953.1 ± 47.3 10030.8 ± 68.2
200 — — 6947 ± 42.4 —
250 8961.2 ± 49.3 9383.9 ± 122.0 8525.8 ± 148.3 7077.4 ± 38.9
NAT (ng/g)
23 4611 ± 29.5 4611 ± 29.5 4430.1 ± 28.8 4430.1 ± 28.8
23 4622.5 ± 268.1 4622.5 ± 268.1 4894.2 ± 5.4 4894.2 ± 5.4
100 4627.4 ± 5.1 4660.9 ± 7.9 5585.9 ± 6.1 5893.8 ± 86.0
150 4812.6 ± 169.9 6485.5 ± 81.7 6386.6 ± 44.1 8067.5 ± 49.2
200 6327.2 ± 160.1 10063.8 ± 169.1 10005.5 ± 0.0 10677.6 ± 59.8
200 — — 9543.5 ± 40.1 —
250 10655.9 ± 86.3 4775.7 ± 27.2 8994.5 ± 161.9 1356.7 ± 3.7
NAB (ng/g)
23 204.7 ± 10.8 204.7 ± 10.8 176.4 ± 2.1 176.4 ± 2.1
23 194.8 ± 0.8 194.8 ± 0.8 190.1 ± 0.8 190.1 ± 0.8
100 199.2 ± 1.4 196.0 ± 1.2 223.8 ± 0.1 224.5 ± 1.7
150 207.6 ± 9.3 290.2 ± 1.6 249.1 ± 2.7 340.8 ± 0.5
200 282.6 ± 7.1 629.9 ± 7.1 423.4 ± 2.4 582.1 ± 2.9
200 — — 396.8 ± 3.1 —
250 637.3 ± 2.5 1683.8 ± 11.1 474.9 ± 6.5 1734.9 ± 15.4
NNK (ng/g)
23 855.6 ± 32.3 855.6 ± 32.3 791.7 ± 1.7 791.7 ± 1.7
23 866.4 ± 0.4 866.4 ± 0.4 754.8 ± 0.0 754.8 ± 0.0
100 844.9 ± 9.5 866.9 ± 6.7 858.8 ± 13.7 884.9 ± 1.8
150 898.8 ± 29.3 1039.6 ± 11.1 963.6 ± 3.1 1145.3 ± 1.3
200 1040.9 ± 38.6 2551.8 ± 14.5 1298.4 ± 2.9 2065.1 ± 14.2
200 — — 1216.6 ± 13.5 —
250 3517.8 ± 1.8 4749 ± 2.4 1711.0 ± 27.5 2916.5 ± 3.8

Parameters a, b and R 2 for the MS/MS quantitation.

Analyte a b R2
NNN 33.5151 0.1798 0.9997
NAT 40.8507 0.0479 0.9998
NAB 35.1528 0.0768 0.9992
NNK 35.7655 0.0889 0.9996

Initial TSNA levels in the tobacco samples in ng/g (tobacco as is).

Sample acronym NNN (ng/g) NAT (ng/g) NAB (ng/g) NNK (ng/g)
FC-Lo 365.2 566.5 33.2 283.1
FC-Up 354.1 458.3 32.6 227.4
Bu-Lo 4688.3 4616.8 199.7 861.0
Bu-Up 4780.3 4662.1 183.2 773.3
Or (1) 156.6 54.8 6.14 43.0
Or (2) 126.4 33.5 4.79 32.6

Tobacco samples, moisture (%), alkaloid level (%), and the conditions to which the samples were exposed.

Sample acronym Tobacco description Moisture (%) Total alkaloids (%) Temperatures (°C) Time of exposure (min)
FC-Lo Lower stalk flue-cured 6.44 2.07 100 150 200 250 2 5
FC-Up Upper stalk flue-cured 6.71 2.85 100 150 200 250 2 5
Bu-Lo Lower stalk Burley 6.36 2.51 100 150 200 250 2 5
Bu-Up Upper stalk Burley 7.11 3.46 100 150 200 250 2 5
Or (1) Oriental blend (1) 6.64 1.13 100 150 200 250 2 5
Or (2) Oriental blend (2) 6.23 1.02 100 150 200 250 2 5

Variation of TSNAs in Oriental tobaccos upon heating (in ng/g).

Temperature (°C) Oriental (1), Or(1) Oriental (2), Or(2)
2 min 5 min 2 min 5 min
NNN (ng/g)
23 156.9 ± 1.3 156.9 ± 1.3 130.2 ± 0.5 130.2 ± 0.5
23 156.3 ± 1.3 156.3 ± 1.3 127.0 ± 0.3 127.0 ± 0.3
23 — — 122.0 ± 2.7 122.0 ± 2.7
100 203.7 ± 1.9 218.0 ± 1.8 146.2 ± 0.1 147.6 ± 1.5
150 211.4 ± 1.6 273.4 ± 1.5 194.1 ± 0.2 225.3 ± 1.5
200 282.1 ± 3.4 489.2 ± 13.0 205 ± 0.8 325.3 ± 2.6
200 307.0 ± 1.0 — — 319.5 ± 5.8
250 386.7 ± 11.5 146.2 ± 1.0 289.1 ± 5.2 —
250 — — 298.3 ± 0.5 —
NAT (ng/g)
23 55.0 ± 0.9 55.0 ± 0.9 33.3 ± 0.1 33.3 ± 0.1
23 54.6 ± 0.7 54.6 ± 0.7 33.8 ± 1.3 33.8 ± 1.3
100 69.7 ± 0.4 77.7 ± 1.2 39.0 ± 1.0 39.1 ± 0.7
150 73.2 ± 1.5 122.1 ± 0.2 53.5 ± 0.8 67.7 ± 2.0
200 130.8 ± 1.5 284.4 ± 4.5 64.7 ± 1.0 121.7 ± 2.1
200 139.3 ± 0.9 — — 118.6 ± 0.5
250 207.3 ± 3.8 12.9 ± 0.4 105.6 ± 2.7 —
250 — — 108.8 ± 1.7 —
NAB (ng/g)
23 6.2 ± 0.3 6.2 ± 0.3 4.9 ± 0.1 4.9 ± 0.1
23 6.1 ± 0.3 6.1 ± 0.3 4.7 ± 0.1 4.7 ± 0.1
100 6.7 ± 0.3 7.1 ± 0.6 4.8 ± 0.1 5.1 ± 0.0
150 6.9 ± 0.5 10.3 ± 0.1 5.7 ± 0.3 6.7 ± 0.0
200 10.3 ± 0.2 28.0 ± 0.2 7.0 ± 0.0 18.1 ± 0.4
200 11.2 ± 0.4 — — 17.6 ± 0.0
250 18.4 ± 0.4 25.6 ± 0.1 13.9 ± 0.7 —
250 — — 15.3 ± 0.1 —
NNK (ng/g)
23 41.3 ± 0.6 41.3 ± 0.6 32.9 ± 0.4 32.9 ± 0.4
23 44.7 ± 1.2 44.7 ± 1.2 32.4 ± 0.4 32.4 ± 0.4
100 54.2 ± 0.8 54.7 ± 0.4 38.2 ± 2.0 38.2 ± 0.4
150 55.2 ± 1.4 57.5 ± 0.4 45.2 ± 0.8 49.0 ± 1.5
200 56.8 ± 2.0 119.8 ± 0.4 48.4 ± 0.6 76.8 ± 0.1
200 59.8 ± 0.7 — — 76.8 ± 0.3
250 85.5 ± 1.4 91.5 ± 2.8 62.0 ± 0.3 —
250 — — 65.9 ± 0.2 —

Other parameters for the MS/MS detection.

Compound Ion for Q1 Ion for Q3 Retention time (min) DP1(V) CE2 (V) CXP3 (V)
NNN 178.1 148.1 4.73 30 15 9
NNN-d4 182.1 152.1 4.70 30 16 9
NAT 190.1 160.1 5.35 28 15.5 10
NAT-d4 194.1 164.1 5.31 28 16 10
NAB 192.1 162.1 5.40 26 18 11
NAB-d4 196.1 166.1 5.37 26 17 10
NNK 208.1 122.1 5.16 30 17.6 13
NNK-d4 212.1 126.1 5.15 30 16 12
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