Analytical Challenges of Tobacco and Nicotine Testing

What is Nicotine?

Nicotine is the addictive active component in tobacco and tobacco-based products. These products include cigarettes, cigars, smokeless tobacco products (e.g., chewing tobacco, snuff, and snus), and nicotine-containing e-liquids used in vaping. Nicotine has two enantiomeric forms – one form rotate plane-polarized light to the negative direction and designated as the (-) enantiomer or levorotatory, and the other form rotates plane-polarized light to the positive direction and designated as the (+) enantiomer, or dextrorotatory. The naturally-occurring enantiomer of nicotine is levorotatory – designated as (-)-nicotine – with a specific rotation of [a]D=-166.4o. Nicotine can be converted into salt form by the addition of a weak acid and nicotine salts are increasingly used in liquid formulations to reduce gag reflex or choking reactions due to the harsh basic nature of free nicotine.

Nicotine's Chemical Structure

It should be noted that, while nicotine is traditionally associated with recreational use products, nicotine used for tobacco cessation products like nicotine patches are considered to be pharmaceuticals and are regulated as such. Nicotine associated with recreational use is regulated, on the Federal level, with reduced stringency compared to pharmaceutical products, yet slightly more rigid compared to regulations surrounding the food and beverage space; some states like Massachusetts and Minnesota have specific regulations as well.

How is nicotine regulated?

The FDA-CTP is tasked to regulate the tobacco and nicotine space under the Family Smoking Prevention and Tobacco Control Act signed into law in 2009. While cigarettes, cigars, and smokeless tobacco products are heavily regulated, the nicotine vaping space has remained largely unregulated until the FDA pushed for submission of pre-market authorization applications (PMTA) on September 9, 2020.

As of January 5, 2021, the FDA-CTP has already issued warning letters to 10 firms for continuing to sell their tobacco and nicotine products without PMTAs; these 10 firms account carry over 100,000 nicotine and tobacco brands of which can no longer be sold – highlighting the FDA’s intentions to seriously regulate the nicotine vaping space. The FDA-CTP has established the Tobacco Industry Reporting Requirements which predominantly entails the regular submission of analytical data on Harmful and Potentially Harmful Constituents in (HPHCs) tobacco and tobacco-based products currently sold in the market. These data are guaranteed to be reliable for regulatory submission when generated using an approved analytical method.

Approved Analytical Methods for Nicotine Quantification

Approved analytical methods for nicotine quantification include titration[LR-X1]  and Gas Chromatography with Flame Ionization or Mass Spectroscopy detectors (GC-FID or GC-MS)[LR-X2] . The titration method is almost exclusively applied to highly concentrated, almost pure, nicotine such as USP grade nicotine which are the starting materials for formulated nicotine products. GC-FID and GC-MS are typically applied to nicotine analysis in tobacco and other formulated nicotine products like e-liquids. Titration – conveniently carried out using automatic titrators like the Titroline 7800 – can produce reliable results on samples with simple matrices – e.g., almost pure substances – but e-liquids are much too complex for titration methodologies and can produce inaccurate results. In addition, some e-liquids use nicotine salts which would increase bias on titration results.

On the other hand, the GC method has been validated by the cooperation Center for Scientific Research Relative to Tobacco (CORESTA) and later revalidated by ISO for the analysis of nicotine in pure and formulated nicotine products, hence generally accepted as the gold standard for nicotine analysis. It should, however, be noted that GC methods may occasionally provide over-estimates of nicotine in highly concentrated nicotine samples such as USP-grade nicotine – and this is mainly due to dilution errors – and as such USP still recommends titration assay for these concentrated materials. GC is a great approach for formulated nicotine products as it allows for the separation of components.

OI Analytical's FS3700

A method employing a continuous flow analyzer has been proposed as an alternative method for nicotine analysis due to its high analytical throughput, and relative specificity to nicotine and has been validated by CORESTA. Although not yet formally accepted by FDA-CTP, studies have shown that the continuous flow method strongly correlates with the GC method with a slope and correlation coefficient close to 1.00. Because of this, the continuous flow method is gaining traction towards acceptance by state and federal regulatory bodies. Today numerous continuous flow platforms like the FS3700 have configured methods for nicotine analysis applicable for e-liquids and processed tobacco products. 

While the industry grapples with evolving regulation, there is yet another potential challenge –the analysis of tobacco-free nicotine (TFN); a synthetic form of nicotine that does not fall under current FDA regulations. TFN is a racemic mixture of R- and S-stereo isomers of nicotine while only the S-stereo isomer can be fully absorbed and metabolized by the mammalian system. Natural nicotine is purely the S-stereo isomer and is traditionally extracted from the tobacco plant (Nicotiana tabaccum). This extract is then purified to produce the concentrated starting materials for e-liquid manufacturing. Products derived from tobacco-derived nicotine are the ones under strict regulation as of current FDA guidelines. However, some companies have found creative ways around this regulation by formulating their products from TFN thereby evading FDA purview. There are formulations that use both TFN and tobacco-derived nicotine, and because tobacco-based products are regulated, the mixture can be subject to regulation as well. The challenge is proving that the product is a mix.

Typically, the ratio of the two stereoisomers in TFN is 1:1; if this ratio is off-set to more S-isomer (negative optical rotation) or R-isomer (positive optical rotation), then the formulation is likely mixture of TFN and tobacco-derived nicotine. This poses a challenge to the analytical chemists in the tobacco/nicotine space and, for the time being, uses chiral GC or LC columns to separate and quantify both the R- and the S-isomers.

The presence of purely R- or purely S-form of nicotine can cause an optical rotation of plane-polarized light. Typically, if the ratio of the two isomers is 1:1 the optical rotation caused by these two isomers together would cancel out and there would be no observable optical rotation. A polarimeter like the ADP600 (Bellingham and Stanley) can measure optical rotation to four decimal places in the highly sensitive ultra-violet region; this degree of precision could be sufficient to quantify the ratio of R- to S-isomer in a nicotine product. Although this approach is not currently considered by the FDA to address this question of TFN or TFN + tobacco-based nicotine mixture, the polarimer may provide a straightforward solution to this conundrum.

While the Federal regulations surrounding tobacco and nicotine product remain in constant flux, the analytical lab in the tobacco and nicotine space will continue to face challenges in producing reliable data for regulatory compliance. The applications experts at Xylem Lab Solutions can help provide solutions to these analytical challenges. 

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