**Preface to "Method Development and Applications for Reduced-Risk Products in Separation Science"**

Cigarette smoking is the most hazardous form of tobacco consumption due to the inherent risks of combusting tobacco and inhaling the smoke. Many in public health, including the FDA, agree that a continuum of risk exists among tobacco products, with cigarettes at the highest end and non-combustible tobacco products at the lower end of that continuum. Non-combustible products are lower on the continuum of risk because many of the harmful and potentially harmful constituents (HPHCs) found in cigarette smoke are either absent or present at very low levels. Switching to such products, therefore, may offer a harm reduction opportunity for adult smokers who cannot or will not quit smoking. In addition to the traditional smokeless tobacco products, non-combustible products also include innovative tobacco products such as oral tobacco-derived nicotine (OTDN) products, heated tobacco products (HTPs), and electronic cigarettes (also referred to as e-vapor products; EVPs).

Industry, academic, and government researchers are developing and validating analytical methods to extract, separate, identify, and quantitate a variety of analytes from these innovative tobacco products using a wide range of analytical techniques. These analytes include constituents such as nicotine, degradants and impurities, flavors, non-tobacco ingredients, HPHCs, and other currently unknown constituents.

In this Special Issue, we received nine contributions that covered the latest analytical methods that have been developed and applied for the chemical characterization or exposure assessment to tobacco product constituents of innovative non-combustible products. The developed methods included 1) characterizing the nicotine dissolution release profiles and determining nicotine degradants and HPHCs in OTDN pouches; 2) identifying HPHCs, targeted, and unknown compounds in EVPs; and 3) determining potential biomarkers at trace levels in urine and blood samples in these innovative products.

This Special Issue is representative of the importance of analytical sciences research in characterizing innovative non-combustible products for guiding product design, determining relative product performance, ensuring consistency during the manufacturing process, informing toxicological risk assessment, and enabling regulatory reporting.

The current advances in the development and applications of the analytical methods reported in this Special Issue can be used to inform the harm reduction potential of innovative non-combustible products for adult smokers.

I would like to take this opportunity to express my most profound appreciation to the MDPI Book staff, the editorial team of *Separations*, especially Mr. Ethan Xu, the assistant editor of this Special Issue, all of the talented authors, and the hardworking and professional reviewers.

> **Fadi Aldeek** *Editor*

### *Editorial* **Method Development and Applications for Reduced-Risk Products**

**Fadi Aldeek \* and Mohamadi A. Sarkar**

Altria Client Services LLC, 601 East Jackson Street, Richmond, VA 23219, USA; mohamadi.a.sarkar@altria.com **\*** Correspondence: fadi.aldeek@altria.com

#### **1. Introduction**

Cigarette smoking remains the leading cause of preventable premature death and disease in the U.S. There is an overwhelming scientific consensus that cigarette smoking is addictive and causes lung cancer, heart disease, chronic obstructive pulmonary disease, and other serious diseases [1]. While there are thousands of constituents in cigarette smoke, ref. [2] certain representative classes of chemicals characterized as harmful and potentially harmful constituents (HPHCs) have been studied extensively and attributed to the harm caused by the inhaled smoke of combusted tobacco [3]. Many people in the public health sector have acknowledged that a continuum of risk exists among tobacco products, with conventional combustible cigarettes at the highest end of that spectrum, and noncombustible products on the lower end [4–6]. In recent years, there has been rapid growth in the availability of innovative, non-combustible products, including oral tobacco-derived nicotine (OTDN) products, heated tobacco products (HTPs), and electronic cigarettes (also referred to as e-vapor products; EVPs). Because they are non-combustible, such products contain far fewer combustion-related HPHCs [7–9]. As a result, substantial reduction in the biomarkers for exposure to HPHCs have been reported among adult smokers who completely switch to such products [10,11]. Such large reductions in exposure to HPHCs are accompanied with favorable changes in biomarkers indicative of smoking-related disease outcomes [12]. Consequently, there is a growing body of evidence suggesting that such products likely present a substantial reduction in disease risks [13], and many people in the public health sector recognize the potential of such non-combustible products for reducing harm [6,14,15]. Therefore, switching to non-combustible alternatives presents a significant opportunity to decrease the burden of disease associated with smoking combustible cigarettes, particularly among adult smokers who are unable or unwilling to quit.

There is a growing body of research dedicated to characterizing non-combustible products. Many researchers from industry, academia, and government are working to develop and validate analytical methods to extract, separate, identify, and quantitate a variety of analytes from innovative tobacco products using a wide range of analytical techniques. Understanding the basic properties of these products is important to better characterize innovative oral and inhalable tobacco products. The oral non-combustible categories include traditional smokeless tobacco and OTDN products. Traditional smokeless tobacco products contain tobacco leaves and exist in three different forms including chewing tobacco (loose leaf, plug, or twist); snuff (finely ground tobacco that can be dry, moist, or packaged in pouches (e.g., snus)); and dissolvable (finely ground tobacco pressed into shapes such as tablets, sticks, or strips) products [16]. OTDN products, on the other hand, are tobacco-leaf free and are available in various forms including nicotine pouches, lozenges, gums, and dissolvable products [17,18]. These products may contain a number of ingredients that include tobacco-derived nicotine, pH adjusters (e.g., sodium carbonates), filler materials (e.g., modified cellulose, microcrystalline cellulose), sweeteners, stabilizers, and flavorings.

**Citation:** Aldeek, F.; Sarkar, M.A. Method Development and Applications for Reduced-Risk Products. *Separations* **2022**, *9*, 78. https://doi.org/10.3390/ separations9030078

Received: 16 March 2022 Accepted: 16 March 2022 Published: 18 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Inhalable non-combustible products including EVPs and HTPs are compositionally different than cigarettes. Unlike traditional cigarettes, EVPs do not contain tobacco plant material or paper. They are mainly composed of a mixture of propylene glycol and glycerol in various ratios and flavors, and may or may not contain nicotine. In contrast, HTPs contain tobacco leaves but the tobacco is heated instead of burned, thereby lowering the temperature from >900 ◦C to ~500 ◦C. Due to the absence of tobacco leaves and paper in EVPs and the process of heating the tobacco in HTPs, many of the HPHCs in mainstream smoke are either not present or are present at significantly lower levels than smoking cigarettes [19,20].

The accurate determination and quantitation of constituents and chemicals in these products is needed for guiding product design, determining relative product performance, ensuring consistency during the manufacturing process, informing toxicological risk assessment, and regulatory reporting. This also allows for the characterization of inherent risks of innovative products, which helps determine whether the use of such products is potentially less harmful than smoking cigarettes. In this Special Issue, we discuss the latest analytical methods for chemical characterization of a variety of oral and inhalable non-combustible products.

#### **2. Summary of Published Articles**

This Special Issue includes research papers which address the latest analytical methods used for the identification and characterization of a variety of constituents and analytes in innovative oral and inhalable non-combustible tobacco products, using state-of-the-art techniques and instrumentations. The various contributions presented in this Special Issue are summarized based on the type of products evaluated and related methods reported.

Recently, nicotine pouches have emerged as a new category of innovative OTDN products. In this Special Issue, we received four contributions from different groups on methods that have been developed and validated to determine the nicotine release profiles, nicotine degradants, and HPHCs from a variety of nicotine pouch products. In these contributions, the authors have systematically used the developed methods to compare OTDN to traditional smokeless tobacco products. In the first manuscript, Aldeek et al. evaluated the nicotine release from 35 nicotine pouch products that are currently marketed in seven flavors with five different nicotine levels [21]. This is an important method to characterize the nicotine release from these pouches. The authors implemented a wellestablished dissolution method using the U.S. Pharmacopeia flow-through cell dissolution apparatus 4 (USP-4) that the same group previously developed for the evaluation of the nicotine release from traditional smokeless tobacco products [22]. The dissolution method was used for product-to-product comparison. The percent nicotine release profiles obtained from the 35 nicotine pouches under the same experimental conditions were found to be equivalent across all nicotine levels and flavors analyzed, indicating a similar rate of nicotine release from these oral nicotine pouch products. The authors further compared the percent nicotine release profiles from these nicotine pouches to a variety of other commercially available nicotine pouches and traditional pouched smokeless tobacco products. The authors state that the differences in percent nicotine release rates within the OTDN category could be associated with the inherent product characteristics (e.g., pouch paper and ingredients).

In the second manuscript, Knopp et al. developed a biorelevant dissolution method to study the nicotine release from OTDN nicotine pouches and portioned smokeless tobacco products (e.g., pouched snus) [23]. The in vitro release of nicotine was investigated in biorelevant volumes of artificial saliva using a custom-made dissolution apparatus. The apparatus consisted of a sinker that was prepared by 3D printing using polylactic acid material. The nicotine released was quantitated by a validated high-pressure liquid chromatography ultra-violet spectroscopy (HPLC-UV) method. The percent nicotine release profiles obtained from the OTDN and snus pouches were found to be distinct, indicating the ability of this method to discriminate between these two product categories. Additionally, the authors compared the in vitro dissolution to in vivo data from a previously conducted clinical study [24]. Data showed a strong in vitro/in vivo correlation, indicating that the method reported in this publication is not only sensitive enough to discriminate between nicotine pouch and snus products, but could also serve as a predictive tool for product development and/or a monograph for oral tobacco/nicotine product equivalence studies.

The stability of nicotine depends on the inherent components of the product (e.g., fillers, pH, stabilizers, other ingredients, and moisture content) as well as the external environment (e.g., exposure to light and high temperatures). Therefore, developing methods to assess the nicotine stability in these products by monitoring the nicotine degradation compounds and select impurities is very important. These methods are useful to monitor the stability of nicotine in these products and for quality control purposes (e.g., to evaluate the purity of nicotine added to the product). In the third manuscript, Avagyan et al. developed a selective, accurate, and repeatable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the determination of seven nicotine-related degradants and impurities [25]. The seven nicotine degradants in this method were nicotine-N'-oxide, cotinine, nornicotine, anatabine, anabasine, ß-nicotyrin, and myosmine. Most of the analytes were detected in the nicotine pouch products; however, they were found to be at lower levels compared to traditional tobacco products.

In the fourth manuscript, Jablonski et al. used fully validated CORESTA recommended methods to determine 17 selected HPHCs (including tobacco-specific nitrosamines, carbonyls, benzo[a]pyrene, nitrite, and metals) from 21 nicotine pouch products [26]. The selected pouches were obtained from seven different commercially available brands at the maximum nicotine level and a variety of flavors. The authors assessed two types of pouch products described as "white powder-based pouches" and "plant-based" pouches. The white powder-based pouches were similar to those described above, whereas the plantbased pouches were made from non-tobacco plant materials with pharmaceutical grade nicotine added during the production process. HPHCs in the 21 nicotine pouches were compared to those found in four traditional smokeless tobacco products (two CORESTA reference products and two commercially available products). The authors reported that the HPHCs levels, most notably metals, in the plant-based pouches were higher than those observed in powder-based products. In some plant-based pouches, these levels were even higher than those seen in traditional pouch smokeless tobacco products. However, the overall HPHCs levels observed in these plant-based nicotine pouches were at or below those levels observed in traditional pouch smokeless tobacco products.

The presence of unique constituents in the aerosol of EVPs is an important consideration in overall risk assessment of such products and is of interest to regulators and public health researchers. EVPs include both the e-liquid (containing nicotine and other ingredients) and aerosolizing apparatus, whether sold as a unit or separately. Due to the unique parts and components of EVPs, the constituents are distinct and specific to the product type (e.g., pod-based, open system, etc.). Therefore, in addition to the HPHCs, unknown compounds in the aerosol need to be characterized. The majority of analytical work on EVPs has focused on targeting known chemicals of interest based on changes to the device, formulation, power, temperature, or sampling approaches [27]. In this Special Issue, we received three contributions highlighting the development of targeted and non-targeted analytical methods for the determination of HPHCs and unknowns in EVPs. In the first report, Jin et al. evaluated the traditional 2,4-dinitrophenylhdrazine (2,4-DNPH) derivatization and quantitation of formaldehyde in e-liquid and aerosol of EVPs [28]. Formaldehyde is an HPHC listed by the FDA as a carcinogen and a respiratory toxicant [3]. Previous reports stated that formaldehyde is often underreported in EVPs due to a possible reaction with propylene glycol and glycerin in the aerosol which causes the formation of hemiacetals [29]. The research presented in this study provided a thorough experimental design to clearly demonstrate that hemiacetals formed in the aerosol readily hydrolyze to free formaldehyde and consequently form formaldehyde hydrazone in the typical 2,4-DNPH acidic trapping solution for quantitation. This study showed that the

commonly used 2,4-DNPH method is an appropriate method for the derivatization and accurate quantitation of formaldehyde in the aerosol generated by EVPs.

In the second manuscript, Chen et al. developed a comprehensive, targeted analysis using gas chromatography coupled to mass spectrometry (GC-MS) for the determination of 53 aerosol constituents from EVPs of currently marketed products [30]. The aerosol generation was conducted using non-intense and intense puffing regimens. Only 10 out of the targeted 53 analytes were quantifiable. The authors have compared their data to constituents collected from aerosols generated by both traditional cigarettes and a commercially available HTP that has been authorized for marketing in the U.S. The aerosol generated by the evaluated EVPs had detectable levels of ten targeted analytes including known degradants of propylene glycol and glycerin (e.g., acetaldehyde and formaldehyde) and nicotine-related compounds. The majority of tobacco-related HPHCs were not detectable in the aerosols. The levels of select HPHCs (other than nicotine) measured in the EVPs were found to be 96–99% lower than the same HPHCs reported in the cigarette smoke. However, the reduction levels of these select HPHCs in the EVPs ranged from 61% to 99% when compared to the levels found in HTP aerosol. The authors attributed the low levels of HPHCs in the EVPs' aerosols to the controlled temperature used in the device which is designed to reduce byproducts of combustion.

To address the potential gaps in understanding left by targeted analysis of EVPs, Crosswhite et al. developed and optimized liquid chromatography high resolution mass spectrometry (LC-HRMS) and GC-MS semi-quantitative methods to study unknown chemicals in generated aerosols [31]. These two methods were developed to account for the different physicochemical properties of possible chemical compounds including polarity, volatility, hydrophilicity, etc. The authors used differential analyses based on nine aerosol collection replicates of each studied EVP and each collection condition (intense and nonintense puffing regimens) to characterize compounds that differed from collection blanks. They relied on statistical tools to extract relevant information from a highly complex dataset. The authors reported all compounds at or above concentrations of 0.5 μg/g which were considered related to the sample. A total of 91 compounds were identified using these two methods in both non-intense and intense puffing regimens. This number was strikingly low when compared to the number of compounds (>5000) found in cigarette smoke [32]. Of the detected compounds, 47% were confirmed using reference standards. The authors showed that the studied aerosols from EVPs were approximately 50-fold less complex when compared to cigarette smoke.

We have also received two articles describing the development of LC-MS/MS methods for the identification of biomarkers of exposure specific to EVPs and other non-combustible products. Burkhardt et al. developed an LC-MS/MS method for measuring human exposure to 1,2-propylene glycol and glycerol, the main e-liquid constituents in EVPs [33]. These constituents were analyzed in plasma and urine samples from a clinical study comparing five nicotine product user groups (users of combustible cigarettes, EVPs, HTPs, oral tobacco products, and nicotine replacement therapy (NRT) products) and a control group of non-users. The results demonstrated elevated propylene glycol levels in urine and plasma in EVPs users compared to users of other products. The data showed a correlation between the propylene glycol and nicotine equivalents in the plasma and urine of EVP users. The nicotine equivalents were calculated by measuring the levels of nicotine and ten nicotine metabolites using a method developed by Piller et al. [34]. The authors also reported a dose–response relationship between urinary and plasma propylene glycol and intensity of vaping. The authors proposed that propylene glycol can be used as a potential biomarker to monitor compliance to EVP use when assessing switching behavior among smokers.

The same group, in a second article by Rogner et al., developed and validated another highly sensitive LC-MS/MS method for the determination of 3-hydroxybenzo[a]pyrene (3-OH-BaP), a metabolite of benzo[a]pyrene (BaP), in urine samples from smokers and non-combustible products users [35]. BaP is listed by FDA as an HPHC and classified by IARC as a human carcinogen which is formed during the incomplete combustion of tobacco [3]. The method was validated with a very low limit of quantitation (50 pg/L) to account for trace levels of 3-OH-BaP in urine samples. The detected levels of 3-OH-BaP in urine samples were found to be significantly higher in cigarette smokers compared to non-combustible product users. The data presented by the authors showed the suitability of 3-OH-BaP as a biomarker for BaP and could be applied in clinical studies evaluating innovative non-combustible tobacco products.

#### **3. Conclusions**

The nine articles published in this Special Issue covered the latest analytical methods developed and applied for the chemical characterization or exposure assessment to tobacco product constituents of innovative non-combustible products (i.e., EVPs, HTPs, and OTDN products). The developed methods included (1) characterizing the nicotine dissolution release profiles and determining nicotine degradants and HPHCs in OTDN pouches; (2) identifying HPHCs, targeted, and unknown compounds in EVPs; and (3) determining potential biomarkers at trace levels in urine and blood samples in a variety of EVPs, HTPs, and OTDN products. The contributors to this Special Issue systematically compared the amount and release characteristics of select HPHCs, degradants, and unknown compounds found in innovative non-combustible products to combustible cigarettes or traditional smokeless tobacco products. This Special Issue is representative of the importance of analytical sciences research in characterizing innovative non-combustible products for guiding product design, determining relative product performance, ensuring consistency during the manufacturing process, informing toxicological risk assessment, and enabling regulatory reporting. The current advances in the development and applications of the analytical methods reported in this Special Issue can be used to inform the harm reduction potential of innovative non-combustible products for adult smokers.

**Author Contributions:** Conceptualization, F.A.; writing—review and editing, F.A. and M.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Fadi Aldeek thanks all the authors for their excellent contributions. The efforts of the reviewers are acknowledged as contributing greatly to the quality of this Special Issue.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Dissolution Testing of Nicotine Release from OTDN Pouches: Product Characterization and Product-to-Product Comparison**

**Fadi Aldeek \*, Nicholas McCutcheon, Cameron Smith, John H. Miller and Timothy L. Danielson**

Altria Client Services LLC, 601 East Jackson Street, Richmond, VA 23219, USA; Nicholas.McCutcheon@altria.com (N.M.); Cameron.R.Smith@altria.com (C.S.); John.H.Miller@altria.com (J.H.M.); Timothy.L.Danielson@altria.com (T.L.D.) **\*** Correspondence: fadi.aldeek@altria.com; Tel.: +1-804-335-3119

**Abstract:** In recent years, oral tobacco-derived nicotine (OTDN) pouches have emerged as a new oral tobacco product category. They are available in a variety of flavors and do not contain cut or ground tobacco leaf. The on!® nicotine pouches fall within this category of OTDN products and are currently marketed in seven (7) flavors with five (5) different nicotine levels. Evaluation of the nicotine release from these products is valuable for product assessment and product-to-product comparisons. In this work, we characterized the in vitro release profiles of nicotine from the 35 varieties of on!® nicotine pouches using a fit-for-purpose dissolution method, employing the U.S. Pharmacopeia flow-through cell dissolution apparatus 4 (USP-4). The nicotine release profiles were compared using the FDA's Guidance for Industry: *Dissolution Testing of Immediate Release Solid Oral Dosage Forms*. The cumulative release profiles of nicotine show a dose dependent response for all nicotine levels. The on!® nicotine pouches exhibit equivalent percent nicotine release rates for each flavor variant across all nicotine levels. Furthermore, the nicotine release profiles from on!® nicotine pouches were compared to a variety of other commercially available OTDN pouches and traditional pouched smokeless tobacco products. The percent nicotine release rates were found to be dependent on the product characteristics, showing similarities and differences in the nicotine release profiles between the on!® nicotine pouches and other compared products.

**Keywords:** on!® nicotine pouches; nicotine; dissolution; release profile; validation; product assessment; smokeless tobacco product

#### **1. Introduction**

Over recent years, oral tobacco products have provided alternatives to smoking cigarettes [1,2]. The use of oral tobacco products is considered by many to have potentially reduced risks of harm compared to smoking cigarettes [3–5]. Oral tobacco products exist in two major categories: traditional smokeless tobacco and modern oral nicotine products. Typically, traditional smokeless tobacco products come in three different types, including chewing tobacco (loose leaf, plug, or twist), snuff (finely ground tobacco that can be dry, moist, or packaged in pouches (e.g., snus)), and dissolvable (finely ground tobacco pressed into shapes such as tablets and sticks) products [6]. While traditional smokeless tobacco products contain tobacco leaves, modern oral nicotine products are tobacco leaf-free that contain tobacco-derived nicotine and food grade ingredients [7]. In the last decade, modern oral tobacco-derived nicotine (OTDN) products have been commercialized in various solid forms, including lozenges, gums, and dissolving tablets [8–10]. More recently, nicotine pouches have emerged as a new category of OTDN products. These products are preportioned pouches similar to snus but replace the tobacco leaf with non-tobacco filler and tobacco-derived nicotine.

The scientific evidence regarding the long-term health effects of OTDN pouches has not yet been established; however, the vast body of literature on other oral tobacco products, such as moist smokeless tobacco products, suggests that nicotine pouches will

**Citation:** Aldeek, F.; McCutcheon, N.; Smith, C.; Miller, J.H.; Danielson, T.L. Dissolution Testing of Nicotine Release from OTDN Pouches: Product Characterization and Product-to-Product Comparison. *Separations* **2021**, *8*, 7. https:// doi.org/10.3390/separations80 10007

Received: 15 October 2020 Accepted: 2 January 2021 Published: 7 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional clai-ms in published maps and institutio-nal affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pose significantly lower risks than cigarettes [11]. Based on our review of statements from authoritative bodies regarding the long-term health effects of nicotine and available scientific literature on nicotine replacement therapy (NRT) as well as moist smokeless tobacco products, we believe that OTDN pouch products are not risk free and can lead to dependence. Nicotine, while not benign, has substantially lower health risks compared to smoking cigarettes [12,13].

Tobacco product manufacturers are currently selling OTDN pouch products under different brand names such as ZYN®, Velo, and on!® [7]. These products come in a variety of flavors and different nicotine contents per pouch. The on!® nicotine pouch products, for example, are currently marketed with seven flavor variants (e.g., Citrus, Wintergreen, Mint, Coffee, Berry, Cinnamon, and Original) and 5 different nicotine levels (1.5, 2, 3.5, 4, and 8 mg per pouch), providing an overall portfolio of 35 combinations of flavor variants and nicotine levels. These products are consumed by placing the pouch between the gum and upper lip, allowing for the dissolution of nicotine to occur in the saliva before being absorbed in the oral cavity and entering the bloodstream [14].

The market for oral nicotine pouches has been increasing in recent years as adult tobacco consumers are looking for alternatives to more traditional tobacco products, such as cigarettes [4]. Therefore, research evaluating the release of nicotine from these pouches is needed for product characterization and product-to-product comparisons.

Dissolution testing is commonly used by the pharmaceutical industry to assess product quality, demonstrate equivalency in constituent release, guide formulation design, and develop in vivo/in vitro correlation (IVIVC) [15–19]. Dissolution testing measures in vitro drug release as a function of time, which may reflect the reproducibility of the manufacturing process and, in some cases, relates to the active ingredient's in vivo release [20–23]. Despite the numerous well-established and standardized methods described in the pharmacopoeias, only a few dissolution methods have been developed for the comparison of OTDN, using a variety of dissolution apparatus and analytical methods [24–27]. Recently, we developed and validated a fit-for-purpose method for the dissolution testing of nicotine from a variety of traditional smokeless tobacco products using a USP-4 flow-through cell apparatus. This method quantitatively determines the nicotine release into artificial saliva from a variety of smokeless tobacco products selectively and precisely. This discriminatory dissolution methodology was successfully applied to study the dissolution release profiles from a variety of traditional reference and commercial smokeless tobacco products. We demonstrated the ability of this method to be used as an important tool for tobacco product assessment and product-to-product comparisons, and also that the nicotine release profile is dependent on the form and cut of the studied traditional smokeless tobacco products [28].

In this study, we built on our initial findings and expanded the scope of our validated method to include oral nicotine pouch products, on!®. We characterized the dissolution release of nicotine from 35 on!® nicotine pouch products across the seven flavors and five nicotine levels by comparing the cumulative and percent of total nicotine release profiles. We further calculated the difference factor (f1) and similarity factor (f2) using a methodology referenced in the Guidance for Industry from FDA's Center for Drug Evaluation and Research (CDER) [29,30]. Furthermore, the nicotine release profiles from on!® nicotine pouches were compared to a variety of OTDN pouches and traditional smokeless tobacco products to better understand the nicotine release rates within and across product categories.

#### **2. Materials and Methods**

The dissolution testing was carried out using a USP-4 flow-through cell apparatus (SOTAX, Westborough, MA, USA) following our previous methodology [28]. The determination of nicotine was performed using Acquity I-Class Ultra Performance Liquid Chromatography coupled to a Photodiode Array detector (UPLC-PDA) (Waters, Milford, MA, USA). The UPLC was fitted with a BEH C18 analytical column (2.1 × 100 mm, 1.7 μm) and a BEH C18 VanGuard pre-column (2.1 × 5 mm, 1.7 μm) (Waters, Milford, MA, USA) [28]. The artificial saliva was prepared according to the method described in the German Institute for Standardization (DIN) recipe listed in the German standard DIN V Test Method 53160-1 2002-10 [31]. The USP-4 fractions collection and UPLC solutions and standards preparation were performed following our previously published report [28].

#### *2.1. Test Products*

The 35 on!® nicotine pouch products, currently marketed with seven flavor variants (Citrus, Wintergreen, Mint, Coffee, Berry, Cinnamon, and Original) and 5 different nicotine levels (1.5, 2, 3.5, 4, and 8 mg per pouch) were provided by the manufacturer. Similarly, the Skoal® Bandits and Skoal® pouches (Wintergreen flavored traditional pouched smokeless tobacco products) were provided by the manufacturer. The ZYN® nicotine pouch products used in this study, with different flavor variants (Coffee, Wintergreen, and Cool Mint) and nicotine levels (3 and 6 mg per pouch), were purchased from retail stores.

#### *2.2. Dissolution Fractions Collection*

The USP-4 apparatus used in this study consisted of an array of seven flow-through cells, a cell holder including a water bath, a reservoir and pump for artificial saliva, and a fractions collection rack. The pump delivered a constant flow of artificial saliva (4 mL/min) through the flow-through cells. The flow-through cells were mounted vertically with a filter system that prevents the pouches from exiting the cell. The cells were immersed in the water bath, and the temperature was maintained at 37 ± 0.5 ◦C. A 5 mm ruby bead check valve was placed in the bottom of each sample cell, and approximately 6.6 g of 1 mm glass beads was added to the conical portion of the cell to ensure a laminar flow. Pouched products were weighed, and a single pouch of on!® (~0.265 g), ZYN® (~0.393 g), or traditional pouched smokeless tobacco product (~0.72–1.55 g), was added directly into each vessel. The cell was then filled with approximately 6.6 g of 3 mm glass beads to maintain the pouch position in the center of the flow-through cell. The dissolution testing was conducted according to the guidance issued by the FDA using 12 replicates of one product and taking a dissolution profile at a maximum of 15-min intervals [29,30]. Each replicate was dissolved into 9 fractions. The collection time was 4 min for fractions 1–5, resulting in a final collection volume of 16 mL for each fraction, and 10 min for fractions 6–9, resulting in a final collection volume of 40 mL in each fraction for a total dissolution time of 60 min.

#### *2.3. Quantitative Analysis of Nicotine*

Upon collection of all 9 fractions from each sample replicate, 0.1 mL of each dissolution fraction was added to an autosampler vial, followed by the addition of 0.1 mL of ethyl benzoate as an internal standard (1 mg/mL) and 0.8 mL of artificial saliva. The nicotine concentration in μg/mL was quantitated in all fractions collected from the 12 replicates following the analytical UPLC-PDA method described previously [28]. The concentration of the nicotine based on sample pouch (nicotine amount (mg) released), was determined using the calculated concentration of nicotine (μg/mL), weight of the sample analyzed, and volume of the dissolution fraction.

#### *2.4. Cumulative and Percent of Total Release Profiles*

The cumulative concentrations of nicotine (nicotine amount (mg) released) from each tested product were calculated by summing the averaged nicotine released for each fraction time point from all 12 replicates. The sum of the averaged cumulative nicotine amount corresponds to the total amount of nicotine released up to each time point. The percentage relative to the total nicotine released at each time point (rate) was then calculated and plotted to provide the total release profile. The relative percentage to the total nicotine released was calculated by dividing the amount of nicotine released up to each time point for each fraction by the cumulative amount released in 60 min.

#### *2.5. F1 and F2 Calculations*

The difference factor (f1) and similarity factor (f2) were calculated by adopting a methodology referenced in the Guidance for Industry from FDA's Center for Drug Evaluation and Research (CDER) [29,30]. These two factors can be calculated mathematically by the following equations [32,33]:

$$\mathbf{f}\_1 = \left\{ \left[ \sum\_{t=1}^n |R - T| \right] / \left[ \sum\_{t=1}^n R \right] \right\} \times 100 \tag{1}$$

$$\text{sf}\_2 = 50 \cdot \log \left[ \frac{100}{\sqrt{1 + \frac{\sum\_{t=1}^{t=n} [Rt - Tt]^2}{n}}} \right] \tag{2}$$

*Rt* and *Tt* are the cumulative percentage dissolved at each of the selected n time points of the two products. The factor f1 is proportional to the average difference between the two profiles, whereas factor f2 is inversely proportional to the average squared difference between the two profiles, with emphasis on the larger difference among all the time points. Following the FDA's guidance document, at least 12 replicates should be used for each profile determination. The dissolution measurements of the two products should also be made under identical test conditions. For curves (kinetic release profiles) to be considered equivalent, f1 values should be close to 0 and f2 values should be close to 100. Generally, f1 values up to 15 (0–15) and f2 values of 50 or greater (50–100) demonstrate equivalence of the two curves, reflecting a similar performance of the two products.

#### **3. Results and Discussion**

Previously, we developed and validated a dissolution method to quantitatively evaluate the rate of nicotine release from traditional smokeless tobacco products using USP-4 flow-through cell dissolution apparatus and UPLC-PDA. We based our approach on consensus methodology already existing in the field of pharmaceutical products, including the choice of apparatus, dissolution medium, and the analytical method used for the nicotine quantitation [26,34,35]. We described approaches for product-to-product comparisons between various nicotine-containing traditional loose and pouched traditional smokeless tobacco products [28]. Here, we expanded this methodology to measure the rate of nicotine release for the on!® nicotine pouches portfolio, consisting of 35 products (7 flavors at 5 different nicotine levels).

#### *3.1. Method Validation*

Our USP-4 flow-through cell/UPLC-PDA method was initially validated to study the dissolution release of nicotine from loose and pouched traditional smokeless tobacco products. To study the nicotine release profile from on!® nicotine pouch products, we conducted a supplemental validation to expand the scope of our original method. The supplemental validated elements of the method were accuracy, precision, specificity, and fraction stability. Accuracy of the analytical method was measured by calculating the recovery from two fortification levels in pooled fractions collected from 1.5 mg and 8 mg on!® nicotine pouch products of all flavor variants. Dissolution fractions from the beginning (fractions 1–5) and end (fractions 6–9) of the collection were combined into two pools: pool #1 (fractions 1–5) and pool #2 (fractions 6–9). The fortification levels were 50 and 200 μg/mL for pool #1 and 10 and 50 μg/mL for pool #2. Three replicates of each fortified sample were analyzed to determine accuracy. To determine the % recovery, the measured nicotine value from the unfortified samples was subtracted from each of the fortified samples. The corresponding results were divided by the fortified amounts to determine % recovery. All fortification levels and matrix types had calculated nicotine recovery values between 85 and 107%. Intra-day precision was determined by analyzing 3 replicates each of on!® Mint 1.5 mg and 8 mg pouches within a single day and was found to be <3% Relative Standard Deviation (RSD). Intermediate precision was measured by analyzing 3 replicates

each of the same product over the course of three days (*n* = 9) and was found to be <4% RSD. The specificity of the method was validated by examining the chromatograms in all fractions and artificial saliva (used as a blank). The chromatograms were free of matrix interference, showing the ability of the method to quantitate nicotine in this sample matrix. Finally, the stability of the dissolution fractions was assessed over a period of 14 days in amber glass bottles with a screw cap at 0–4 ◦C. An initial analysis was made for time zero (day 1) and compared to the latter time points. The day 1 fractions were prepared and analyzed immediately in triplicate after dissolution. The average concentration of the aged samples (triplicates) on each day was calculated and compared to the concentrations of day 1 samples. The percent change from the initial measurement was calculated for all aged samples and was found to be less than 5% after 14 days of storage in the above conditions.

#### *3.2. Nicotine Release from on!® Pouches*

Following method validation for oral nicotine pouches, we characterized the in vitro release profiles of nicotine from the 35 varieties of on!® nicotine pouch products. As an example of the release profiles measured, Figure 1 shows the cumulative release profiles (Figure 1A) and percent of total release (Figure 1B) of nicotine from on!® Mint pouches at five different nicotine levels (1.5, 2, 3.5, 4, and 8 mg per pouch). As expected, the cumulative nicotine released from the on!® pouches increases as the nicotine content of the product increases. The percent of total release profiles of nicotine from the on!® Mint pouches at various nicotine levels were equivalent (Figure 1B). More rapid nicotine dissolution was observed for all five products with a total percent release of ~80% in the profile region between zero and 20 min. The total percent of release for all products (>95%) was achieved within 40 min before the nicotine dissolution profiles reached a plateau. Despite differences in total nicotine content, on!® Mint pouches at various nicotine levels exhibit similar kinetic profiles. Similar observations were seen for the other flavor variants of on!® nicotine pouches including Citrus, Wintergreen, Coffee, Berry, Cinnamon, and Original across all five nicotine levels (Table 1).

To further confirm the above observations, we analyzed the nicotine release profiles by calculating the difference factor (f1) and similarity factor (f2) by adopting a methodology referenced in the Guidance for Industry from FDA's Center for Drug Evaluation and Research (CDER) [29,30]. Table 1 shows the f1 and f2 values obtained by using the 4 mg on!® nicotine pouches as the reference products for all flavor variants. In this study, we have chosen the on!® 4 mg as a comparator as it represents the mid-range nicotine concentration of all products. The f1 and f2 values for the 35 on!® nicotine pouches at different nicotine strengths and within each flavor variant demonstrate equivalency of the products with calculated f1 lower than 15 and f2 higher than 50. These data indicate that the total amount of nicotine content in on!® pouches does not affect the nicotine release profile.

To assess the influence of the flavor on the nicotine release rate, we evaluated the nicotine release profiles from all flavored on!® pouch products at each nicotine level. As an example, Figure 2 shows the cumulative release (Figure 2A) and percent of total release profiles (Figure 2B) of nicotine from the 3.5 mg on!® pouches with seven different flavor variants. The cumulative nicotine release profiles show that similar amounts of nicotine are released from the pouches (Figure 2B). The overlapping percent of total release profiles of nicotine indicate equivalency between the seven flavor variants of on!® pouch products at the 3.5 mg nicotine level. Moreover, the calculated f1 and f2 values demonstrated equivalency between these products (Table 2) using the on!® Mint nicotine pouches as a comparator. This shows that the flavor in the 3.5 mg on!® nicotine pouches do not influence the release profile of nicotine under our experimental conditions.

**Figure 1.** (**A**) Cumulative release and (**B**) percent of total dissolution release profiles of nicotine collected from Mint on!® pouches across all nicotine levels (n = 12) (Error Bars ± 1 SD).

**Table 1.** f1 and f2 values for on!® nicotine pouch comparisons across all nicotine levels for each flavor. The on!® 4 mg pouches for each flavor were used as the reference products for all comparisons.


#### **Table 1.** *Cont.*


**Figure 2.** (**A**) Cumulative release and (**B**) percent of total dissolution release profiles of nicotine collected from all flavored on!® pouches at 3.5 mg nicotine level (n = 12) (Error Bars <sup>±</sup> 1 SD).


**Table 2.** f1 and f2 values for on!® 3.5 mg nicotine pouch comparisons across all seven flavors. Mint on!® 3.5 mg nicotine pouches were used as a reference product for all comparisons.

*3.3. Comparison with Smokeless Tobacco and Other OTDN Pouch Products*

To better understand the release rates of on!® nicotine pouches and how they compare to traditional smokeless tobacco products and other OTDN pouch products, we compared the nicotine release profiles of on!® nicotine pouches to Skoal® Bandits and Skoal® pouches (commercially available traditional pouched smokeless tobacco products) and ZYN® nicotine pouches, another OTDN pouch products.

The on!® nicotine pouches are free of tobacco and do not have the same matrix content as the traditional pouched smokeless tobacco products. In addition, they are smaller in size and have a lower amount of nicotine per pouch compared to traditional pouched smokeless tobacco. Figure 3A shows the cumulative release profiles of nicotine from the Wintergreen flavored on!® 3.5 mg compared to Wintergreen flavored traditional smokeless tobacco pouch products, Skoal® Bandits and Skoal® pouches. The pouch weights for each product are 0.263 g, 0.72 g, and 1.55 g for on!®, Skoal® Bandits, and Skoal® pouches, respectively. The amount of total nicotine released (nicotine amount (mg) released) from Skoal® Bandits and Skoal® pouches as compared to the on!® product is attributed to the differences in nicotine concentration per pouch (Figure 3A). Despite the different pouch weight, nicotine concentration per pouch, and pouch composition, the percent nicotine released at each collection time point for the on!® and Skoal® Bandits pouches were found to be equivalent, as indicated by the overlapping release profiles (Figure 3B). However, the rate of nicotine release from the Skoal® pouches was found to be slower than on!® and Skoal® Bandits pouches (Figure 3B). In the profile region between zero and 20 min, a rapid dissolution was observed for on!® and Skoal® Bandits pouches, with a total percent release of 80% nicotine, whereas only 65% of the nicotine was released for Skoal® pouches. These observations were confirmed by calculating the f1 and f2 values. The calculated f1 and f2 values were 8.1 and 61.0 when comparing on!® to Skoal® Bandits, indicating equivalency between these products, and 21.1 and 46.0 when comparing on!® to Skoal® pouches, showing a difference in the nicotine release rates between these two products. These data illustrate that on!® nicotine pouches show similar or faster nicotine release profiles than the traditional pouched smokeless products tested here.

We also compared the performance of on!® to ZYN® pouches, a product marketed by Swedish Match North America. For this comparison, we selected the 3.5 mg and 8 mg on!® Mint, Wintergreen, and Coffee and 3 mg and 6 mg ZYN® Cool Mint, Wintergreen, and Coffee nicotine pouch products. As an example, Figure 4A shows the cumulative release profiles of nicotine from the 3.5 mg Mint on!® and 3 mg Cool Mint ZYN® pouches. Figure 4B shows the cumulative release profiles of nicotine from the 3.5 mg Wintergreen on!® and 3 mg Wintergreen ZYN® pouches. As expected, similar amounts of nicotine were released from both the on!® and the ZYN® products. The total release profiles of nicotine from the 3.5 mg on!® demonstrated a slightly slower release rate than the 3 mg ZYN® pouches (Figure 4C,D). However, when we calculate the f1 and f2 values, comparing on!® Mint to ZYN® Cool Mint, the nicotine release rate demonstrated equivalency, with f1 and f2 values of 9.2 and 60.1, respectively. In contrast, the on!® Wintergreen to ZYN® Wintergreen comparison resulted in a difference in the nicotine release rate with f1 and f2 values of 16.1 and 48.7, respectively. This observation was also seen when comparing other flavor variants and nicotine levels between on!® and ZYN® pouches (Table 3), indicating that the nicotine release profile and performance of these products could be similar but not necessarily statistically equivalent. Any measured and calculated differences between products within this OTDN category could be associated with inherent product characteristics (e.g., pouch paper and ingredients).

**Figure 3.** (**A**) Cumulative release and (**B**) percent of total dissolution release profiles of nicotine collected from Wintergreen flavored on!® 3.5 mg, Skoal® Bandits, and Skoal® pouches (n = 12) (Error Bars <sup>±</sup> 1 SD).



**Figure 4.** (**A**,**B**) Cumulative release and (**C**,**D**) percent of total dissolution release profiles of nicotine collected from Mint and Wintergreen on!® 3.5 mg and Cool Mint and Wintergreen ZYN® 3 mg pouches (n = 12) (Error Bars <sup>±</sup> 1 SD).

#### **4. Conclusions**

In this report, we evaluated the release profile of nicotine from 35 on!® nicotine pouch products, which are currently marketed in seven flavor variants with five different nicotine levels. Our data show similar nicotine release profiles among the thirty-five (35) on!® products. Factor of difference (f1) and factor of similarity (f2) calculations confirmed similar product performance for all products. Nicotine release rate was not dependent on flavor and nicotine levels. Furthermore, we showed similarities and differences in the nicotine release rate from on!® nicotine pouches when compared to a few selected traditional pouched moist smokeless tobacco and non-traditional ZYN® pouch products. We believe that the data presented will provide useful information for product characterization and product-to-product comparisons. In addition, the dissolution data provided herein could be used to support clinical studies and establish future in vitro/in vivo (IVIV) correlations.

**Author Contributions:** Conceptualization, F.A.; validation, F.A., N.M. and C.S.; formal analysis, F.A.; investigation, F.A., N.M. and C.S.; resources, J.H.M.; data curation; writing—original draft preparation, F.A.; writing—review and editing, F.A., J.H.M. and T.L.D.; visualization, F.A.; supervision, F.A., J.H.M. and T.L.D.; project administration, F.A., T.L.D.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are openly available in [repository name e.g., FigShare] at [doi], reference number [reference number].

**Acknowledgments:** The authors thank Mohamadi Sarkar, Jack Marshall, Christopher McFarlane, Karl Wagner, and James Skapars from Altria Client Services for the helpful discussions.

**Conflicts of Interest:** The authors declare no competing financial interest.

#### **References**

