1. bookTom 31 (2022): Zeszyt 1 (March 2022)
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Does the Level of NNK in Tobacco and Tobacco Products Depend on the Level of Pseudooxynicotine or of Nicotine-1′-N-Oxide?

Data publikacji: 16 Apr 2022
Tom & Zeszyt: Tom 31 (2022) - Zeszyt 1 (March 2022)
Zakres stron: 25 - 34
Otrzymano: 09 Nov 2021
Przyjęty: 25 Jan 2022
Informacje o czasopiśmie
License
Format
Czasopismo
eISSN
2719-9509
Pierwsze wydanie
01 Jan 1992
Częstotliwość wydawania
4 razy w roku
Języki
Angielski
INTRODUCTION

4-(Methylamino)-1-(3-pyridyl)-1-butanone (pseudooxynicotine or PON) is present in tobacco and tobacco products being a result of nicotine metabolization by specific bacteria such as those of the genus Pseudomonas and by the fungus Cunninghamella echinulate (1,2,3). Oxidation of nicotine caused by flavin-containing monooxygenases generates nicotine-1′-N-oxide, the compound being also obtained from non-enzymatic nicotine oxidation. Nicotine-1′-N-oxide can be isomerized by heating to form 2-methyl-6-(pyridine-3-yl)-1,2-oxazinane that can lead to the formation of PON (4). The interest in the levels of nicotine-1′-N-oxide and in particular of PON in tobacco and tobacco products is related to the formation of 4-(N’-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By nitrosation, PON can generate NNK by the following reaction:

NNK is one of the potentially carcinogenic tobacco specific nitrosamines (TSNAs) (5), and an important compound in the Harmful and Potentially Harmful Constituents (HPHCs) list of U.S. Food and Drug Administration (FDA) (6). With the purpose of detecting the factors contributing to the content of NNK in tobacco and tobacco products, present study attempted to evaluate the contribution of the levels of PON or of nicotine-1′-N-oxide to the NNK levels. A good correlation between PON and NNK or nicotine-1′-N-oxide and NNK would provide valuable information regarding the potential precursors of NNK in tobacco. For this purpose, PON and nicotine-1′-N-oxide were measured by two different newly developed techniques in several tobaccos and tobacco products, while NNK was measured using a method reported in the literature (7). Following these measurements, various correlations between the levels of NNK, PON and nicotine-1′-N-oxide were evaluated and are further discussed.

EXPERIMENTAL
Materials

Nicotine, nicotine-d3, nicotine-1′-N-oxide, pseudooxynicotine dihydrochloride, PON-d3 dihydrochloride, PON-d4 dihydrochloride, 4-(N’-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and NNK-d4 were obtained from Toronto Research Chemicals Inc. (TRC) (North York, ON, Canada). Several other chemicals including ammonium acetate, ammonium formate, formic acid, ammonium hydroxide solution, ammonium bicarbonate, citric acid, disodium phosphate, and acetonitrile were obtained from Sigma/Aldrich (St. Louis, MO, USA). Pure water (18.2 MΩ/cm) was obtained from a Barnstead water purification unit (Thermo Fisher Scientific, Waltham, MA, USA). 20-mL scintillation vials, 2-mL and 4-mL liquid chromatography (LC) vials with screw-caps and septa were used. For filtration 0.45-µm PVDF (polyninylidene fluoride) filters were used (Whatman Autovial, GE Healthcare, Little Chalfort, UK).

Instrumentation

Some general preparation instrumentation, such as a halogen moisture analyzer HE53 (Mettler Toledo, Greifensee, Switzerland) and a wrist action shaker (Burrell Co., Pittsburgh, PA, USA) were used for all analyses. Otherwise, each method applied for measuring PON, nicotine-1′-N-oxide, NNK, and also nicotine used different instrumentation further described in each method below.

Methods description

All samples were analyzed “as is” without drying. Drying by heating may generate some TSNAs (7) and thus should be avoided. Freeze drying was another option to generate dry samples, but the operation requires more time and additional sample manipulation. A simpler solution was to analyze the samples “as is” and measure their moisture. For this purpose, on a separate portion from the samples, the oven moisture was measured and the results were reported on “dry weight basis”. The measurement was performed using a halogen moisture analyzer that generates IR radiation and the heating is continued until the sample weight (minimum 400 mg) shows a variation less than 1 mg in 140 s of heating. Only summary description of the methods used for nicotine determination and NNK determination is provided below. The two methods for the analysis of PON and nicotine-1′-N-oxide are described in more details.

Nicotine analysis

The method for nicotine analysis started with sample extraction. For this purpose, 250 mg tobacco (“as is”) or tobacco products were weighed in a 20-mL scintillation vial. To this vial, 10 mL extraction solution was added. The extraction solution consisted of 100 mM ammonium acetate in water. The extraction was performed for 1 h on a wrist action shaker. The resulting solution was filtered through 0.45-µm pore size polyvinylidene fluoride (PVDF) filters. From the filtered solution 200 µL were placed in 4-mL screw-cap vials. To the vials were added 3.8 mL water. The analysis was performed on a 1100 Series High performance liquid chromatography (HPLC) system from Agilent (Wilmington, DE, USA) consisting of a solvent delivery system, a quaternary pump, autosampler, column temperature controller, and a variable wavelength detector. For the separation a Xterra® RP 18,5 µm, 4.6 × 150 mm column from Waters (Milford MA, USA) was used. The detection was performed by ultraviolet (UV) absorption at 254 nm. The mobile phase was made from solvent A: 0.025 M NH4HCO3 in water brought to pH 9.5 with NH4OH, and solvent B: acetonitrile. The separation was performed in gradient starting with 100% solvent A to 60% A at 10 min, hold for 2 min and then reverse to initial conditions. The total run time was 15 min. The injection volume was 5 µL. The quantitation was performed using calibration with six nicotine standard solutions obtained by sequential dilution having concentrations between 836.5 µg/mL and 26.1 µg/mL. The resulting calibration curve was linear with a correlation coefficient R2 = 1.0000.

NNK analysis

For the analysis of NNK, the same extraction procedure as for nicotine was utilized. The solution obtained from the extraction of 250 mg tobacco or tobacco products was analyzed without dilution. The separation was achieved on an Agilent 1290 HPLC binary system with a binary pump, an autosampler with cooling capability, and a column thermostatted compartment. The instrument was equipped with a Luna® 3 µm C18 150 × 3 mm column from Phenomenex (Torrance, CA, USA). The tandem mass spectrometry (MS/MS) system was an API-5000 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA). The liquid chromatography with tandem mass spectrometry (LC-MS/MS) system was controlled using Analyst 1.6.2 software, and the peak integration was performed with MultiQuant 3.0.1 software. For the HPLC separation a gradient program with the mobile phase A: 100 mM ammonium formate pH 4.9 aqueous solution with 5% acetonitrile and mobile phase B: 0.2% formic acid in acetonitrile. The timetable for the gradient of the HPLC separation is reported in the literature (7). The detection was performed using scheduled multiple reaction monitoring (MRM) in positive mode, the mass spectrometric settings being previously reported (7).

For the quantitation of NNK, seven standard solutions were prepared by sequential dilution having concentrations between 141.10 ng/mL and 2.52 ng/mL. The solutions contained 40.28 ng/mL NNK-d4 as internal standard (I.S.). Calibration curves were constructed by plotting the concentrations of the standard solutions as a function of the peak areas normalized to the area of the corresponding deuterated internal standard. The calibration was linear with correlation coefficients R2 = 0.9998. More details about the method for NNK analysis are given in reference (7). One aspect requiring some comments is related to the presence in tobacco of free and bound NNK (8). Exhaustive extraction of tobacco in rather harsh conditions seems to increase the level of detected NNK.

In situ generation of NNK due to the extraction conditions is not excluded to be in part the cause of additional NNK formation during the harsh extraction procedure. In the present work, only the free NNK obtained by extraction using a buffer solution was measured.

PON and nicotine-1′-N-oxide analysis (Method 1)

For the analysis of PON and nicotine-1′-N-oxide, the same extraction procedure has been used for nicotine. Also the same dilution from 200 µL extract to 4 mL final volume by adding extracting solution was applied to the samples. The extraction of PON and the nicotine-1′-N-oxide with a buffer solution was reported in the literature as efficient (9). To each 1 mL of sample solution (or of standards used for quantitation) were added 20 µL of a solution of nicotine-d3 containing 133 µg/mL. The concentration of the I.S. in the final solution was 2.66 µg/mL (not adjusted to 1.02 mL volume). This solution was further analyzed. The instrument used for the analysis was an Agilent 1290 HPLC system with a binary pump, an autosampler with cooling capability, and a column thermostatted compartment. The MS/MS system was an API-6500 triple quadrupole mass spectrometer. The LC-MS/MS system was controlled using Analyst 1.6.2 software, and the peak integration was performed with MultiQuant 3.0.1 software. The HPLC separation was performed on an Acquity UPLC™ BEH Phenyl 1.7 m column, 3 × 150 mm dimensions from Waters (10). The mobile phase was made from solvent A: 0.02 M NH4COOH in water brought to pH 9.5 with NH4OH, and solvent B: acetonitrile. The gradient program started with 4% solvent B kept constant for 2.0 min followed by an increase to 60% at 6 min. The 60% B solvent was kept constant for 1.0 min followed by decrease to the initial condition at 8 min and kept at 4% B for one more min. The electrospray ionization (ESI) detection was performed by MS/MS in MRM positive mode using the parameters indicated in Table 1.

Parameters for the MS/MS detection.

Compound Ion for Q1 Ion for Q3 Acquisation time (ms) Declustering potential (V) Collision energy (V) Collision exit potential (V)
Pseudooxynicotine 179.1 148.1 100 70 17 13
Nicotine-1′-N-oxide 180.1 133.1 100 70 21 13
Nicotine-d3 (I.S.) 166.1 870 100 30 15 9

Other parameters include curtain gas: 20 mL/min, N2 collision gas: 4 mL/min, ion spray voltage: 4500 V, temperature: 500 °C, ion source gas (1) 40 mL/min, ion source gas (2): 50 mL/min.

A typical chromatogram obtained in the previously described conditions for a Burley tobacco sample showing the peaks of nicotine-1′-N-oxide, PON and of the internal standard nicotine-d3 is given in Figure 1. Several additional peaks corresponding to the same transition as for PON molecule m/z 179.1 → 148.1 but at different retention times than for PON standard are presented in the chromatogram. The nature of these peaks was not determined.

Figure 1

Chromatogram generated in positive MRM mode from a Burley tobacco sample indicating the peaks for nicotine-1′-N-oxide, PON and the internal standard nicotine-d3.

For the quantitation of PON and nicotine-1′-N-oxide, calibration curves were constructed by plotting the concentrations of the standard solutions as a function of the peak areas. For this purpose, seven standards were used. They were obtained by sequential dilutions with water of an initial solution generating standards with concentrations between 1.419 µg/mL and 0.022 µg/mL for PON and between 2.000 µg/mL and 0.031 µg/mL for nicotine-1′-N-oxide for each sample (not adjusted from 1.0 mL to 1.02 mL volume, the difference caused by the addition of the internal standard).

The calibration curves were obtained for both (concentration)/(peak area) as well as for (concentration)/(normalized peak areas by the area of I.S.). Since the use of normalized peak areas in the back-calculation of concentrations did not provide better results, only the correlation standard concentration versus peak area was further used for quantitation. The peak area for nicotine-d3 was used only as a chromatographic standard to verify the integrity of the separation. The best fit for the calibration curve for PON quantitation was quadratic and is expressed using the formula: Y(conc)=a(Std.Area)2+b(St.Area)+c Y\left( {conc} \right) = a{\left( {Std.\,Area} \right)^2} + b\left( {St.\,Area} \right) + c

For nicotine-1′-N-oxide the calibration curve was linear and is expressed using the formula: Y(conc)=b(Std.Area)+c Y\left( {conc} \right) = b\left( {Std.\,Area} \right) + c

Parameters a, b, and c as well as correlation coefficient R2 for the calibration lines are given in Table 2.

Parameters a, b, and c for the quantitation of pseudooxynicotine (PON) and nicotine-1′-N-oxide, and the regression coefficient R2 of the calibration line.

Compound a b c R2
PON −1.3581 E-13 1.33470 E-06 −2.1595 E-02 0.9996
Nicotine-1′-N-oxide 3.04019 E-07 −0.039649 0.9993

The results from the calculations were corrected to “dry weight basis” values.

A summary method validation was further performed for the PON and nicotine-1′-N-oxide analysis. The selectivity of the method is based on using three parameters to differentiate each compound, m/z for the molecular ion (for Q1), m/z for the fragment ion (for Q3), and the retention time. No additional transition was utilized for verifying selectivity. The method accuracy was verified by obtaining the same back-calculation results from the areas in the chromatogram for all the standards. The precision of the method was verified to be very good.

For the lowest standard of PON the back calculation of the concentration for 3 replicates, RSD% was 7.06% with a deviation of the average by + 0.002 µg/mL from the standard value. For the lowest standard of nicotine-1′-N-oxide the back calculation of the concentration for 3 replicates, RSD% was 3.50% with a deviation of the average by + 0.001 µg/mL from the standard value.

The limit of quantitation (LOQ) for both compounds can be taken as equal with the value of the lowest standard, although based on signal to noise (S/N) values, a much lower LOQ would be obtained. The LOQ values resulting by this procedure are LOQ (PON) = 0.022 µag/mL and LOQ (nicotine-1′-N-oxide) = 0.031 µg/mL.

PON analysis (Method 2)

An additional method for the analysis of PON has been developed and tested. This method includes a double extraction in the sample preparation with the intention to eliminate any potential interferences in the PON measurement. In this procedure, 100 mg tobacco or tobacco product were placed in a scintillation vial and extracted with 10 mL of a buffer at pH = 3.0 containing 1:1 (v/v) 0.16 M citric acid and 0.08 M disodium phosphate containing 39.5 µg/mL PON-d3 as the internal standard. After shaken for 10 min, the extract solution was cleaned twice based on the following steps:

1) 100 µL of 5M NaOH and 1 mL hexane were added to 1 mL of the above extract solution and the mixture was vortexed for 30 s, which transferred PON into hexane layer,

2) the hexane layer was transferred to a new vial and 1 mL of the pH 3.0 extracting buffer solution was added and vortexed for 30 s, which re-extracted PON into the aqueous solution,

3) the re-extracted PON in the aqueous buffer was separated and further analyzed.

The instrument used for the analysis was a ACQUITY UPLC™ H-Class system connected to a Xevo TQD triple quadrupole mass spectrometry (Waters Corporation, Milford, MA, USA). The sample separation was achieved using a Waters ACQUITY UPLC™ XBridge BEH C18 2.1 × 50 mm column with 1.7 µm particles at a flow rate of 0.25 mL/min. The elution was made with mobile phases consisting of solvent A 10 mM ammonium acetate with 0.1% NH4OH and solvent B 100% acetonitrile. The ultra performance liquid chromatography (UPLC) gradient program was an initial 2% solvent B, increased linearly to 90% in 3.5 min and further linearly to 100% B in 0.1 min and held for 1 min followed by a sharp decrease to 2% solvent B where it was held for 4 min for re-equilibration. The injection volume was 2 µL. The Waters Xevo TQD used ESI in positive mode with MRM detection. The quantitative fragmentation transitions for MRM were m/z 179 → 106 for PON, and m/z 182 → 106 for PON-d3, → while the qualitative fragmentation transitions for MRM were m/z 179 → 148 for PON, and m/z 182 → 148 for PON-d3. The source and desolvation temperatures were 150 and 500 °C, respectively. The desolvation gas flow was 800 L/hr. The capillary voltage was 0.75 kV. Using these conditions, the typical chromatograms for PON and PON-d3 in a 3R4F sample and in a standard with a concentration of 46.2 ng/mL are shown in Figure 2.

Figure 2

Typical chromatogram of PON and PON-d3 in 3R4F filler and in a standard containing 46.2 ng/mL PON.

For the quantitation of PON, a standard calibration curve was established by injecting a series of individual PON standard solutions of known concentrations in the range between 45 ng/mL and 2200 ng/mL. The PON standard calibration solutions were made by mixing a known amount of PON and an internal standard (PON-d3) into pH = 3.0 buffer. Masslynx software was used to process the data. The ratio of the peak area of PON to that of PON-d3 was calculated. The graph of the peak area ratio versus the concentration ratio of PON to PON-d3 was plotted. The calibration type was linear with 1/x weighting. The regression line was not forced through the origin and generated R2 = 0.999.

The validation of the second method for PON analysis was performed according to literature recommendations (11). The method was validated for the accuracy of PON at different concentrations with the recovery study. The reference cigarette 3R4F ground filler extract solution was used as a matrix for the recovery study. Since tobacco samples usually contain a certain amount of PON, PON-d4 (quantitative transition 183 → 152) was spiked into 3R4F extract solution for the recovery test, and PON-d3 was used again as an internal standard. The low, medium, and high levels of the PON-d4 were spiked into 3R4F and followed the sample preparation procedure as previously described for PON. Three replicates of each concentration level were tested. The average recovery of PON-d4 in the extraction procedure was 102% and it is indicated in Table 3. The very good recovery of PON-d4 is a reasonable proof that PON recovery is also very good.

Method recovery with spiked PON-d4 into 3R4F (n = 3).

Spiked PON-d4 (ng/mL) Recovery (%) RSD (%)
302.7 100 10.1
1210.8 103 4.0
2421.6 102 3.1

For the evaluation of precision of the UPLC-MS/MS method, the amount of PON in 3R4F filler was measured on three replicates each day for three different days. The coefficient of variations (RSD%) of intra-day and inter-day were found to be within 4% and 8%, respectively, indicating the repeatability of this method.

The limit of detection (LOD) and the limit of quantitation (LOQ) were based on the standard deviation of y-intercepts of the regression line and the slope. The PON calibration curve was made by injecting a series of known concentration PON standards into the UPLC-MS/MS. The LOD and LOQ were estimated by the standard deviation of the response (SD) and slope (S) from the standard curve using the formula LOD = 3.3 × SD/S and LOQ = 10 × SD/S. The LOD and LOQ of PON are 0.3 ng/mL and 1 ng/mL, respectively.

RESULTS AND DISCUSSION
Sample evaluated in this study

Various tobacco samples were selected to cover a variety of tobaccos (flue-cured, Burley, Oriental) from different sources (USA or off-shore), different stalk positions, and different ages. The reference materials from University of Kentucky Center for Tobacco Research Reference Products (CTRP) were also included. Besides tobaccos, nine moist snuff samples and two snus samples were also evaluated. The list of samples and their description is given in Table 4.

List of tobaccos, moist snuff, and snus samples evaluated in this study.

No. Sample Description
1 FC L (1) Eastern NC belt, lower stalk (lug) flue-cured (2016)
2 FC U (1) Eastern NC belt, upper stalk (leaf & some tips) flue-cured (2016)
3 FC L (2) South Carolina belt, lower stalk (lug) flue-cured (2016)
4 FC U (2) South Carolina belt, upper stalk (leaf & some tips) flue-cured (2016)
5 FC off L Brazil, lower stalk (lugs & primings) flue-cured (2016)
6 FC off U Brazil, upper stalk (leaf & tips) flue-cured (2016)
7 Bu L (1) Kentucky & Tennessee, lower stalk (flyings & cutters) Burley (2016)
8 Bu U (1) Kentucky & Tennessee, upper stalk (leaf) Burley (2016)
9 Bu L (2) North Carolina & Virginia, lower stalk (flyings & cutters) Burley (2016)
10 Bu U (2) North Carolina & Virginia, upper stalk (leaf) Burley (2016)
11 Bu off L Malawi, lower stalk (flyings & cutters) Burley (2016)
12 Bu off U Malawi, upper stalk (leaf) Burley (2016)
13 O SA U Turkey, good quality middle to upper stalk, Samsun Oriental (2015)
14 O Iz U Turkey, good quality middle to upper stalk, Izmir Oriental (2015)
15 FC L-Prod Low stalk flue-cured blend used in cigarette production (2018)
16 FC U-Prod Leaf flue-cured blend used in cigarette production (2018)
17 Bu L-Prod Low stalk Burly blend used in cigarette production (2018)
18 Bu U-Prod Leaf Burley blend used in cigarette production (2018)
19 O1-Prod Oriental blend used in cigarette production (2018)
20 O2-Prod Oriental blend used in cigarette production (2018)
21 Moist 1 Red Seal Natural Fine Cut (2019)
22 Moist 2 Skoal Long Cut Classic Wintergreen (2019)
23 Moist 3 Long Horn Fine Cut Natural (2019)
24 Moist 4 Grizzly Fine Cut Premium Wintergreen (2019)
25 Moist 5 Grizzly Long Cut Premium Wintergreen (2019)
26 Moist 6 Kodiak Premium Wintergreen (2019)
27 Moist 7 Copenhagen Snuff Original Fine Cut (2019)
28 Moist 8 Copenhagen Long Cut Mint (2019)
29 RT1a 1R6F filler
30 RT2a Flue-cured ground tobacco
31 RT3a Oriental ground tobacco
32 RT4a Burley ground tobacco
33 RT5a High TSNA dark air-cured ground tobacco
34 RT6a Flavored cigar filler
35 RT7a 1R5F filler
36 RT8a Unflavored cigar filler
37 RT9a Dark air-cured ground tobacco
38 RT10a Dark fire-cured ground tobacco
39 1S4a Swedish style Snus
40 1S5a Snus
41 3S3a Moist Snuff
42 3S1a Loose-leaf chewing tobacco

Samples from University of Kentucky Center for Tobacco Research Reference Products

Results regarding nicotine, NNK, PON and nicotine-1′-N-oxide levels

The results for the levels of nicotine, NNK, PON and nicotine-1′-N-oxide are given in Table 5.

Average levels of nicotine, NNK, PON, and nicotine-1′-N-oxide (on “dry weight basis”).

No. Sample Nicotine (mg/g) NNK (ng/g) PON (µg/g) Nic-Ox (mg/g) PON method
1 FC L (1) 22.9 452.2 56.4 103.7 1
2 FC U (1) 25.6 242.9 83.1 134.9 1
3 FC L (2) 16.6 265.3 38.6 50.4 1
4 FC U (2) 24.7 49.2 76.5 62.4 1
5 FC off L 20.4 109.6 62.1 146.9 1
6 FC off U 29.4 72.4 71.8 183.0 1
7 Bu L (1) 26.1 274.7 150.1 224.8 1
8 Bu U (1) 33.0 317.8 155.4 254.2 1
9 Bu L (2) 21.1 1791.9 134.5 209.4 1
10 Bu U (2) 24.6 1583.3 199.2 244.2 1
11 Bu off L 7.6 136.2 76.0 123.7 1
12 Bu off U 7.3 174.0 69.4 110.2 1
13 O SA U 14.0 44.4 102.0 226.5 1
14 O Iz U 7.9 0.5 51.5 141.7 1
15 FC L-Prod 17.9 195.4 94.0 204.5 1
16 FC U-Prod 27.2 171.0 121.7 195.4 1
17 Bu L-Prod 22.4 885.5 205.9 221.3 1
18 Bu U-Prod 30.9 755.2 284.2 232.2 1
19 O1-Prod 10.3 7.0 67.4 150.2 1
20 O2-Prod 8.0 1.2 50.8 135.8 1
21 Moist 1 6.0 210.7 5.1 17.8 1
22 Moist 2 5.4 177.2 3.2 23.6 1
23 Moist 3 5.5 265.4 5.8 78.0 1
24 Moist 4 7.8 564.9 10.6 88.7 1
25 Moist 5 4.6 331.6 5.8 92.8 1
26 Moist 6 3.9 403.9 5.5 135.8 1
27 Moist 7 7.1 310.4 7.8 27.6 1
28 Moist 8 5.5 211.1 3.2 28.2 1
29 RT1 21.4 683.4 62.2 2
30 RT2 29.0 90.3 72.3 2
31 RT3 5.9 <LOQ 31.9 2
32 RT4 38.5 672.7 105.8 2
33 RT5 31.9 1075.5 149.0 2
34 RT6 13.1 1126.4 33.9 2
35 RT7 16.5 927.8 34.9 2
36 RT8 15.5 904.8 63.1 2
37 RT9 50.4 2160.0 80.8 2
38 RT10 40.3 526.3 61.8 2
39 1S4 15.8 168.4 28.6 2
40 1S5 15.0 333.5 51.2 2
41 3S3 25.2 381.2 15.5 2
42 3S1 8.7 72.0 25.5 2

The results for the levels of nicotine and of NNK were obtained as averages of duplicate measurements. All nicotine results had a RSD% below 0.3%. For NNK the RSD% values were all below 3.5%. The PON and nicotine-1′-N-oxide measured by Method 1 were analyzed in duplicate and the RSD% values were between 0.1% and 2.8%. The PON levels measured by Method 2 were analyzed in triplicate and the RSD% values were between 1.1% and 5.8%, with the exception of sample 1S5 that had RSD% = 9.4%.

Correlations between the levels of NNK and PON

The study was further focused on evaluating various correlations between the levels of NNK and its potential precursors. The dependence of NNK levels (in ng/g) on the values of PON (in µg/g) is graphically indicated in Figure 3.

Figure 3

The variation of levels of NNK as a function of the levels of PON for all analyzed samples. The numbers next to each point indicate the sample number (for the clarity of the graph not all numbers are shown).

The results from Figure 3 demonstrate that the NNK levels in the samples do not depend on PON levels, as shown by the very low correlation coefficient R2 = 0.1391. The attempt to separate only the tobacco samples and eliminate the moist snuff, the snus, the dark air-cured, and the fire-cured tobaccos did not improve the correlation, the R2 value showing only a slight increase to R2 = 0.1761.

Considering only the moist snuff samples, the R2 of the dependence is 0.5281, which is still a very low value that does not demonstrate dependence. Selecting only the samples analyzed by Method 1 for PON quantitation (10) or by Method 2 quantitation, the correlation remains very low in both cases.

Correlations between the levels of NNK and nicotine-1′-N-oxide

The dependence of NNK levels (in ng/g) on the values of nicotine-1′-N-oxide (in µg/g) is graphically indicated in Figure 4. Similar to the case of PON, the results from Figure 4 demonstrate that the NNK levels in the samples do not depend on nicotine-1′-N-oxide levels, as shown by the very low correlation coefficient R2 = 0.1443. Also, using specific selection of the type of sample, no significant improvement of the correlation is obtained.

Figure 4

Graph showing the dependence of NNK on nicotine-1′-N-oxide levels for all samples. The numbers next to each point indicate the sample number.

Correlations between the levels of NNK and nicotine

Although the basic source of NNK is nicotine, the correlation between the NNK levels and nicotine levels is also very poor. This is illustrated in Figure 5.

Figure 5

Graph showing the dependence of NNK on nicotine levels for all samples. The numbers next to each point indicate the sample number.

CONCLUSIONS

The attempt to determine a dependence of the levels of NNK on the levels of PON was unsuccessful for a number of tobaccos and tobacco products. This result did not prove that NNK in tobacco or tobacco products is not generated via PON, but it demonstrated that the limiting factor (rate limiting step) in the formation of NNK is not the level of PON.

The same lack of dependence was noticed between the levels of NNK and those of nicotine-1′-N-oxide or nicotine. It can be concluded that other factors are responsible for the effectiveness of NNK formation in tobacco and tobacco products.

Figure 1

Chromatogram generated in positive MRM mode from a Burley tobacco sample indicating the peaks for nicotine-1′-N-oxide, PON and the internal standard nicotine-d3.
Chromatogram generated in positive MRM mode from a Burley tobacco sample indicating the peaks for nicotine-1′-N-oxide, PON and the internal standard nicotine-d3.

Figure 2

Typical chromatogram of PON and PON-d3 in 3R4F filler and in a standard containing 46.2 ng/mL PON.
Typical chromatogram of PON and PON-d3 in 3R4F filler and in a standard containing 46.2 ng/mL PON.

Figure 3

The variation of levels of NNK as a function of the levels of PON for all analyzed samples. The numbers next to each point indicate the sample number (for the clarity of the graph not all numbers are shown).
The variation of levels of NNK as a function of the levels of PON for all analyzed samples. The numbers next to each point indicate the sample number (for the clarity of the graph not all numbers are shown).

Figure 4

Graph showing the dependence of NNK on nicotine-1′-N-oxide levels for all samples. The numbers next to each point indicate the sample number.
Graph showing the dependence of NNK on nicotine-1′-N-oxide levels for all samples. The numbers next to each point indicate the sample number.

Figure 5

Graph showing the dependence of NNK on nicotine levels for all samples. The numbers next to each point indicate the sample number.
Graph showing the dependence of NNK on nicotine levels for all samples. The numbers next to each point indicate the sample number.

Average levels of nicotine, NNK, PON, and nicotine-1′-N-oxide (on “dry weight basis”).

No. Sample Nicotine (mg/g) NNK (ng/g) PON (µg/g) Nic-Ox (mg/g) PON method
1 FC L (1) 22.9 452.2 56.4 103.7 1
2 FC U (1) 25.6 242.9 83.1 134.9 1
3 FC L (2) 16.6 265.3 38.6 50.4 1
4 FC U (2) 24.7 49.2 76.5 62.4 1
5 FC off L 20.4 109.6 62.1 146.9 1
6 FC off U 29.4 72.4 71.8 183.0 1
7 Bu L (1) 26.1 274.7 150.1 224.8 1
8 Bu U (1) 33.0 317.8 155.4 254.2 1
9 Bu L (2) 21.1 1791.9 134.5 209.4 1
10 Bu U (2) 24.6 1583.3 199.2 244.2 1
11 Bu off L 7.6 136.2 76.0 123.7 1
12 Bu off U 7.3 174.0 69.4 110.2 1
13 O SA U 14.0 44.4 102.0 226.5 1
14 O Iz U 7.9 0.5 51.5 141.7 1
15 FC L-Prod 17.9 195.4 94.0 204.5 1
16 FC U-Prod 27.2 171.0 121.7 195.4 1
17 Bu L-Prod 22.4 885.5 205.9 221.3 1
18 Bu U-Prod 30.9 755.2 284.2 232.2 1
19 O1-Prod 10.3 7.0 67.4 150.2 1
20 O2-Prod 8.0 1.2 50.8 135.8 1
21 Moist 1 6.0 210.7 5.1 17.8 1
22 Moist 2 5.4 177.2 3.2 23.6 1
23 Moist 3 5.5 265.4 5.8 78.0 1
24 Moist 4 7.8 564.9 10.6 88.7 1
25 Moist 5 4.6 331.6 5.8 92.8 1
26 Moist 6 3.9 403.9 5.5 135.8 1
27 Moist 7 7.1 310.4 7.8 27.6 1
28 Moist 8 5.5 211.1 3.2 28.2 1
29 RT1 21.4 683.4 62.2 2
30 RT2 29.0 90.3 72.3 2
31 RT3 5.9 <LOQ 31.9 2
32 RT4 38.5 672.7 105.8 2
33 RT5 31.9 1075.5 149.0 2
34 RT6 13.1 1126.4 33.9 2
35 RT7 16.5 927.8 34.9 2
36 RT8 15.5 904.8 63.1 2
37 RT9 50.4 2160.0 80.8 2
38 RT10 40.3 526.3 61.8 2
39 1S4 15.8 168.4 28.6 2
40 1S5 15.0 333.5 51.2 2
41 3S3 25.2 381.2 15.5 2
42 3S1 8.7 72.0 25.5 2

Parameters for the MS/MS detection.

Compound Ion for Q1 Ion for Q3 Acquisation time (ms) Declustering potential (V) Collision energy (V) Collision exit potential (V)
Pseudooxynicotine 179.1 148.1 100 70 17 13
Nicotine-1′-N-oxide 180.1 133.1 100 70 21 13
Nicotine-d3 (I.S.) 166.1 870 100 30 15 9

Method recovery with spiked PON-d4 into 3R4F (n = 3).

Spiked PON-d4 (ng/mL) Recovery (%) RSD (%)
302.7 100 10.1
1210.8 103 4.0
2421.6 102 3.1

List of tobaccos, moist snuff, and snus samples evaluated in this study.

No. Sample Description
1 FC L (1) Eastern NC belt, lower stalk (lug) flue-cured (2016)
2 FC U (1) Eastern NC belt, upper stalk (leaf & some tips) flue-cured (2016)
3 FC L (2) South Carolina belt, lower stalk (lug) flue-cured (2016)
4 FC U (2) South Carolina belt, upper stalk (leaf & some tips) flue-cured (2016)
5 FC off L Brazil, lower stalk (lugs & primings) flue-cured (2016)
6 FC off U Brazil, upper stalk (leaf & tips) flue-cured (2016)
7 Bu L (1) Kentucky & Tennessee, lower stalk (flyings & cutters) Burley (2016)
8 Bu U (1) Kentucky & Tennessee, upper stalk (leaf) Burley (2016)
9 Bu L (2) North Carolina & Virginia, lower stalk (flyings & cutters) Burley (2016)
10 Bu U (2) North Carolina & Virginia, upper stalk (leaf) Burley (2016)
11 Bu off L Malawi, lower stalk (flyings & cutters) Burley (2016)
12 Bu off U Malawi, upper stalk (leaf) Burley (2016)
13 O SA U Turkey, good quality middle to upper stalk, Samsun Oriental (2015)
14 O Iz U Turkey, good quality middle to upper stalk, Izmir Oriental (2015)
15 FC L-Prod Low stalk flue-cured blend used in cigarette production (2018)
16 FC U-Prod Leaf flue-cured blend used in cigarette production (2018)
17 Bu L-Prod Low stalk Burly blend used in cigarette production (2018)
18 Bu U-Prod Leaf Burley blend used in cigarette production (2018)
19 O1-Prod Oriental blend used in cigarette production (2018)
20 O2-Prod Oriental blend used in cigarette production (2018)
21 Moist 1 Red Seal Natural Fine Cut (2019)
22 Moist 2 Skoal Long Cut Classic Wintergreen (2019)
23 Moist 3 Long Horn Fine Cut Natural (2019)
24 Moist 4 Grizzly Fine Cut Premium Wintergreen (2019)
25 Moist 5 Grizzly Long Cut Premium Wintergreen (2019)
26 Moist 6 Kodiak Premium Wintergreen (2019)
27 Moist 7 Copenhagen Snuff Original Fine Cut (2019)
28 Moist 8 Copenhagen Long Cut Mint (2019)
29 RT1a 1R6F filler
30 RT2a Flue-cured ground tobacco
31 RT3a Oriental ground tobacco
32 RT4a Burley ground tobacco
33 RT5a High TSNA dark air-cured ground tobacco
34 RT6a Flavored cigar filler
35 RT7a 1R5F filler
36 RT8a Unflavored cigar filler
37 RT9a Dark air-cured ground tobacco
38 RT10a Dark fire-cured ground tobacco
39 1S4a Swedish style Snus
40 1S5a Snus
41 3S3a Moist Snuff
42 3S1a Loose-leaf chewing tobacco

Parameters a, b, and c for the quantitation of pseudooxynicotine (PON) and nicotine-1′-N-oxide, and the regression coefficient R2 of the calibration line.

Compound a b c R2
PON −1.3581 E-13 1.33470 E-06 −2.1595 E-02 0.9996
Nicotine-1′-N-oxide 3.04019 E-07 −0.039649 0.9993

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