Nicotine-related impurities in e-cigarette cartridges and refill e-liquids

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1 Journal of Liquid Chromatography & Related Technologies ISSN: (Print) X (Online) Journal homepage: Nicotine-related impurities in e-cigarette cartridges and refill e-liquids Jason W. Flora, Celeste T. Wilkinson, Kathleen M. Sink, Diana L. McKinney & John H. Miller To cite this article: Jason W. Flora, Celeste T. Wilkinson, Kathleen M. Sink, Diana L. McKinney & John H. Miller (2016) Nicotine-related impurities in e-cigarette cartridges and refill e- liquids, Journal of Liquid Chromatography & Related Technologies, 39:17-18, , DOI: / To link to this article: Published with license by Taylor & Francis Group, LLC Jason W. Flora, Celeste T. Wilkinson, Kathleen M. Sink, Diana L. McKinney, and John H. Miller Accepted author version posted online: 14 Dec Published online: 09 Jan Submit your article to this journal Article views: 1097 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at

2 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 2016, VOL. 39, NOS , Nicotine-related impurities in e-cigarette cartridges and refill e-liquids Jason W. Flora, Celeste T. Wilkinson, Kathleen M. Sink, Diana L. McKinney, and John H. Miller Research and Development, Altria Client Services, LLC, Richmond, Virginia, USA ABSTRACT The nicotine used in e-cigarettes and refill e-liquids is extracted from tobacco, and its purity can vary depending upon manufacturer and grade. The US and European Pharmacopoeias make recommendations for the purity of nicotine intended for pharmaceuticals; however, there is no official purity recommendation for nicotine used in e-cigarettes. To date, there are few published reports on nicotine-related impurities in e-cigarettes and refill e-liquids. The objective of this work was to develop a sensitive, selective, and robust analytical method for the quantitation of nicotine-related impurities in e-vapor products and to evaluate the nicotine-related impurities in a variety of commercial e-cigarette cartridges (n ¼ 10) and refill e-liquids (n ¼ 10). Nicotine-N-oxide, nornicotine, mysomine, and cotinine were observed to increase with time during stability studies. This method was also applied to estimate the transfer efficiency of nicotine-related impurities to the aerosol. Most of the impurities were observed to transfer efficiently. However, nicotine-noxides showed low transfer efficiency and demonstrated thermal degradation. This selective and sensitive method is suitable to provide quantitative data for risk assessments and for use in e-cigarette product and refill e-liquid stability studies as one of the stability-indicating measures. KEYWORDS e-cigarette; e-liquid; electronic nicotine delivery system; LC MS; nicotine; nicotine-related impurities GRAPHICAL ABSTRACT Introduction E-cigarettes, also known as e-vapor products and electronic nicotine delivery systems (ENDS), are an emerging product category in the global market. These products are available in a variety of configurations including small devices resembling cigarettes that are disposable or have rechargeable batteries with disposable cartridges or larger formats with rechargeable batteries and disposable prefilled or refillable tanks. When a user puffs on an e-cigarette, a liquid is heated, aerosolized, and inhaled. This liquid (often called e-liquid) typically contains propylene glycol and/or glycerin, water, nicotine, and flavors. The nicotine used in e-liquids is extracted from tobacco, and the purity of the extracted nicotine can vary depending upon manufacturer and grade (e.g., pharmaceutical). [1] Not only can nicotine extracts contain natural impurities such as other tobacco alkaloids, but they can also contain degradation products. [2,3] The US and European Pharmacopoeias make recommendations for the purity of nicotine intended for pharmaceutical products, but no official purity recommendation for the nicotine used in e-cigarettes and refill e-liquids has been made. [4,5] US Pharmacopeia (USP)-grade nicotine requires single impurities to be less than 0.5% (5 mg/g) and total impurities to be less than 1% (10 mg/g). [4,6] Nicotine impurities are specified in the European Pharmacopoeia monograph 1452 (Figure 1) as nicotine-n-oxides, cotinine, nornicotine, anatabine, myosmine, anabasine, and β-nicotyrine. [5] CONTACT Jason W. Flora Jason.W.Flora@altria.com Altria Client Services, LLC, 615 Maury Street, Richmond, VA 23224, USA. Color versions of one or more of the figures in this article can be found online at Published with license by Taylor & Francis Group, LLC Jason W. Flora, Celeste T. Wilkinson, Kathleen M. Sink, Diana L. McKinney, and John H. Miller This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

3 822 J. W. FLORA ET AL. Figure 1. Specified nicotine degradation products and natural impurities from the European Pharmacopoeia monograph [5] The United States Food and Drug Administration (FDA) recently issued the final rule to deem ENDS to be subject to the Federal Food, Drug, and Cosmetic Act, as amended by the Family Smoking Prevention and Tobacco Control Act. This provides FDA authority to regulate e-cigarettes and e-liquids (among other tobacco products). [7] FDA also recently published draft guidance on the premarket tobacco product application for ENDS. [8] Within this guidance, FDA specifies the constituents that should be measured in e-liquids and aerosols. Anabasine is the only nicotine impurity specified. However, FDA states that in addition to the list provided in the draft guidance, manufacturers should also measure other toxic chemicals contained within the product or delivered by the product, such as a reaction product from leaching or aging and aerosol generated through the heating of the product. [8] To date, there are a few published reports on the nicotinerelated impurities in e-cigarettes and refill e-liquids. [1,3,6,9,10] Trehy et al. [9] investigated the nicotine-related impurities in e-cigarette cartridges, refill e-liquids, and the aerosol from products made by four e-cigarette manufacturers and found that the nicotine-related impurities varied among e-cigarette manufacturers. Lisko et al. [6] also looked at some of the nicotine-related impurities (alkaloids) in e-cigarette cartridges and refill solution and found that in some cases these minor alkaloids exceeded the USP limits. Etter et al. [1] observed that nicotine-related impurities in refill e-liquids from several commercially available products varied greatly and in some cases were well above 0.5% (the USP limit) of the nicotine content for individual specified impurities. Etter et al. was the first to publish quantitative results for all the nicotine-related impurities specified in the European Pharmacopoeia (Figure 1) for the e-vapor product category. [5] While Lisko et al. used gas chromatography with tandem mass spectrometry detection (GC/MS/MS) for alkaloid analysis, both Trehy et al. and Etter et al. conducted their analysis using liquid chromatography (LC) with ultraviolet (UV) detection similar to methods found in the pharmacopeias for the analysis of nicotine-related substances. [4 6] GC/MS techniques are typically very sensitive and selective, however, quantitation is difficult for thermally unstable compounds. LC with UV detection is a robust detection method, but it has limited sensitivity and can potentially be subject to interferences from flavor compounds. Medana et al. [3] recently published an LC approach using mass spectrometry (MS) detection for the analysis of nicotine, cotinine, nitrosonornicotine (NNN), nornicotine, myosmine, and anabasine in commercial e-liquids sold in Italy, China, Poland, and Germany. Using this method, they observed that NNN was below their detection limit, the nicotine-related impurities were below 0.3% of the nicotine concentration, and nicotine concentrations were not consistent with values listed by the manufacturer. [3] Another recent publication discussed the characterization of potential impurities and degradation products in e-liquids and aerosols. [10] The work included nicotine-related impurities measured by LC MS in commercially available MarkTen 1 e-cigarettes. In the study, all specified nicotine-related impurities were less than 0.3% of the nicotine concentration. [10,11] The research described herein builds upon those previous observations as related to nicotine-related impurities in e-cigarette cartridges and refill e-liquids by providing a comprehensive description of a sensitive and highly selective method using LC MS for the quantitative analysis of specified nicotine-related impurities. This method was fully validated following International Council for Harmonization (ICH) guidelines for quantitative analysis of all seven nicotine-related impurities listed in the European Pharmacopeia for e-cigarettes and refill e-liquids (Figure 1). [5,12] An objective of this work was to provide an evaluation of nicotine-related impurities in commercially available e-cigarette cartridges, prefilled tanks, and refill e-liquids of varying age and packaging configurations to demonstrate that the method is fit for purpose. This method can be used to evaluate both e-cigarettes and refill e-liquids for quality control studies and during stability studies, where nicotine-related impurities are considered stability-indicating measures. This method was also applied to estimate the transfer efficiency of nicotine-related impurities to the aerosol.

4 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 823 Experimental Test products Disposable e-cigarette cartridges and refill e-liquids used for this investigation were commercially available products, which were either purchased at retail locations or ordered from the Internet. The 20 test products (Table 1) included refill e-liquids, disposable prefilled tanks, disposable e-cigarette devices, and disposable e-cigarette cartridges from multiple manufacturers. Test products included commercial MarkTen 1 XL e-cigarettes (Nu Mark LLC, Richmond, VA), disposable cartridges with flavors Classic (2.4% nicotine by weight (NBW); product 18), and Menthol (3.5% NBW; product 19). All products contained propylene glycol and/or glycerin and water as the primary formulation components (determined by laboratory analysis, data not included). Nicotine levels for all test products ranged from 1.2 to 4.8% NBW, as listed by the manufacturer. Product packaging varied across manufacturers, as did the inclusion of shelf-life information such as sell by dates, expiration dates, and do not use after dates; only 12 of the 20 test products provided any shelf-life information. Only products 9 and 11 contained production dates on their labels stating and , respectively. All products were tested in January, Nicotine-related impurities method This method was developed and validated to quantitatively determine the amounts of nicotine-n-oxides (cis and trans; CAS # ), cotinine (CAS # ), nornicotine (CAS # ), anatabine (CAS # ), myosmine (CAS # ), anabasine (CAS # ), and β-nicotyrine (CAS # ) in e-cigarettes and refill e-liquids containing propylene glycol and/or glycerin, water, flavors, and tobacco-derived nicotine using LC MS. This method uses four deuterium-labeled internal standards to enhance method accuracy and robustness. Target analytes measured by this method, along with their retention times and internal standard assignments, are compiled in Table 2; molecular structures of the analytes are shown in Figure 1. All standard materials were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Some nicotine-related impurities such as nicotine-n-oxides are known to be thermally unstable, which can prevent the use of gas chromatography (GC) for this analysis. [13] Method validation was conducted consistent with ICH Guideline Q2(R1). [12] The limit of quantitation (LOQ) for this method is considered the lowest concentration of the calibration standard, which varies by analyte. The LOQs were confirmed by creating reference standards that produced a minimum signal-to-noise ratio of 10. The limits of detection (LODs) values were estimated by dividing the empirically derived LOQs by 3. The calibration ranges, LOQs, and estimated LODs for the prepared samples are shown in Table 3. Sample preparation refill e-liquids A 100-mg sample of the refill e-liquids was weighed directly into a 2-mL amber autosampler vial, and 1 ml of extraction solution was added to the vial. The extraction solution consisted of 70:30 methanol:water containing 2.0 µg/ml of each deuterated internal standard listed in Table 2. Vials were capped and vortexed for approximately 20 s. A 1-µL injection was analyzed by LC MS, as described in Liquid chromatography and Mass spectrometry. Sample preparation disposable cartridges and prefilled tanks E-cigarette cartridges were detached from the battery, if necessary. Cartridges were placed in 2-mL microcentrifuge tubes with the mouthpiece orifice facing downward. Cartridges were centrifuged at 4800 rpm ( 1000 g) for 30 s to collect the e-liquid in the conical section of the microcentrifuge tubes. Prefilled tank models were disassembled and prefilled e-liquid was aspirated using a glass transfer pipette. In both cases, a 100-mg sample of the e-liquid was weighed directly into a 2-mL amber autosampler vial and 1 ml of extraction solution was added to the vial. The extraction solution consisted of 70:30 methanol: Table 1. Test product types, packaging, and shelf-life information. Product number Product type NBW (%) Shelf-life information a Product packaging 1 Refill e-liquid 1.2 NA Glass bottle with plastic overwrap 2 Refill e-liquid 1.2 NA Glass bottle with plastic overwrap 3 Refill e-liquid Plastic bottle with plastic overwrap 4 Refill e-liquid Plastic bottle with plastic overwrap 5 Refill e-liquid Glass bottle 6 Refill e-liquid Glass bottle 7 Refill e-liquid 1.2 NA Glass bottle with plastic overwrap 8 Refill e-liquid 1.2 NA Glass bottle with plastic overwrap 9 Refill e-liquid Plastic bottle with plastic overwrap 10 Refill e-liquid Plastic bottle with plastic overwrap 11 Disposable prefilled tank Blister pack with rubber mouthpiece cover 12 Disposable prefilled tank 5.0 NA Blister pack with plastic backing 13 Disposable prefilled tank Plastic box with rubber or plastic endcaps 14 Disposable device Plastic box with rubber or plastic endcaps 15 Disposable device Plastic box with rubber or plastic endcaps 16 Disposable device 4.5 NA Plastic box with plastic overwrap 17 Disposable cartridge 4.8 NA Blister pack with foil backing 18 Disposable cartridge Blister pack with foil backing 19 Disposable cartridge Blister pack with foil backing 20 Disposable cartridge 1.6 NA Plastic overwrap, blister with foil backing NBW, nicotine by weight; NA, not available. a Shelf-life information included Expiration, Sell By, or Do Not Use After dates on the packaging.

5 824 J. W. FLORA ET AL. Table 2. Analyte Stable isotope-labeled internal standards for each target analyte. Analyte retention time (min) Internal standard Internal standard retention time (min) Myosmine 4.61 Myosmine-d Nornicotine 4.10 Nornicotine-d Cotinine 2.94 Cotinine-d Anabasine 5.24 Anabasine-d Nicotine-N-oxide 1.39 Cotinine-d Anatabine 4.59 Myosmine-d β-nicotyrine 6.58 Myosmine-d Table 4. LC gradient for quantitative analysis of nicotine-related impurities. Mobile phase a (%) Time (min) A B Flow rate (ml/min) Gradient curve N/A a Mobile phase A was 10 mm ammonium acetate, ph 10.0; mobile phase B was methanol. water containing 2.0 µg/ml of each deuterated internal standard listed in Table 2. Vials were capped and vortexed at 1500 rpm (single-tube manual vortexer) for approximately 20 s. A 1-µL injection was analyzed by LC MS as described in Liquid chromatography and Mass spectrometry. Sample preparation aerosol analysis Aerosol was collected as described in Aerosol collection. The glass wool Cambridge filter pad (CFP) containing captured aerosol was removed from the filter holder and placed into a 20-mL glass vial, and 10 ml of extraction solution was added to the vial. The extraction solution consisted of 70:30 methanol:water containing 2.0 µg/ml of each deuterated internal standard listed in Table 2. The glass vials were capped, and the pad was extracted by vortexing at 1800 rpm (multitube vortexer) for approximately 30 min. A 1-µL injection was analyzed by LC MS as described in Liquid chromatography and Mass spectrometry. Liquid chromatography Liquid chromatography was conducted on a Waters ACQUITY 1 UPLC instrument (Milford, MA) with a Waters Acquity X-Bridge C18 (2.5 µm) mm 2 column. Mobile phase A and the weak wash solvent were 10 mm ammonium acetate (ph 10) and mobile phase B and the strong wash solvent were Optima TM -grade methanol (Fisher Scientific, Pittsburgh, PA). The analytical column selected is stable over an extended ph range, which allows for resolution of the anabasine and nicotine peaks. Sample temperature was held at 15 C, and column temperature was at ambient conditions. LC mobile phase flow rate was set to 0.3 ml/min. LC conditions are shown in Table 4. Mass spectrometry All nicotine-related impurities analyses were conducted using a Waters Quattro Micro Mass spectrometer (Milford, MA) in positive electrospray ionization mode with the conditions listed in Table 5. The nicotine-related compounds were detected in Table 3. Calibration range, limits of quantitation (LOQs), and estimated limits of detection (LODs) for each target analyte using LC MS. Analyte Calibration range (µg/ml) LOQ (µg/g of formulation) LOD (µg/g of formulation) Myosmine Nornicotine Cotinine Anabasine Nicotine-N-oxide Anatabine β-nicotyrine the LC eluent using multiple reaction monitoring (MRM) at the settings shown in Table 6. Product ions were not used for myosmine, β-nicotyrine, and anabasine because they exhibited greater signal-to-noise as molecular ions. The acquisition span was set to 0 Da, the interchannel delay time was set to 0.01 s, and the interscan delay time was set to 0.02 s. The MS stores the acquisition between 0 and 8 min of the LC run. Aerosol collection All aerosol collections were conducted under International Organization for Standardization smoking environmental conditions [14] with temperature at C and relative humidity at 60 5%. Aerosol collection was performed using a square wave puff profile, 825 ml/min volumetric air flow, ml puff volume, s puff duration, and s puff interval. Before collecting aerosol from the test products, the collection apparatus was evaluated for breakthrough of analytes by including a sorbent tube to capture any analytes that may pass through the filter pad. The collection train consisted of a 44-mm CFP followed by a XAD-4 polymeric sorbent tube (7 70 mm 2 ) from SKC Inc. (Eighty Four, PA). The XAD-4 sorbent tube with 80/40 mg bed was selected, because it is recommended for use in the NIOSH method 2551 for sampling of airborne nicotine. Aerosol from the two models of MarkTen 1 XL e-cigarettes was collected from six replicates per model, with freshly charged batteries on the e-cigarettes. A linear 5-port smoking machine (KC Automation, Richmond, VA) was used to acquire 100 puffs from each cartridge onto a 44-mm glass wool CFP. Three clearing puffs were taken following the final puff of the collection. Masses of the e-cigarette, the filter pads, and the sorbent tubes were measured before and after aerosol collection. As this was an estimate of transfer efficiency, this extraction procedure was not fully validated. No breakthrough was observed in the sorbent tubes. Nicotine in e-cigarettes and refill e-liquids This method was developed and validated to quantitatively determine the concentration of nicotine (CAS# ) in Table 5. Mass spectrometer parameters used for quantitative analysis of nicotine-related impurities. Parameter Setting Source temperature 110 C Desolvation temperature 375 C Desolvation gas (nitrogen) 700 L/h Cone gas (nitrogen) 30 L/h Electrospray voltage 3.50 kv

6 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 825 Table 6. Multiple reaction monitoring settings used for quantitative analysis of nicotine-related impurities. Analyte Precursor ion (m/z) Product ion (m/z) Cone voltage (V) Collision energy (V) Myosmine Nornicotine Myosmine-d Nornicotine-d β-nicotyrine Anatabine Anabasine Anabasine-d Cotinine Nicotine-N oxide Cotinine-d e-cigarettes, refill e-liquids, and aerosols. For e-liquid preparation, 250 mg of formulation was added to 10 ml of n-propanol containing quinoline (Acros) as the internal standard and shaken for 10 s. The e-cigarette extracts were analyzed by GC with a flame ionization detector. The LOQ was 2.0 mg/g formulation; LOD was not determined. When manufacturers provided nicotine concentrations on a weight per volume basis, the concentration was converted based upon the density of the formulation. Density was determined by the measured propylene glycol, glycerin, and water content of the formulations (data not provided). All densities were less than 1.16 g/ml. Stability study protocol A stability study was conducted on the commercial MarkTen 1 XL e-cigarettes (products 18 and 19) to determine their shelflife before commercialization. The stability study involved testing three production lots in their final commercial packaging. Each production lot is defined as a formulation that is produced and filled on different production lines or produced on different production lines and filled on the same line with a changeover performed between lots. As a part of the stability study, the analytical test method detailed herein was utilized to measure all specified nicotine-related impurities at 1, 2, 4, and 6 months after production. During the study, all products were stored under ICH guideline for Stability Testing of New Drug Substances and Products under the long-term storage conditions (25 2 C and 60% RH 5%). [15] Three replicates were analyzed for each sample. of all other sample matrix components. Samples were fortified (n ¼ 3) at 2 µg/ml for all analytes and accuracy ranged from to %. The %RSDs for all analytes extracted from cartridges ranged from 1.43 to 12.37%. Instrument precision was evaluated by a single aliquot of a calibration standard (0.10 µg/ml) analyzed 10 times. The %RSD for the 10 replicate injections was between 3.70 and 11.3% for all analytes. Three individually prepared samples of e-liquids and cartridge samples were analyzed to determine repeatability. The %RSDs for the three preparations of each sample matrix was between 0.75 and 14.68%. Nine individually prepared samples (3 per day) of e-liquids and cartridge samples were analyzed over three days to determine intermediate precision. The %RSDs were between 2.28 and 13.09%. Method specificity was evaluated using three unfortified e-liquids and three laboratory-fortified samples. Fortification of target analytes was conducted at two different concentration levels (1 and 2 µg/ml). For cartridges, fortification of target analyte was conducted at one concentration level (2 µg/ml). No matrix interference for target analytes was observed. Figure 2 provides an example of the chromatograph obtained for an e-liquid sample using this methodology. Sample extract stability was evaluated at 0 and 72 h at refrigerated conditions (0 4 C) using sample extracts of refill e-liquids and disposable cartridge samples. Three replicates of each sample matrix were analyzed. The percent difference in results between 0 and 72 h was within 10%. Stock stability was evaluated by preparing fresh solutions containing all the analytes from old stocks prepared approximately 1 5 months from the control fresh stock. Stock stability for up to 5 months was determined for all analytes by verification within 15% of the control fresh stock solutions. Intermediate and calibration standards stability were also evaluated for intermediate and calibration solutions prepared approximately 6 7 months (stored at 20 C) from the fresh control solutions. Stability for up to 7 months was determined for intermediate solutions by verification within 14% of the fresh control solutions. Calibration standard stability for up to 6 months was determined for all analytes by verification within 10% of the fresh control solutions. System suitability was assessed during method validation to ensure that instrument performance was acceptable. For this Results and discussion The working range of the calibration model was demonstrated by a quadratic calibration plot using six standards covering the method dynamic range with a 1/X fit. The coefficient of determination (R 2 ) was for all analytes. To determine the accuracy of the method for e-liquids, samples were fortified at two different concentration levels (1 and 2 µg/ml) for all analytes. The mean accuracy for all analytes evaluated at two different fortification levels (n ¼ 3) in e-liquids ranged from to % and the %RSDs for all analytes ranged from 0.81 to 12.85%. Fortification of cartridge samples was performed at one level to determine if the analytical method accurately measures the analyte concentration in the presence Figure 2. Chromatography for myosmine detected in MarkTen 1 Classic formulation with 2.4% nicotine by weight (product 18).

7 826 J. W. FLORA ET AL. analysis, the system suitability criteria were based on detector response (area) and resolution between the anabasine and nicotine peaks. Anabasine and nicotine peaks elute were close to each other and share the same parent ion. The coelution of anabasine and nicotine has been previously observed when following the USP method. [9,16] To ensure that acceptable signal-to-noise ratio would be obtained for the LOD for analysis of samples, a criterion of 50% of the response was set for the lowest calibration standard. In addition, the resolution between anabasine and nicotine peaks should meet a minimum resolution of 0.79 to have separation between these analytes. Resolution criterion was calculated as follows: Retention time nicotine retention time anabasine Resolution ¼ Peak end time anabasine peak start time anabasine ðminþ ðminþ ðminþ ðminþ It is recommended that system suitability be checked against this specification before analysis to ensure that the system performance meets the intended purpose. To evaluate if the method was fit for purpose, a variety of commercial products were tested in January, These products included disposable cartridges and refill e-liquids that were commercially available and either purchased at retail locations or ordered from the Internet and are shown in Table 1. Test samples included commercial MarkTen 1 XL e-cigarettes (Nu Mark LLC, Richmond, VA), disposable cartridges with flavors Classic with 2.4% nicotine by weight (NBW) (product code 18), and Menthol with 3.5% NBW (product code 19). All products contained propylene glycol and/or glycerin and water as the primary formulation components. Nicotine levels ranged from 1.2 to 4.8% NBW as reported on the packaging or manufacturer s website. Product packaging varied across manufacturers as did the inclusion of shelf-life information such as sell by dates, expiration dates, and do not use after dates. Total percent nicotine calculations were based upon the nicotine levels reported by the manufacturers (e.g., label claim), and nicotine was quantitatively measured for all samples to confirm these levels. Nicotine levels in e-vapor products have been previously discussed in the scientific literature where the values reported by the manufacturers are not always consistent with the levels measured in the formulations. For example, Goniewicz et al. [17,18] showed that nicotine values for nine of the 20 commercially available e-cigarette cartridges tested differed by more than 20% from those reported by the manufacturers. The same phenomenon was also observed for three of 15 refill e-liquids. [17,18] An earlier study by Trehy et al. [9] observed that nicotine content labeling issues were also present with most of the products they tested. While some studies have reported nicotine levels higher than label claims (e.g., Trehy et al. [9] and Farsalinos et al. [19] ), consistent with Lisko et al., [6] for the samples measured in this study, all nicotine values were lower than reported values. Laugesen et al. [20] also observed differences between labeled and measured nicotine concentration, and they concluded that these differences were due to a lack of quality control. However, it should also be considered that in some situations, differences may simply be due to product age and/or poor packaging and storage conditions. It is assumed that the age of the products used in our study varied considerably as only two products contained production dates on their packaging. Only 12 of the 20 products tested were sold with shelf-life information (Table 1); four of the 12 products showed expiration dates that had passed at the time of testing. Of the 20 products tested in this study (Figure 3), six contained nicotine between 90 and 100% of their reported value, four of which contained shelf-life information on their packaging. One of the products (product 10) with 92% of its label claim contained shelf-life information on its packaging and had expired for approximately 6 months. Nicotine contents in 11 of the products tested were between 70 and 90% of the labeled nicotine concentration. Six of these 11 products contained shelf-life information, and only one product had expired by less than 1 month before testing (product 11). Three of the 20 products tested contained less than 50% of the nicotine label claim; two of them had shelf-life information showing that they had expired almost 2 years before analysis (products 14 and 15). Although these discrepancies may be a result of poor quality control among the commercial products Figure 3. Labeled and measured nicotine content in refill e-liquids (products 1 10) and disposable e-cigarettes and cartridges (products 10 20). Blue bars show the reported nicotine levels, and orange bars show the average measured values. *Products 18 and 19 are MarkTen 1 e-cigarettes manufactured by Nu Mark LLC.

8 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 827 Figure 4. Average concentrations of specified nicotine-related impurities in refill e-liquids (products 1 10) and disposable e-cigarettes, cartridges, and prefilled tanks (products 10 20), represented as a percent of the total reported nicotine content. *Products 18 and 19 are MarkTen 1 e-cigarettes manufactured by Nu Mark LLC. in this category, it should also be considered that nicotine loss through evaporation, adsorption into the container material (cartridge or refill bottle), or degradation may also account for values measured below the label claim. For most commercial products tested, specified nicotine degradation products and natural impurities in the e-cigarette cartridges and refill e-liquids were below 0.5% of the total nicotine concentration. [11] Figure 4 shows the specified nicotine-related impurities for all 20 products tested. The plots have been normalized to a scale maximized at 0.8% of the total nicotine content as the highest measured value was 0.77% for nicotine-n-oxides in product 15. For myosmine, anabasine, β-nicotyrine, cotinine, and nornicotine, all measured values were equal to or less than 0.2% of the labeled nicotine content where many were well below 0.1%. Products 10, 13, and 20 had relatively high average anatabine levels at 0.61, 0.49, and 0.44% of the labeled nicotine content, respectively. It had been previously observed that e-cigarettes from some manufacturers contain elevated levels of anatabine and may be associated with the purity and source of nicotine used. [9] For nicotine-n-oxides levels, six of the 20 products contained levels greater than the identification threshold of 0.5% proposed in ICH Guideline Q3B(R2) and the USP maximum for a single impurity [4,11] (products 3, 4, 10, 14, 15, and 17). It is not surprising that high levels were observed in products 14 and 15 as they were almost 2 years past their reported expiration dates, and nicotine-n-oxides are a known oxidation product of nicotine. Product 10 was also expired, and it was tested approximately 6 months after its labeled shelf-life. Products three and four were refill e-liquids that were well within their labeled shelf-life with more than 5 months left until expiration. Product 17 had no shelf-life information on its packaging. Products 10, 13, 14, 15, and 20 also had total specified impurities exceeding 1.0% of the total nicotine content. Products 10, 14, and 15 had expired while product 13 was within its shelf-life and product 20 had no shelf-life information. [4] Consumer packaged goods are typically sold with some indication of product shelf-life printed on the packaging. These shelf-lives are determined by stability studies with endpoints defined by both sensory evaluations for consumer acceptability and biological and chemical profiling to determine if any safety issues arise during these studies. Pharmaceutical products are extensively evaluated in rigorous stability studies to provide evidence on how the quality, safety, and efficacy of the drug product varies with time. [15] Potential degradation products and impurities that arise from exposure and interactions with the closure system are monitored during these stability studies. The nicotine degradation products and impurities measured in this method are adapted from previous investigations on medications used for nicotine replacement therapy (NRTs). [4,5] The primary oxidation products of nicotine are nicotine-n-oxides and cotinine. [2] Pharmacopeia guidelines have specified the nicotine-related impurities that should be measured in NRTs as nicotine-n-oxides, cotinine, nornicotine, anatabine, myosmine, anabasine, and β-nicotyrine. [5] It is unknown how most e-cigarette and e-liquid manufacturers determine the shelf-life of their products. For Figure 5. Nicotine-related impurities during long-term storage shown on a percent total nicotine basis for MarkTen 1 XL Classic with 2.4% nicotine by weight (product 18). Three replicates were made per data point.

9 828 J. W. FLORA ET AL. Table 7. Average (n ¼ 6) estimated transfer percent in aerosol for commercial MarkTen 1 XL e-cigarettes with flavors Classic with 2.4% nicotine by weight (NBW) (product 18) and Menthol with 3.5% NBW (product 19). Myosmine Nornicotine Cotinine Anabasine Nicotine-N-oxides Anatabine β-nicotyrine Markten 1 Classic MarkTen 1 Menthol eight of the 20 products tested in this study, no shelf-life information was provided for the commercial e-cigarettes and refill e-liquids. It is highly recommended that appropriate stability-indicating measures be monitored during rigorous stability studies from which product shelf-life can be determined. The methods should be validated based upon the ICH Guideline Q2(R1). [12] For ENDS products, one such stability-indicating measure should be the specified nicotinerelated impurities discussed in this report along with other appropriate extractables and leachables. Studies should follow ICH Guideline Q1A(R2). [15] Stability studies were conducted on the commercial MarkTen 1 XL e-cigarettes (products 18 and 19) to determine their shelf-life before commercialization. One of the stabilityindicating measures was quantification of the nicotine-related impurities discussed in this report. Under ICH long-term storage conditions (25 2 C and 60% RH 5%) it was observed that all specified impurities remained at levels <0.2% of the total reported nicotine concentration after 6 months. [15] There were no measurable increases of anabasine, β-nicotyrine, or anatabine, whereas slight increases in nicotine-n-oxides, cotinine, myosmine, and nornicotine were observed as shown in Figure 5. The method discussed herein is a targeted approach to quantitatively measure specified nicotine-related impurities and degradation products by LC MS. Measuring these compounds using MRM affords considerable advantages in sensitivity and selectivity. However, this method does not address the identification of unknown compounds. It is recommended that additional nontargeted methodologies be used to evaluate potential unknown compounds that may be formed during stability studies, using GC for volatile and semivolatile compounds and LC for nonvolatile compounds. The previous investigation by Trehy et al. attempted to evaluate nicotine-related impurities in e-liquids and aerosols collected from three commercial e-cigarettes. They observed that in the e-liquid, the nicotine-related impurities varied between e-cigarette manufacturers; however, the LC method with UV detection (diode array) was not sensitive enough to measure the analytes in the aerosol. [9] As the method discussed herein affords the sensitivity to measure low levels of nicotinerelated impurities in the aerosol, the transfer efficiency was estimated for all of the nicotine-related impurities. Estimating transfer efficiency is an important part of understanding the potential exposure to ENDS users. Table 7 shows the estimated transfer efficiency for the nicotine-related impurities detected in the MarkTen 1 XL products included in this study. The estimations were based upon a comparison of all nicotine-related impurity concentrations in the e-liquid and the collected aerosol. Myosmine, nornicotine, cotinine, anabasine, and anatabine estimated transfer efficiencies ranged from 80 to 110%. Nicotine-N-oxide transfer efficiencies were less than 10%, and β-nicotyrine ranged from 141 to 164%. Nicotine-N-oxides are known to be thermally unstable, which prevents the use of standard GC MS methods for quantitative analysis. To investigate the thermal decomposition pathway of nicotine-n-oxides, a standard solution was injected into a GC MS system (injection port temperature set to 260 C), where it was observed that primary thermal decomposition pathways were conversion to nicotine and β-nicotyrine, thus accounting for the high estimated transfer efficiency of β-nicotyrine ( %) shown in Table 7. Decomposition pathways for the other nicotine-related impurities were not investigated. Conclusions Results demonstrate that concentrations of nicotine-related impurities in commercial e-liquids and e-cigarette devices vary. Factors affecting these concentrations can range from purity of the original nicotine used in the e-liquids, packaging factors such as oxygen transfer rate, product age, and storage conditions. Most of these impurities were below 0.3% of the labeled nicotine levels. However, for select products, levels were observed as high as 0.77% for nicotine-n-oxides (seen in product 15). It was also demonstrated that the nicotine-related impurities that increase under long-term storage conditions include nicotine-n-oxides, cotinine, myosmine, and nornicotine. Transfer efficiency to the aerosol was also estimated with most impurities transferring near 100%. Nicotine-N-oxides were shown to thermally degrade to primarily nicotine and β-nicotyrine during the aerosol formation. Nicotine levels were also evaluated in the commercial products to confirm label claims and, consistent with previously published results, inconsistencies were observed for some products. [6,9,18,19,21,22] Most products were within 30% of the label claim, while others exceeded 50%. Using the targeted method described herein using MS with MRM, it was possible to achieve the sensitivity necessary to detect most of the nicotine-related impurities in all the commercial products tested. This selective and sensitive method is suitable to provide quantitative data for risk assessments and for use in e-vapor product and refill e-liquid stability studies as one of the stability-indicating measures. Acknowledgment The authors acknowledge the editorial assistance of Eileen Y. Ivasauskas of Accuwrit Inc. References [1] Etter, J. F.; Zather, E.; Svensson, S. Analysis of Refill Liquids for Electronic Cigarettes. Addiction 2013, 108(9), [2] Smyth, T. J.; Ramachandran, V. N.; McGuigan, A.; Hopps, J.; Smyth, W. F. Characterisation of Nicotine and Related Compounds Using

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