BAT Science - Methods for measuring constituents in smokeless tobacco products

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Methods for measuring constituents in smokeless tobacco products


British American Tobacco manufacture and sell snus in Sweden, and have held several small scale test markets in other countries. Our snus products are manufactured to standards commonly used among Swedish manufacturers, which includes limits on the levels of some constituents voluntarily applied for Swedish-style snus products.

The relevant constituents are total tobacco specific nitrosamines, nitrosodimethylamine (NDMA), benzo(a)pyrene, lead, cadmium and the sum of Aflatoxin B1,B2,G1 and G2.  In proposing future regulation of smokeless tobacco products within the European Union, the trade body of the European Smokeless Tobacco Council, of which we are a member, has advocated that ISO standards for measurement of smokeless tobacco constituents should be developed as a matter of urgency [ 1 ].

In our research programme we have begun to develop methods and make measurements on a much wider set of smokeless tobacco constituents, as we noted in our submissions to the US Food and Drug Administration (the FDA). Previous submissions to the FDA include an overview of our scientific research programme [ 2 ] and more recently we submitted to the Subcommittee some of our research relevant to the identification, prioritisation and measurement of tobacco and tobacco smoke constituents [ 3 ]. In some cases, multi-analyte analytical methods have been developed that allow for examination of a range of constituents from one analytical run.

We currently use the following analytical methods:

  • Nicotine is determined by extracting tobacco with potassium hydroxide and methanol and analysed by gas chromatography-mass spectrometry (GC-MS) using Selective Ion Monitoring (SIM).
  • Tobacco specific nitrosamines (TSNAs) are determined by extracting tobacco in methanol, centrifuged and analysed by liquid chromatography-tandem mass spectrometry (LC-MS-MS).
  • The non-tobacco-specific nitrosamines n-nitrosodimethylamine, n-nitrosodiethanolamine and n-nitrosomorpholine are determined as follows: N-nitrosodimethylamine is determined using di-ethyl ether extraction of tobacco, partitioning into acidified water, and analysis by LC-MS/MS; N-nitrosodiethanolamine is determined using water extraction of tobacco, centrifuging, filtering, solid phase clean-up, derivatisation and analysis by GC/MS;  N-nitrosomorpholine is determined by extraction of tobacco by a citrate-phosphate buffer, solid phase clean-up, concentration and analysis using GC-TEA (thermal energy analyser).
  • The non-tobacco-specific nitrosamine n-nitrosopiperidine is analysed by Labstat International using an in-house method.
  • Nitrate nitrogen is determined by aqueous extraction of tobacco prior to reduction of the nitrate to nitrite with hydrazinium sulphate, followed by complexation and colourimetric determination.
  • Nitrite is determined by extraction using aqueous ammonium chloride buffer, dialysis and reaction to form a diazo compound; this is then analysed spectroscopically.
  • Benzo(a)pyrene is measured by extracting a sample of tobacco in cyclohexane and acetonitrile. The sample is analysed by High Performance Liquid Chromatography (HPLC) with fluorescence detection.
  • A suite of polycyclic aromatic hydrocarbons are determined by Labstat International using an in-house method.
  • Formaldehyde, acetaldehyde, and crotonaldehyde were determined by Labstat International using an in-house method.
  • Ethyl carbamate analysis is undertaken by Eurofins Sweden.  A tobacco sample is extracted in water. Internal standard is then added and the sample mixed and then centrifuged. The samples are then analysed by ultra-high performance liquid chromatography with tandem mass spectrometry.
  • Metals (arsenic, beryllium, cadmium, chromium, lead, mercury, nickel and selenium) are determined by digesting tobacco under pressure using aqueous nitric acid prior to analysis by ICP-MS.
  • Polonium-210 is determined by alpha spectrometry after addition of Po-208 as internal tracer, prior to wet oxidation and homogenisation using acid, and auto deposition onto a silver disc.
  • Uranium-235 & 238 are determined by alpha-spectrometry following addition of U-232 internal tracer, ashing, dissolution in acid, co-precipitation, ion-exchange chromatography, and electro-deposition onto stainless-steel discs.

We are currently in the process of reviewing all our existing smokeless research methodologies to ensure scientific robustness prior to posting in our library under the methods section.

  1. ESTOC proposed regulation of smokeless tobacco products within the EU – part 2, February 8, 2010.
  2. Submission by Group Research and Development of British American Tobacco (Investments) Limited, Docket No. FDA-2009 N-0294.  Regulation of Tobacco Products: Request for comments, 29th September 2009.
  3. Submission by Group Research and Development of British American Tobacco (Investments) Limited, Docket No. FDA-2010 N-001.  Information for consideration at the June 8-9, 2010 meeting of the Tobacco Constituents Subcommittee of the Tobacco Products Scientific Advisory Committee, 27th May 2010.
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