Variations of TSNA Levels in Tobaccos Upon Heating at Moderate Temperatures

Moldoveanu, Serban C.; Adams, Marlene; St.Charles, Frank K.

doi:10.2478/cttr-2020-0009

Figures & Tables

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

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.

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.

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

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

Initial levels of TSNAs (data taken from Table 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 5 min at 200 °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).

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

Variations of TSNA Levels in Tobaccos Upon Heating at Moderate Temperatures

Figures & Tables

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Concentrations of deuterated TSNAs in the samples containing internal standard solution_

Concentrations of TSNAs in the standard solutions_

Solvent gradient employed for the HPLC-MS/MS analysis of TSNAs_

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

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

Parameters a, b and R 2 for the MS/MS quantitation_

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

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

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

Other parameters for the MS/MS detection_

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