Final Report on FDA Analyses

The final report on the initial FDA Analyses of Electronic Cigarettes.

Date: May 4, 2009 To: Michael Levy, Supervisor Regulatory Counsel, CDER, Office of Compliance, Division of New Drugs and Labeling Compliance

From: B.J. Westenberger, Deputy Director, CDER/OPS/OTR, Division of Pharmaceutical Analysis

Subject: Evaluation of e-cigarettes

Background: The Center for Drug Evaluation and Research through the Office of Compliance (OC) has requested that the Division of Pharmaceutical Analysis (DPA) evaluate two brands of electronic cigarettes (e-cigarettes) for nicotine content and other impurities. An e-cigarette is advertised as an alternative to smoked tobacco products. It is a battery-powered device that provides inhaled doses of nicotine by delivering a vaporized propylene glycol/nicotine mixture as shown in Figure 1. The Center is concerned that in addition to nicotine delivery, the vapor may also provide other potentially harmful volatile components. DPA was asked to quantitate the amount of nicotine present in each brand and to evaluate each brand for the presence of tobacco specific nitrosamines (TSNA), certain tobacco specific impurities, ethylene glycol (EG) and diethylene glycol (DEG).

Test Products:

• Njoy e-cigarette (Reference 1) with various cartridges
• Smoking Everywhere Electronic Cigarette (Reference 2) with various cartridges
• Nicotrol Inhaler, 10mg cartridge was used as a control for some test methods

Conclusions: Nicotine is present in both products. The Smoking Everywhere Electronic Cigarette cartridges listed as containing no nicotine in some cases had very low amounts of nicotine present. Tobacco specific nitrosamines and tobacco specific impurities were detected in both products at very low levels. DEG was identified in one cartridge, Smoking Everywhere 555 High. See Table 1 for results of analyses of entire cartridges after extraction. A sparging apparatus (see figure in Attachment A) and headspace GC (HS-GC) analysis were utilized to simulate actual use of these products. With the sparging apparatus, nicotine was detected in cartridges claiming to contain nicotine and quantitated by LC-UV; cotinine was also found in some products by this procedure. Repeat testing of 3 different cartridges with the same label (menthol high) gave varying results from 26.8 to 43.2 mcg nicotine/100 mL puff. HS-GC detected nicotine in both products and ?-Nicotyrine was detected in all Njoy cartridges (see Table 2).
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Experimental: See Attachment A for further experimental details.
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Nicotine content was analyzed by HPLC-UV and GC-MS. Quantification was done by HPLC-UV using two different extractions: a methanol extraction, and a 10% acetonitrile/ 1% phosphoric acid in water extraction.
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Tobacco specific impurities and diethylene glycol were analyzed by GC-MS.
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Diethylene glycol presence was confirmed with proton NMR.
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Detection of nicotine and tobacco specific impurities during use of these products was estimated by simulating use temperatures and analyzing volatiles using head space GC-MS (HSGC-MS) and utilizing a sparging apparatus (see figure in attachment).

Results and Discussion:

• Whole Cartridge: Nicotine content by HPLC-UV Results, similar for both methanol extraction and 10% acetonitrile/1% phosphoric acid in water extractions, are shown in Table 1.
• Whole Cartridge: Tobacco Specific Nitrosoamines by LC-MS/MS
  The four major TSNAs include: N-nitrosonicotine (NNN), N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were analyzed for using LC-MS/MS and the results are shown in Table 1.
• Whole Cartridge: Tobacco Specific Impurities by GC-MS and GC-MS/MS
  Nicotine was detected in both products in all cartridges including samples identified as containing no nicotine. Samples were screened for possible tobacco specific impurities: cotinine, nicotine-N-oxide, nornicotine, anatabine, anabasine, pseudooxynicotine, myosmine, ?-nicotyrine, and 1-methyl-3-nicotinoylpyrrolidine (MNP). Nicotine-N-oxide, nornicotine, anatabine, pseudooxynicotine and MNP were not observed in any of the samples. Results from cotinine, anabasine, myosmine, and ?-nicotyrine are shown in Table 1.
• Whole Cartridge: Diethylene Glycol by GC-MS
  Diethylene Glycol was detected in one sample (Smoking Everywhere 555 High cartridge) at approximately 1%.

Simulated Use: Nicotine and tobacco Specific Impurities by Head Space GC-MS (HSGC-MS) and sparging apparatus
HSGC is likely to be less sensitive than the GC-MS technique that takes advantage of injecting all of the soluble components and then volatilizing them at 280 °C; however, the head space analyzer can be set to a specific temperature to mimic what may be volatilized during use of the products.

The temperature of the heating element in each e-cigarette was determined by inserting a thermocouple and then activating the e-cigarette by drawing air through it. These temperatures ranged from 40 to 65°C. HSGC-MS analysis was conducted at 60°C to simulate the temperature that would be encountered during activation of an e-cigarette. Nicotine was detected in both products for all cartridges containing low, medium and high levels of nicotine but was not observed in cartridges identified as containing no nicotine. Screening for the possible tobacco specific impurities cotinine, nicotine-N-oxide, nornicotine, anabasine and myosmine was negative. ?-Nicotyrine was detected in all Njoy cartridges but was not detected in the Smoking Everywhere cartridges. The sparging apparatus was used to quantify the amount of nicotine released during use of these electronic cigarettes (Table 2). Levels found were consistent with the labeling (low, medium and high); however, the cartridge labeled “no” still delivered some nicotine. The cartridges labeled “high” delivered more nicotine than the approved Nicotrol product. Repeat testing of 3 different cartridges with the same label (menthol high) gave varying results from 26.8 to 43.2 mcg nicotine/100 mL puff.

References:
(1) http://www.njoythefreedom.com/
(2) http://www.smokingeverywhere.com/
(3) C.N. Man, L.H. Gam, S. Ismail, R. Lajis, R. Awang, J. Chromatogr. B 844 (2006) 322–327.
(4) S. S. Yang et al., J. Chromatogr. A, 942 (2002) 33-39.
(5) USP 31/NF 26, Official 12/1/08-4/30/09 NF Monographs: Diethylene Glycol Monoethyl Ether: Assay; pg 1126

See Attached PDF for tables and the full report