Jana
Ticha
* and
Christopher
Wright
British American Tobacco, Research and Development, Southampton, UK. E-mail: jana_ticha@bat.com
First published on 10th August 2016
In 2012, the FDA Tobacco Products Scientific Advisory Committee published a list of 93 harmful and potentially harmful constituents (HPHCs) of tobacco products and tobacco smoke. This list includes many of the “Hoffmann analytes”—the most frequently cited substances regarding the negative health effects of cigarette smoking. Proposed changes to US tobacco product regulation require reporting of HPHC concentrations in smoke. Fit-for-purpose analytical methods for measurement of HPHCs are a priority for regulatory agencies and the tobacco industry, but the chemical diversity of these substances dictates labor-intensive analyses by established techniques. Here, a semi-quantitative analysis of organic compounds on the Hoffmann list was developed using high-resolution proton nuclear magnetic resonance (HR 1H NMR). The data acquisition protocol was validated and used to build a database of analytes in methanolic tobacco smoke condensate (TSC) of Kentucky 3R4F research cigarettes. Among 33 Hoffmann analytes amenable to NMR measurement, 20 were detected directly in TSC. For the 13 undetected substances, fortification experiments were conducted to identify the concentrations at which they were detectable. Among 34 further FDA HPHCs analyzed, 13 were detectable in 3R4F TSC via overspiking experiments. The chemical shifts of these 13 compounds plus the 20 Hoffman analytes establish a database of 33 smoke toxicants measureable in a single NMR analysis. This approach is compatible with standardized smoke collection procedures and allows rapid and consistent measurement of the selected substances in TSC. It will facilitate the chemical evaluation of large numbers of TSC samples with relatively high throughput and acceptable results reproducibility.
Researchers and public health organizations have proposed numerous lists of substances in mainstream smoke that should be prioritized with regard to human health, including 44 substances subsequently named the ‘Hoffmann analytes’.7 Hoffmann analytes include substances prioritized by World Health Organisation (WHO) Framework Convention on Tobacco Control, i.e. acetaldehyde, acrolein, carbon monoxide, benzene, 1,3-butadiene, formaldehyde, N-nitrosonornicotine (NNN), N-nitrosonornicotine ketone (NNK), benzo[a]pyrene (B[a]P) and nicotine. In June 2009, the Family Smoking Prevention and Tobacco Control Act became law in the United States and assigned authority to the FDA to regulate the manufacture, distribution and marketing of tobacco products to protect public health.14 It also imposed obligations on the US tobacco industry to measure and report chemical constituents of tobacco products and cigarette smoke. In 2012, the FDA Tobacco Products Scientific Advisory Committee published a list of 93 harmful and potentially harmful constituents (HPHCs) defined as chemical compounds present in tobacco products or tobacco smoke that are known to cause, or potentially to cause, serious illnesses such as cancer, cardiovascular diseases and chronic obstructive pulmonary disease.15 The FDA HPHC and Hoffman analytes lists overlap in 35 substances. The Hoffmann analytes not included in the current HPHCs list are 3-aminobiphenyl, butyraldehyde, hydroquinone, N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT), nitric oxide, nicotine-free-dry-particulate-matter (NFDPM, “tar”), pyridine and resorcinol.
Under section 904 of the Tobacco Control Act, US tobacco product manufacturers and importers have been obliged to report the concentration of a subset of 20 HPHCs in tobacco products or tobacco smoke by brand since June 2012, and will have to report the remaining HPHCs in due course.16 The development of appropriate analytical methods for the measurement of HPHCs in tobacco and smoke is therefore a priority for both regulatory agencies and the tobacco industry, but the diversity of these chemical substances currently dictates a labor-intensive analysis by various specialized techniques.9 For example, current determination of the 44 Hoffmann toxicants typically requires 14 separate analyses, including HPLC/UV, HLPC/MS, GC/MS, IC and ICP/MS. Furthermore, the established methods are fully targeted and cannot provide any additional information, such as data on an additional member of a homologous series, without modification or repeat analysis.17–26 Moreover, the FDA list may be reviewed and altered over time, requiring further modifications to established methods.
Recently, attention has been turning from the targeted measurement of cigarette smoke constituents to chemical screening that can increase the characterization of tobacco smoke. This latter strategy provides significantly more comprehensive information than the targeted measurement of specific groups of substances and generates data that can be retrospectively re-analyzed (or ‘mined’) to provide information in the light of new knowledge (e.g., to determine whether a previously ‘insignificant’ substance was present in historical samples). Recent screens of tobacco smoke have applied predominantly chromatographic approaches that limit the number and range of substances discernible and thereby provide only partial information.21,27 While it is likely that no single analytical technique will be able to measure all of the constituents of cigarette smoke, one that is capable of measuring a large homologous series of multiple compound classes may offer the best approach to tobacco smoke screening.
High-resolution (HR) proton nuclear magnetic resonance (1H NMR) spectroscopy is a comprehensive non-destructive orthogonal technique for both non-targeted and targeted analysis of substances with a wide range of physico-chemical properties. It provides information about the physical and chemical properties of atoms by exploiting the capability of certain nuclei to absorb and emit energy in response to radiofrequency perturbation in the presence of a static magnetic field (B0). A key feature of the NMR spectrum is that the signals arising from a molecule are resolved on the frequency axis (chemical shift). The overall NMR pattern of a mixture is characterized by the sum of all responses relating to each individual substance in the mixture. Thus, within a single NMR measurement it is possible to gain information on the substances present in the mixture at one time, and the data can be further and retrospectively interrogated with regard to additional substances of interest. NMR is also quantitative, because the areas of the signals are directly proportional to the number of active nuclei that give rise to them. Although NMR is generally perceived as less sensitive than other spectroscopic techniques, it is being continually improved by technological developments such as the maximum available magnetic field strength and the application of cryoprobes, which have facilitated detection down to the micromolar range.28–30
HR NMR has several key advantages. It is a non-destructive technique and does not normally require time-consuming sample preparation steps. It is independent of chromatographic methods but complementary to them, and facilitates the confirmation of other spectroscopically derived data. It has the potential to screen the composition of liquid-state samples for a wide range of chemical classes. Since its inception in the 1950s, HR 1H NMR has emerged as an essential tool for chemical research and quality control in the fields of chemistry, biochemistry, physics and medicine.31–35
For example, it has been successfully used to detect unknown contaminants in potable water,36 carbonated37 and alcoholic38,39 beverages. In particular, Charlton et al.36 demonstrated that NMR spectroscopy is an ideal technique for the non-targeted detection of unknown contaminants, showing that mixtures of pesticides, industrial solvents, toxins and explosives could be identified under the same experimental conditions. Lachenmeier et al.40 highlighted a need for non-targeted screening methods in food industry and potential of NMR as routine analytical tool for both, non-targeted and targeted methods. Advantages of NMR were also reported in the analysis of electronic cigarettes (e-cigarettes), for example Hahn et al. used NMR for the analysis of several components of e-cigarette liquids to estimate the risk of consumer exposure.41
Of the 44 Hoffmann analytes that must be measured in cigarette smoke in certain jurisdiction, 33 are indicated as amenable to NMR analysis. However, few studies have applied NMR to cigarette smoke measurement, and those published focus on mainstream smoke particulate matter.42 Pankow et al. published studies concerning the ratio of protonated nicotine and the effect of protonated nicotine formation on pH in particulate matter of selected commercial and reference cigarettes43 and also in the Eclipse “cigarette” product that heats the tobacco instead of burning it by using a carbon rod.44
The aim of the present study was therefore to demonstrate the applicability of HR 1H NMR spectroscopy to the screening and quantification of selected Hoffmann substances and other HPHCs in mainstream tobacco smoke by providing not only a rapid alternative to chromatographic techniques but also a confirmatory spectroscopic technique.
Parameter | Mean value (mg per cigarette) | |
---|---|---|
3R4F | CM6 | |
a Approved monitor no. 6 (CM6). Abbreviations: TPM, total particulate matter; NFPDM, nicotine-free dry particulate matter (TPM with nicotine and water subtracted; ‘tar’). | ||
Weight | 1060 | 974 |
TPM45 | 11.0 | 17.54 |
Nicotine46 | 0.73 | 1.39 |
NFDPM45 | 10.27 | 14.28 |
CO47 | 12.0 | 14.83 |
Puff count48 | 9.0 | 9.15 |
Fig. 1 Sample generation workflow. Particulate phase collected on a CFP was extracted and combined with the content of two impingers that were kept at approximately −70 °C. The final extract was stored at −70 °C to minimize the loss of volatile substances. Adapted from Adamson et al.50 |
Hoffmann analyte | MW (g mol−1) | Abundance in 3R4F51 | Approximate abundancea (g per cig.) | Expected concentration in TSCb (mg l−1) | Concentration added stdc (mg l−1) | |
---|---|---|---|---|---|---|
a Approximate abundance of these substances in the smoke of one cigarette expressed in grams. b Expected concentration in the 3R4F TSC expressed in mg l−1. c Concentration of the standard solutions expressed in mg l−1. | ||||||
Nicotine | 162.23 | 0.73 | mg per cig | 7 × 10−4 | 125.09 | 156.39 |
Acetaldehyde | 44.05 | 469 | μg per cig | 5 × 10−4 | 125.07 | 156.34 |
Isoprene | 68.12 | 347 | μg per cig | 3 × 10−4 | 62.55 | 78.2 |
Acetone | 58.08 | 199 | μg per cig | 2 × 10−4 | 12.51 | 15.62 |
1,3-Butadiene | 54.09 | 30.5 | μg per cig | 3 × 10−5 | 6.26 | 7.84 |
Acrolein | 56.06 | 52.2 | μg per cig | 5 × 10−5 | 6.25 | 7.79 |
Toluene | 92.14 | 64.5 | μg per cig | 6 × 10−5 | 6.26 | 7.83 |
Catechol | 110.1 | 38.9 | μg per cig | 4 × 10−5 | 6.25 | 7.82 |
Hydroquinone | 110.11 | 31.1 | μg per cig | 3 × 10−5 | 6.25 | 7.82 |
Formaldehyde | 30.03 | 25 | μg per cig | 3 × 10−5 | 1.25 | 1.56 |
Acrylonitrile | 53.06 | 10.7 | μg per cig | 1 × 10−5 | 1.25 | 1.54 |
Propionaldehyde | 58.08 | 45 | μg per cig | 5 × 10−5 | 1.25 | 1.57 |
Crotonaldehyde | 70.09 | 10.7 | μg per cig | 1 × 10−5 | 1.25 | 1.54 |
Butyraldehyde | 72.11 | 33.6 | μg per cig | 3 × 10−5 | 1.25 | 1.59 |
2-Butanone | 72.11 | 52.4 | μg per cig | 5 × 10−5 | 1.25 | 1.59 |
Benzene | 78.11 | 43.4 | μg per cig | 4 × 10−5 | 1.25 | 1.56 |
Pyridine | 79.1 | 4.5 | μg per cig | 5 × 10−6 | 1.25 | 1.58 |
Phenol | 94.11 | 7.6 | μg per cig | 8 × 10−6 | 1.25 | 1.6 |
Styrene | 104.15 | 4.2 | μg per cig | 4 × 10−6 | 1.25 | 1.56 |
o-Cresol | 108.14 | 2.49 | μg per cig | 2 × 10−6 | 1.25 | 1.51 |
m-Cresol | 108.14 | 2.09 | μg per cig | 2 × 10−6 | 1.25 | 1.51 |
p-Cresol | 108.14 | 4.69 | μg per cig | 5 × 10−6 | 1.25 | 1.51 |
Resorcinol | 110.1 | 0.78 | μg per cig | 8 × 10−7 | 0.13 | 0.15 |
Quinoline | 129.16 | 0.27 | μg per cig | 3 × 10−7 | 0.13 | 0.15 |
NNN | 177.2 | 113.5 | ng per cig | 1 × 10−7 | 7 × 10−3 | 7 × 10−3 |
NAT | 189.21 | 125.4 | ng per cig | 1 × 10−7 | 1 × 10−3 | 100.09 |
NNK | 207.23 | 103.6 | ng per cig | 1 × 10−7 | 1 × 10−3 | 100.09 |
NAB | 191.23 | 14.1 | ng per cig | 1 × 10−8 | 1 × 10−3 | 100.01 |
1-Aminonaphthalene | 143.19 | 13.6 | ng per cig | 1 × 10−8 | 1 × 10−3 | 1 × 10−3 |
2-Aminonaphthalene | 143.19 | 7.7 | ng per cig | 8 × 10−9 | 1 × 10−3 | 1 × 10−3 |
3-Aminobiphenyl | 169.22 | 1.93 | ng per cig | 2 × 10−9 | 2 × 10−3 | 2 × 10−3 |
4-Aminobiphenyl | 169.22 | 1.15 | ng per cig | 1 × 10−9 | 1 × 10−4 | 100.01 |
B[a]P | 252.31 | 6.22 | ng per cig | 6 × 10−9 | 1 × 10−3 | 100.17 |
Standards for 22 Hoffmann analytes were prepared in deuterated methanol at a concentration 1.25 times higher than that expected for the target compound in the TSC (Table 2, first 22 rows), while those for five others (NAT, NAB, NNK, B[a]P, 4-aminobiphenyl) were prepared at a concentration of 100 mg l−1, several orders of magnitude higher than the concentration expected for these analytes (Table 2).
The final solutions for the overspiking experiments were obtained by adding a 10% (v/v) aliquot of a previously prepared standard solution to the TSC. The concentrations of the standard solutions for the overspiking experiments of HPHCs ranged from 20 to 100 mg l−1.
The following acquisition parameters were used for data collection: 90° pulse length, 7.75 μs; spectral width, 19.9947 ppm; acquisition mode, digital quadrature detection; unrecorded FIDs (Free Induction Decay), 16; recorded FIDs, 512; offset frequency, 4.945 ppm; relaxation delay, 10 seconds; mixing time, 200 ms; and acquisition time, 3.277 seconds. Spectra were acquired at 300 K. These parameters gave a total experimental time of approximately 2 hours.
Statistical analysis was carried out with Metabolab, a custom-written graphical user interface for Matlab version 7.4.0.287 (R2007a) (Mathworks, UK).52
Data were binned using the undecimated wavelet transform to remove noise and to perform peak alignment.53
Previous studies using NMR spectroscopy within the tobacco sector have generally involved investigating individual compounds and their dynamics, primarily in mainstream smoke/aerosol particulate matter.43,44 Isolated studies such as Barsanti et al.42 have generated useful initial information about the composition of tobacco smoke particulates using NMR spectroscopy. The NMR assignments presented here provide a useful resource to support further studies where previously little information about the NMR chemical shifts in relation to mainstream cigarette smoke was known.
The TSC of 3R4F cigarettes was selected as an initial reference sample to facilitate a comparison between the chemical shifts observed for TSC and those observed for analytical standards. An example of the NMR spectrum of the 3R4F TSC is shown in Fig. 2 (see also ESI Fig. S1†).
Eleven Hoffmann analytes that were expected to be present in TSC at concentrations equivalent to 1 μg per cigarette or lower (Table 2, resorcinol to 4-aminobiphenyl) were evaluated and found to be below the current detection limits of the 1H NMR system. Using standard additions, six of these substances (resorcinol, quinoline, NNN, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl) gave sufficiently intense NMR signals to enable their detection in the TSC.
Standard addition experiments at higher concentrations were conducted for the other 5 compounds (NAT, NAB, NNK, B[a]P and 4-aminobiphenyl) in order to record their characteristic chemical shifts for future reference.
In total, 20 of the 33 target Hoffmann substances were identified in the TSC from the 3R4F cigarette, and their detailed chemical shift data were recorded in deuterated methanol in both the presence and absence of TSC (ESI Table S2†). These data form the basis of an initial database of NMR chemical shifts for toxicants in 3R4F TSC. Thirteen substances (1,3-butadiene, butyraldehyde, m-cresol, resorcinol, quinoline, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, NAB, NAT, NNK, NNN and B[a]P) were not detected in the mainstream cigarette TSC. Among these, however, only 1,3-butadiene and butyraldehyde were expected to be present above the LOD of the method (estimated as 10 μg per cigarette).
1,3-Butadiene was not detected in TSC, probably because of evaporative losses during sample transport. When it was spiked directly into the TSC at a concentration of 7.84 mg l−1, 1,3-butadiene was detected, allowing its semi-quantification. Butyraldehyde did not display any uniquely identifiable peaks within the NMR spectrum of TSC (ESI Table S2†).
The repeatability of the analytical procedure was assessed by analyzing five replicates of 3R4F TSC. The relative standard deviation (RSD, %) of the signals of the NMR spectra was calculated to determine the spread of the results.
The spectral profiles of the replicates were averaged by using the method of adaptive binning with undecimated wavelets.53Fig. 3 shows a stacked plot of the five replicate spectra, along with a plot of the observed RSD. An acceptable RSD of less than 5 was obtained for chemical shifts between 0 and 5.5 ppm, and those between 6 and 10 ppm (Fig. 3 and ESI Fig. S2†).
Fig. 3 Stacked plot of the NMR spectra of five replicates of 3R4F smoke condensate, along with a plot of the relative standard deviation. |
However, higher RSDs were observed for chemical shifts between 5.5 and 6 ppm. This NMR region was characterized by the resonances of 1,3-butadiene, as confirmed by overspiking experiments, the volatility of which might explain the variation observed between replicates.
Nevertheless, the low RSD values obtained in all other NMR regions showed that high experimental reproducibility was achieved. This reproducibility was further confirmed by the consistency of the chemical shifts obtained for the five replicates of 3R4F TSC (ESI Table S3†). The acquisition of consistent replicate data for 3R4F thus confirms the robust nature of the preparation procedure and the stability of the NMR instrumentation. The analytical method established using the 3R4F NMR protocol was then applied to TSC prepared from CM6, which differs in blend composition; mainstream smoke tar and nicotine yield (Table 1). The CM6 TSC chemical shifts of the resonance peaks in each spectrum were compared with those reported in the 3R4F database.
The same 20 Hoffmann analytes detected in 3R4F TSC during development of the protocol were found in CM6 TSC. Similarly, 13 analytes present below the LOD were not detected directly in CM6 TSC. Fig. 4 and ESI Fig. S3† show pairwise comparisons of the NMR spectra of 3R4F and CM6. The results of the protocol validation are reported in detail in ESI Tables S3–S5;† semi-quantitative data for the Hoffmann analytes in 3R4F and CM6 TSC are summarized in Table 3.
Fig. 4 Stacked plot of the NMR spectra of 3R4F and CM6 smoke condensates. The NMR spectral profiles of these two samples showed a difference of 10% when peak areas where compared. |
Hoffmann analyte | Conc. in 3R4F51 (mg per cig) | NMR semi-quantitative data (mg per cig) | |
---|---|---|---|
3R4F | CM6 | ||
Acetaldehyde | 0.469 | 0.630 | 0.333 |
Isoprene | 0.347 | 0.373 | 0.273 |
Nicotine | 0.730 | 0.592 | 0.438 |
Acetone | 0.199 | 0.220 | 0.128 |
1,3-Butadiene | 0.031 | 0.101 | 0.088 |
Acrolein | 0.052 | 0.042 | 0.020 |
Toluene | 0.065 | 0.078 | 0.109 |
Catechol | 0.039 | 0.058 | 0.057 |
Hydroquinone | 0.031 | 0.026 | 0.025 |
Formaldehyde | 0.025 | 0.021 | 0.012 |
Acrylonitrile | 0.011 | 0.009 | 0.006 |
Propionaldehyde | 0.045 | 0.167 | 0.105 |
Crotonaldehyde | 0.011 | 0.009 | 0.007 |
2-Butanone | 0.052 | 0.076 | 0.045 |
Benzene | 0.043 | 0.032 | 0.022 |
Pyridine | 0.005 | 0.018 | 0.017 |
Styrene | 0.004 | 0.009 | 0.007 |
TSC for NMR was generated as a mainstream smoke extract in deuterated methanol that was directly analyzed by NMR. The NMR and chromatographic yields51 show good agreement with ratios close to 1, thereby demonstrating the potential of NMR as a rapid comprehensive screening technique (Table 3).
Slightly higher ratios were observed in the case of 1,3-butadiene and some carbonyl substances, possibly owing to the very low temperature of the impingers, which was maintained at −70 °C during whole-smoke generation, sample extraction, storage, shipment and NMR analysis in an attempt to minimize evaporative losses.
The 3R4F NMR data were further theoretically evaluated as a complementary technique to enable multi-residue analysis of mainstream tobacco smoke. As indicated above, 20 smoke constituents were directly detected in TSC and 13 analytes require further concentration to be directly detected. Employing the current sample preparation, 1H HR NMR would allow simultaneous measurement of carbonyls, phenols, volatile hydrocarbons and nitrogen heterocycles of the Hoffmann list with only two analytes requiring concentration or modification in sample preparation, resorcinol (10×) and quinoline (50×), Table 4. (It should be noted that quinoline and butyraldehyde belong to Hoffmann analytes but are not on FDA HPHC list.) Thus, 20 out of 24 substances in 4 classes can be measured directly replacing 4 analyses with one.
Group | Analytes | Concentration required? |
---|---|---|
a No isolated NMR signal. Not on FDA FCTC list. b Evaporative losses/handling issues. | ||
Carbonyls | Methyl ethyl ketone | No |
Acetaldehyde | No | |
Acetone | No | |
Acrolein | No | |
Butyraldehyde | Seea | |
Croton aldehyde | No | |
Formaldehyde | No | |
Propion aldehyde | No | |
Phenols | Catechol | No |
Hydroquinone | No | |
m + p-Cresol | No | |
o-Cresol | No | |
Phenol | No | |
Resorcinol | 10× | |
Organics | Acrylonitrile | No |
Volatile hydrocarbons | 1,3-Butadiene | Seeb |
Benzene | No | |
Isoprene | No | |
Toluene | No | |
Styrene | No | |
Nitrogen heterocyclics | Pyridine | No |
Quinoline | 50× | |
Nicotine | No |
The very low abundance genotoxic substances, i.e. B[a]P, NNN and NNK necessities more selective analysis.
In total, 13 compounds were identified directly in the extract and further eight were tentatively identified (Table 5). The assignment of the NMR signals of eight of the target FDA substances (i.e., benzo(a,h)anthracene, 2,5-dimethylanyline, dibenzo(a,l)pyrene, coumarine, acrylamide, benz(a)anthracene, N-nitrosodiethanolamine and benzo(k)fluoranthene) did not clearly confirm the presence of these compounds in the TSC. In other words, when the chemical shifts were recorded, it was concluded that the number of peaks attributed to these eight substances was not sufficient to confirm their presence with reasonable certainty (Table 5).
# | Compound | Detected in 3R4F TSC |
---|---|---|
1 | Acetamide | Yes |
2 | Nitrobenzene | No |
3 | Dibenzo(a,h)pyrene | No |
4 | Benzo(a,h)anthracene | Uncertain |
5 | Caffeic acid | Yes |
6 | Dibenzo(a,e)pyrene | No |
7 | 2,5-Dimethylanyline | Uncertain |
8 | Dibenzo(a,l)pyrene | Uncertain |
9 | o-Anisidine | Yes |
10 | Chrysene | Yes |
11 | 5-Methyl chrysene | Yes |
12 | 2,3-Benzofuran | Yes |
13 | Pyrrolidine | Yes |
14 | N-Methylethylamine | Yes |
15 | Coumarine | Uncertain |
16 | Acrylamide | Uncertain |
17 | 4-(Nitrosomethylamino)-1-(3-pyridyl)-1-butanone | Yes |
18 | Benz(a)anthracene | Uncertain |
19 | Naphtalene | No |
20 | Urethane | No |
21 | Indeno(1,2,3-cd)pyrene | No |
22 | Anabasine | Yes |
23 | Ethylbenzene | Yes |
24 | N-Nitrosodiethylamine | No |
25 | N-Nitrosodiethanolamine | Uncertain |
26 | Benzo(b)fluoranthene | No |
27 | Vinylacetate | No |
28 | N-Nitrosomorpholine | Yes |
29 | o-Toluidine | Yes |
30 | Propylene oxide | No |
31 | 2-Nitropropane | No |
32 | Benzo(k)fluoranthene | Uncertain |
33 | N-Nitrosopyrrolidine | No |
34 | N-Nitrosopiperidine | No |
The chemical shifts of the compounds that were identified were recorded in detail. In addition to 3R4F TSC prepared from 5 and 10 cigarettes, CM6 TSC was evaluated for the presence of the chemical shifts of these 13 HPHCs in order to monitor sample variation in relation to the type of tobacco blend in the smoke extract (ESI Table S6†).
Among 33 potentially NMR-amenable toxicants selected from the Hoffmann list, 20 (66%) were identified directly in the 3R4F cigarette TSC by their chemical shifts.
Using some modification of the sample collection and extraction, a further 2–5 Hoffmann analytes (quinoline, resorcinol, NAT, NNN and NNK) are estimated to be detected directly in TSC. In addition, 13 HPHCs out of a further 34 compounds from the FDA list were successfully detected in the 3R4F TSC by using an overspiking technique.
As a part of the validation process, the HR 1H-NMR protocol was successfully used to detect the same toxicants in the TSC of other type of cigarette (CORESTA monitor test piece, CM6). The sample preparation required for NMR is simple, and the total time needed to screen the substances in TSC is approximately 2 hours. The LOD of this method for the selected substances in TSC was estimated to be approximately 10 μg per cigarette.
In summary, the presented results demonstrate the feasibility of HR 1H NMR spectroscopy as a rapid, non-destructive method for assessing a wide range of toxicants in tobacco mainstream smoke and providing comprehensive data packages complimentary to chromatographic methods.
The primary focus of the work presented here was the simultaneous detection of multiple toxic analytes with a focus on establishing a NMR spectroscopy method capable of at least semi-quantification of the analytes, thus reducing the time and effort taken in comparison to the use of established methods.
The method performs acceptably for substances present at μg per cigarette levels in mainstream smoke, but would require significant increase in sensitivity to apply to genotoxic substances that are present at low ng per cigarette levels in mainstream smoke.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ay00849f |
This journal is © The Royal Society of Chemistry 2016 |