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. 2023 Mar 16;36(4):685–690. doi: 10.1021/acs.chemrestox.3c00004

Long-Term Storage Study of the Certified 1R6F Reference Cigarette

Huihua Ji †,*, Laura Fenton †, Stacey Slone ‡, Siqi Guan †, Ying Wu †
PMCID: PMC10114065  PMID: 36926865

Abstract

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The first certified reference cigarette, 1R6F, was produced by the Center for Tobacco Reference Products at the University of Kentucky in 2015 and certified in 2016. 1R6F reference cigarettes have been stored at −20 °C since they were manufactured. 1R6F has been widely used as a control cigarette or a monitor for nonclinical investigational purposes in tobacco product analysis and scientific research. However, there is little published data to demonstrate the stability of the 1R6F cigarette. In this paper, we report the results of a long-term storage study of the 1R6F cigarette tobacco filler and the resulting mainstream smoke. 1R6F cigarettes were stored under different conditions (room temperature, refrigerator (4 °C), and freezer (−20 °C)) for 3 years since April 2017. The constituents in the cigarette tobacco filler (oven volatiles, nicotine, N′-nitrosornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)) and the mainstream smoke (nicotine, NNN, NNK, benzo[α]pyrene, carbon monoxide, total particulate matter) were analyzed. Some physical parameters (resistance to draw and ventilation) were also measured. Analysis of our data showed that no significant differences in these major constituents were detected after storage of the 1R6F cigarette at −20 °C for 3 years.

1. Introduction

It is well-known that cigarette smoking is harmful to many body organs and causes negative human health issues such as cancers, heart disease, stroke, lung disease, and diabetes.1 Approximately 14% of adults aged 18 years or older (an estimated 34.2 million) were cigarette smokers in the United States in 2018.2 In March 2012, the US Food and Drug Administration (FDA) established a list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke.3 In August 2016, the FDA issued a final rule to regulate all tobacco products that included cigarettes, cigarette tobacco, cigars, roll-your-own tobacco, smokeless tobacco products, and any other tobacco products. The FDA requires tobacco companies to report the levels of HPHCs found in their tobacco products and tobacco smoke. In addition, any new tobacco products need to be submitted as a premarket tobacco product application (PMTA) to the FDA before the new tobacco product can be marketed.4 The use of reference materials is essential to PMTA. Reference tobacco products are crucially important to report, compare, and interpret the levels of HPHCs in tobacco products. The reporting or publication of data might be misinterpreted without the inclusion of certified reference materials in the communication. Currently, more and more studies have documented the application of reference cigarettes in their research.5−10

Based on the International Organization for Standardization (ISO) Guide, “Reference material is the material that is sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process”.11 Stability is a crucial factor for reference material. The University of Kentucky Center for Tobacco Reference Products (CTRP) has provided reference cigarettes for almost 50 years. These reference cigarettes are widely used as control samples for nonclinical investigational purposes of tobacco research including analytical method development and modified risk tobacco product development. In 2014, CTRP obtained a service agreement with the FDA to produce a certified reference cigarette. The first certified reference cigarette, 1R6F, was manufactured in March 2015. The 1R6F reference cigarettes were manufactured at one time and over 50 million cigarettes were produced, which is enough to last for many years. One crucial property of reference material that is intended to be supplied for many years is its stability in long-term storage. However, there are presently no data showing the stability of 1R6F cigarettes in long-term storage. In this paper, 1R6F cigarette tobacco filler and the resulting mainstream cigarette smoke (including some key constituents in the HPHCs list) were analyzed to evaluate the stability of the reference material under long-term storage.

In our study, the constituents in cigarette tobacco filler (oven volatiles, nicotine, and tobacco-specific N-nitrosamines (TSNAs) including N′-nitrosornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)) and the constituents in mainstream cigarette smoke (nicotine, NNN, NNK, benzo[α]pyrene (B[α]P), carbon monoxide (CO), total particulate matter (TPM)) and puff counts were analyzed at different storage times and storage conditions (room temperature (22 °C), refrigerator (4 °C), and freezer (−20 °C)). Physical parameters including resistance to draw and ventilation were also measured. Our results showed that there were no significant differences in these major constituents of 1R6F cigarettes that had been stored at −20 °C for three years.

2. Experimental Section

2.1. Samples

The 1R6F certified reference cigarettes were purchased from the CTRP at the University of Kentucky. 1R6F, the first certified reference cigarette, was designed to be representative of American market blended tobacco cigarettes. The 1R6F was produced only for research purposes and is not intended for human consumption.

The reference material (RT1–1R6F ground filler) was purchased from the CTRP at the University of Kentucky.

2.2. Reagents

Standard nicotine, B[α]P, and the internal standard B[α]P-d12 were purchased from Sigma-Aldrich (St. Louis, MO). Standards for TSNAs (NNN and NNK) and N-nitroso-di-n-hexylamine (NDHA) were purchased from Toronto Research Chemicals (Ontario, Canada). Dibasic sodium phosphate (Na2HPO4), sodium hydroxide (NaOH), and citric acid were purchased from Sigma-Aldrich (St. Louis, MO). Quinoline, LC-MS grade acetonitrile, HPLC grade methyl tertiary-butyl ether (MTBE), HPLC grade methanol, 99.8% toluene, HPLC grade methylene chloride, and LC-MS grade water were purchased from Fisher Scientific (Fairlawn, NJ, USA).

2.3. Sample Preparation

Two cases of 1R6F certified reference cigarettes were purchased from the CTRP at the University of Kentucky in April 2017. The samples were divided into three groups when they were received from the CTRP. Each group of cigarettes was stored in two Ziploc-sealed plastic bags and stored under different conditions. One group of cigarettes was stored at room temperature (∼22 °C), the second group of cigarettes was stored in a refrigerator (4 °C), and the third group of cigarettes was stored in a frost-free freezer (−20 °C). After each storage interval (0, 1, 2, 4, 6, 9, 12, 18, 24, 30, and 36 months), a subsample of cigarettes from each group was removed from the storage place and conditioned for cigarette smoking and tobacco filler analysis. Before the 1R6F cigarettes were analyzed, they were transferred stepwise from these storage conditions until they reached room temperature. Cigarettes stored at −20 °C were transferred to 4 °C for 24 h then room temperature for at least 2 h or until they had reached equilibrium; cigarettes stored at 4 °C were transferred to room temperature for at least 2 h or until they had reached equilibrium. The reference cigarettes from the three different storage conditions were then conditioned for 48 h at 22 °C and 60% relative humidity according to ISO 3402:1999,12 prior to the analysis of the tobacco filler (except oven volatiles) and the smoking of the cigarettes.

2.4. Smoking Cigarettes and 1R6F Filler Analysis

The sampled cigarettes for smoking were conditioned for 48 h at 22 °C and 60% relative humidity prior to smoking according to ISO 3402:1999. 1R6F cigarettes were smoked under ISO smoking conditions and the standard non-intense smoking regime ISO 3308:2012 (35 mL puff volume, 2 s puff duration, and 60 s puff frequency) using a Cerulean SM450 linear smoking machine (United Kingdom).13,14 The smoke condensate was collected on a Cambridge filter pad. The mainstream smoke analyses included the measurement of nicotine, NNN, NNK, B[α]P, CO levels, TPM, and puff counts. Each data point for CO, TPM, and puff count was a mean value from 25 replicates that was the combined data from all smoke determinations for each constituent, while each data point for nicotine, NNN, NNK, and B[α]P was a mean value from five replicates. Each replicate is the mean value of five cigarettes smoked in a continuous sequence in the same port.

1R6F filler analyses included oven volatiles, nicotine, NNN, and NNK. The tobacco filler was removed from the cigarette by cutting the paper of the cigarette. The filler from 15 cigarettes was combined and well mixed to use for chemical analysis. The oven volatiles were measured with three replicates, and nicotine, NNN, and NNK in the filler were analyzed with two replicates. Physical parameters (resistance to draw and ventilation) were also measured.

2.5. Constituents Analysis

Alkaloid levels were measured with a gas chromatography-flame ionization detector using a protocol developed by the Kentucky Tobacco Research and Development Center at the University of Kentucky. The samples were extracted with NaOH and MTBE. A PerkinElmer Auto System XL GC with a backflush system fitted with a Zebron capillary GC ZB-5 column (30 m × 0.53 mm, 1.50 μm film thickness from Phenomenex Inc.) was coupled with FID for alkaloid determinations. The initial column temperature of 200 °C was increased to 230 °C at 20 °C/min and held for 1 min. The temperatures of the injector and detector were 250 and 275 °C, respectively.

TSNAs were measured with a gas chromatography-thermal energy analyzer using a protocol established by a collaborative study under the guidance of the Tobacco Science Research Conference Analytical Methods Committee. The samples were extracted with methylene chloride containing NDHA as an internal standard. An Ellutia 200 series GC with an 815 TEA detector (Witchford, United Kingdom) was equipped with a DB-5 GC column (30 m × 0.53 mm, 1.50 μm film thickness from Agilent J&W) for analysis. The initial column temperature was 125 °C for 30 s, was increased to 220 °C at 6 °C/min for 2 min and then to 280 °C at 20 °C/min, and held for 10 min. The temperatures of the injector, the pyrolizer of the TEA, and the interface oven were 225 °C, 500 °C, and 280 °C, respectively.

B[α]P was measured using the method adapted from the Cooperation Centre for Scientific Research Relative to Tobacco recommended method – CRM 58.15 An Agilent 7890B GC system (Santa Clara, CA) was coupled with an Agilent 7000C Triple Quad mass spectrometer. Chromatography was performed on a DB-17 GC capillary column (0.25 μm film, 0.25 mm × 30 m, Agilent Technologies). The column temperature was initially held at 200 °C for 1 min, after which it was programmed to increase to 280 °C at 25 °C/min, then from 280 to 320 °C at a rate of 40 °C/min and held there for 5.5 min. Helium was the carrier gas. The inlet and MS source temperatures were maintained at 300 and 250 °C, respectively. The injection volume was 1 μL in the pulsed splitless mode. Agilent 7000C Triple Quad mass spectrometer was operated in an electron ionization source with single ion monitoring mode.

Considering the possibility of the variation from the analytical procedure, the reference material (RT1- 1R6F ground filler) as a control sample was analyzed for these parameters at each time period. The data was compiled from in-house data collection and also compared to the 1R6F ground filler reference material data sheet.16

2.6. Physical Parameter Measurements

The physical parameters resistance to draw, filter pressure drop, filter region ventilation (Vf), and overall ventilation (Vo) were measured using an OMI-FLEX test station from Borgwaldt-Hauni GmbH (Hamburg, Germany).

2.7. Statistical Methods

The mean and standard deviation for each analyte were calculated for each condition at each time point. These summary statistics were visually compared with a ±15% confidence band. In addition, simple linear models, yi = β0 + β1Monthi + ε, were run for each analyte and condition on the mean values at each time point to get estimates of the β1. The goal is for the estimated linear equation to fit within the ±15% confidence band over the 36-month study period, and the value of δ for each analyte was calculated using the bound. Equivalence tests were then performed using the 95% confidence interval of the β1.17

3. Results and Discussion

Typically, to evaluate the stability of an analyte during storage, the approach can be either to compare the stored samples to corresponding freshly prepared samples side-by-side or to compare the values from the stored samples to a reference value. The latter method gives a direct estimate and avoids errors from pre-analytical manipulations, such as the spiking process.18 The reference value can be either the theoretical value or the value determined at the initial time (t = 0 value) before storage in the experiment. The initial time data (t = 0 value) was used as the reference value in this study. The 1R6F certificate of analysis (COA) value and uncertainty were also considered.19

The constituents in cigarette tobacco filler (oven volatiles, nicotine, NNN, and NNK) and the mainstream cigarette smoke (nicotine, NNN, NNK, B[α]P, CO, and TPM) and puff counts were analyzed for the different storage times and storage conditions (room temperature, 4 °C, and −20 °C). Resistance to draw and ventilation were also measured. Nicotine, B[α]P, NNN, and NNK are on the FDA list of HPHCs. Nicotine, a highly addictive chemical, is the most significant alkaloid in tobacco products.20 TSNAs, especially NNN and NNK, are well-known carcinogens present in tobacco products.21 NNN and NNK have shown carcinogenicity in animals and potential carcinogenicity in humans.22 B[α]P, formed during the incomplete combustion of organic matter, has carcinogenic activity because it can form an adduct with deoxyguanosine to covalently modify DNA.23

3.1. 1R6F Cigarette Tobacco Filler Results

The data for oven volatiles, nicotine, NNN, and NNK levels in 1R6F tobacco filler that was stored at room temperature, 4 °C, and −20 °C for 3 years are shown in Figure 1. The oven volatiles were measured before the cigarettes were conditioned. The data indicate that the oven volatiles showed a significant change in 1R6F cigarettes that were stored at ambient room temperature, but the volatiles were stable at 4 °C and −20 °C storage conditions (Figure 1A). When 1R6F cigarettes were stored at ambient room temperature, the oven volatiles were decreased by ∼24% after 1 year of storage and then slightly increased in the later testing dates. There are four main factors affecting the evaporation of liquid: temperature, the surface area occupied by the liquid, humidity of the surroundings, and air circulation or wind speed.24,25 In our study, the products have the same surface area and similar air circulation in the storage area. Therefore, the humidity of the surroundings and temperature are the two main factors affecting evaporation in our study. The relative humidity in the building ranges from 20% to 75% during the year. In the wintertime, the relative humidity in the laboratory is very low. The lower humidity and higher temperature cause more moisture loss in the cigarettes. When the 1R6F cigarettes were stored at 4 °C and −20 °C, the relative humidity range was much narrower (55%–70%) compared to room temperature. The lower temperatures (of 4 °C and −20 °C) result in lower kinetic energy of the individual water molecules, hence more difficult for them to overcome the hydrogen bonding and escape into the atmosphere as water vapor. Consequently, the lower kinetic energy makes the molecule difficult to evaporate.24,25

Figure 1.

Figure 1

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Levels of oven volatiles, nicotine, NNN, and NNK in 1R6F filler tobacco.

The analyses of nicotine, NNN, and NNK in tobacco filler were performed after the 1R6F cigarettes were conditioned at 22 °C and 60% relative humidity for at least 48 h. The moisture content was consistent with ∼12%–13% after the cigarettes were conditioned. Therefore, the moisture variation at room temperature did not affect the results of the chemical analyses. To evaluate the stability of 1R6F in long-term storage, the concentrations of each analyte determined at the different storage conditions were compared to their initial time value (t = 0). Many factors can affect the amounts of the various analytes detected during storage. The “practical” criteria for the evaluated 1R6F storage study data need to make allowances for both the precision of the analytical method and the natural properties of the tobacco products. Variability in some 1R6F constituents is to be expected because 1R6F is an agricultural product and there is some variability in the raw materials. Also, the relative precision in different analytical methods is different. In general, the value of 15% is commonly recommended as the acceptance criteria for the precision of the analytical method validation.18,26−28 A sample would be considered unstable if the difference between the values of the stored samples and the reference value is greater than the variation in the precision of the analytical method.26 Hence, a 15% evaluation criterion was used to evaluate the stability of 1R6F cigarettes. Most of the results for nicotine, NNN, and NNK in 1R6F cigarette filler fit within the 15% range in the long term storage study (Figure 1B–D). This indicates that nicotine, NNN, and NNK in 1R6F cigarette fillers are stable when stored at room temperature, 4 °C, and −20 °C for 3 years.

3.2. 1R6F Mainstream Smoke Results

The mainstream smoke analysis data (nicotine, NNN, NNK, B[α]P, CO, TPM, and puff counts) of 1R6F cigarettes that were stored at room temperature, 4 °C, and −20 °C for 3 years are shown in Figures 2 and 3. The data of nicotine, NNN, NNK, B[α]P, CO, TPM, and puff counts in the mainstream smoke fit in the 15% range. All these results indicated that there were no significant changes for puff counts, CO, TPM, nicotine, NNN, NNK, and B[α]P in the mainstream smoke of cigarettes stored under room temperature, 4 °C, and −20 °C conditions for 3 years.

Figure 2.

Figure 2

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CO level, TPM, and puff counts in the mainstream smoke of 1R6F cigarettes.

Figure 3.

Figure 3

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Nicotine, B[α]P, NNN, and NNK levels in the mainstream smoke of 1R6F cigarettes.

3.3. Equivalence Testing

For the filler analyses, equivalence testing supported the consistency of nicotine, while NNK and NNN did not quite reach significance with p’s < 0.10. Within the mainstream smoke analyses, the consistency of all of the analytes with the exception of NNN was supported by equivalence testing. While NNN is influenced by the unexpectedly lower result at 18 months, the p-value for the equivalence test is relatively close with p < 0.11.

3.4. Physical Parameters

Data for the physical parameters (resistance to draw, filter pressure drop, Vf, and Vo) of the 1R6F cigarettes stored at −20 °C are presented in Table 1. Data for each parameter were the mean of 20 individual cigarettes. The physical parameter data for 1R6F cigarettes stored at room temperature and 4 °C are very similar to cigarettes stored at −20 °C (data are not shown). This suggests that there were no significant changes in the physical parameters of 1R6F cigarettes during the storage period.

Table 1. Physical Parameters of 1R6F Cigarettes Stored at −20°Ca.

months Resistance to draw (mmWg) Filter pressure drop (mmWg) Vf (%) Vo (%)
COA 107 ± 4 137 ± 4 NA 33 ± 6
0 102.00 ± 14.84 141.90 ± 4.76 37.22 ± 9.04 39.63 ± 7.33
1 93.30 ± 19.49 138.10 ± 7.18 41.94 ± 11.68 43.81 ± 8.81
2 101.95 ± 8.80 139.45 ± 5.00 37.29 ± 5.54 40.76 ± 4.09
6 101.35 ± 7.44 135.95 ± 6.75 36.04 ± 3.27 39.19 ± 2.40
9 104.90 ± 4.97 137.80 ± 6.00 34.37 ± 2.02 38.32 ± 2.06
12 107.00 ± 3.81 140.00 ± 4.79 34.14 ± 2.13 38.02 ± 2.09
18 106.15 ± 4.82 139.95 ± 5.75 34.61 ± 1.92 38.91 ± 1.74
24 106.85 ± 3.77 140.35 ± 4.30 NA NA
30 104.70 ± 4.05 137.40 ± 4.89 33.80 ± 1.54 37.33 ± 1.64
36 106.40 ± 3.73 138.90 ± 4.22 33.60 ± 2.25 37.30 ± 2.14
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a

Vf, filter region ventilation. Vo, overall ventilation. NA, not available.

3.5. Small Volatile Compounds

The stability of small volatile compounds, such as carbonyl compounds, was not determined in the current study. A project including the stability test for these compounds is ongoing. Nevertheless, a study reported in the literature demonstrated that these small volatile compounds in tobacco products are not stable when stored at room temperature and 4 °C.29 Therefore, we recommend that the best temperature for long-term storage of the 1R6F certified reference cigarette is −20 °C.

4. Conclusion

The certified 1R6F reference cigarettes have been stored at −20 °C since they were manufactured in 2015 and certified in 2016. The results for the contents of oven volatiles, nicotine, NNN, and NNK in the 1R6F cigarette filler are comparable after storage at 4 °C and −20° for 3 years. However, the oven volatiles decreased significantly when the 1R6F cigarettes were stored at room temperature. There were no significant changes in puff counts, CO, TPM, nicotine, B[α]P, NNN, and NNK in the mainstream smoke of 1R6F cigarettes stored at room temperature, 4 °C, and −20 °C. Our experimental data demonstrated that 1R6F cigarettes are stable for the above-selected constituents when stored at 4 °C and −20 °C for 3 years. Overall, we recommend keeping the certified reference cigarettes at −20 °C for long-term storage. This is the first systematic comparison of the long-term storage stability of the reference cigarette tobacco filler as well as the constituents in the 1R6F cigarette smoke for three storage temperatures: room temperature, refrigerator (4 °C), and freezer (−20 °C). The methodology used in this paper can be applied in the future to other reference cigarettes.

Glossary

Abbreviations

HPHC

harmful and potentially harmful constituents

TSNA

tobacco-specific nitrosamines

NNK

4-(methynitrosamino)-1(3-pyridyl)-1-butanone

NNN

N-nitrosonornicotine

B[α]P

benzo[α]pyrene

NDHA

N-nitroso-di-n-hexylamine

Na2HPO4

disodium hydrogen phosphate

NaOH

sodium hydroxide

MTBE

methyl tertiary-butyl ether

CO

carbon monoxide

TPM

total particulate matter

Vf

filter region ventilation

Vo

overall ventilation

FDA

US Food and Drug Administration

ISO

International Organization for Standardization

CTRP

the Center of Tobacco Reference Products

Author Present Address

§ Nitto Avecia Pharma Services, 6 Vanderbilt, Irvine, California 92618, United States

This project was supported by the U.S. Food and Drug Administration through grant RFA-FD-14-001.

The views expressed in this paper do not necessarily reflect the official policies of the Department of Health and Human Services nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.

The authors declare no competing financial interest.

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