A light at the end of the tunnel – E-cigarette analysis made easy with TD and TOF MS

Smoking has never been far from the headlines over recent months, with the forthcoming ban on smoking in cars carrying children in England and Wales having gained a considerable amount of media attention, and the rise in awareness in indirect impacts of smoking such as third-hand smoke.

A particular focus of attention has been the e-cigarette phenomenon and the potential for harm from these mimics of real cigarettes. As a consequence, the UK Government has proposed a ban on e-cigarette sales to under-18s in England, and there are also moves afoot in the USA, with e-cigarettes proposed to come under the remit of the US FDA.

E-cigarette liquids vary widely in composition, but typically contain a variety of flavourings, with or without nicotine, dissolved in a carrier liquid such as glycerol or propylene glycol (1,2-propanediol). Heating this liquid within the e-cigarette generates a vapour, which following inhalation into the lungs, generates a visible ‘fume’ upon exhalation.

However, a meta-review of e-cigarette constituents, published earlier this year, found that commerical e-cigarette solutions can contain a number of potentially harmful chemicals, including formaldehyde, nitrosamines, and polycyclic aromatic hydrocarbons (PAHs). The presence of such chemicals naturally gives rise to some concern, and has already led analytical scientists to examine e-cigarette vapour itself in more detail.

One such study was carried out last year by Tobias Schripp and co-workers at the Fraunhofer Wilhelm-Klauditz-Institut (WKI) in Braunschweig, Germany. They used chamber sampling with Markes’ UNITY-ULTRA and GC–quadrupole MS to identify the mix of chemicals that is released upon ‘vaping’, and how this changes after exhalation.

However, a presentation by Stuart Martin and Chris Rawlinson (of tobacco company British American Tobacco – BAT) at a scientific conference on Smoke Science & Product Technology in September 2013 makes a compelling case that quadrupole MS detectors are not sufficiently sensitive to adequately characterise vapour from e-cigarettes. This is because although e-cigarettes emit a lot less particulate matter than regular tobacco, since no combustion takes place, they still produce a wide range of compounds at trace levels.

Having previously been in conversation with our specialists on how to analyse cigarette smoke, Martin and Rawlinson turned to our TD–GC–TOF MS system to tackle the problem of e-cigarettes. They weren’t disappointed. The concentrating power of TD and sensitivity of the BenchTOF not only allowed them to identify about 130 components (twice as many as before), but greatly shortened the sampling too, because they were able to replace the cumbersome smoking machine with a much simpler syringe drive. This collected just 25 mL of e-cigarette vapour on to a TD tube packed with Tenax TA and SulfiCarb, which was then conveniently desorbed on a Markes TD-100 before transfer to the GC–MS.

Remarkably, the total time for analysis and data-processing was less than 30 minutes – a speed that they say could allow 100 samples to be processed every day, a vast improvement on the eight samples per day obtainable with existing technology. At the same time, they were able to reduce limits of detection from 0.1 µg to less than 5 ng on-tube – a 20-fold increase in sensitivity.

The presentation concludes that, although existing analytical ‘smoking machines’ in conjunction with GC–quadrupole MS, may be suitable for assessing the bulk components of e-cigarettes, they are not suitable for in-depth screening, and present the combination of Markes’ TD and TOF MS technologies as a powerful alternative.

VOCs in the news

As part of our regular sweep of news items in the analytical sciences, we often come across instances where volatile organic compounds (VOCs) are the focus of attention. We thought it might be useful and interesting to bring these together in a regular round-up – so here’s the first!

VOCs used to profile bacteria

VOCs emitted by cultures of ten strains of the diarrhoea-causing bacterium Clostridium difficile have been profiled using a custom-built headspace–TOF MS setup. Paul Monk and colleagues at the University of Leicester, UK, identified 69 VOCs and used them to distinguish between the strains – methanol, p-cresol, dimethylamine, ethylene sulfide, dimethyl sulfide and methyl thioacetate were most of value. The authors say that their method “may have utility as a rapid means of identifying C. difficile infection”.

Picked up by:

• wired.co.uk
• Science World Report (including an interview with Monks’ team)
• Scicasts (again with an interview)

The case for phthalates as endocrine disruptors strengthens

The case for phthalates being endocrine disruptors has been further bolstered by research carried out by John Meeker and Kelly Ferguson at the University of Michigan, Ann Arbor, USA. They used HPLC–MS–MS to assess urinary levels of 13 phthalate metabolites – primarily oxygenated and singly-hydrolysed derivatives of phthalate esters. Significant reductions of testosterone were found in both men and women of different ages. Notably, substantial increases in metabolites of bis(2-ethylhexyl) phthalate (dioctyl phthalate) in 6–12-year-old boys were associated with a 29% drop in testosterone.

Picked up by:

• Science Daily

Apple tightens regulations on hazardous solvents

Benzene and n-hexane have been banned from use as cleaning agents and degreasers in the final assembly process at 22 of Apple’s iPhone and iPad production plants. Their new Regulated Substances Specification additionally stipulates that “All cleaning agents and degreasers used at final assembly process facilities for the manufacturing of Apple products shall be tested for benzene, n-hexane and chlorinated organic solvent content at a certified lab prior to use in production”, and that permitted levels in the breathing zone of workers must be <100 mg/m³ (28 ppm) for n-hexane, and <0.32 mg/m³ (0.1 ppm) for benzene.

Picked up by:

• BBC News

US National Academy of Sciences concludes that formaldehyde causes cancer

The long-running debate in the US over whether formaldehyde is carcinogenic took moved forward in August with the publication of a report by the US National Academy of Sciences (NAS), where they conclude that the answer is “yes it is”.

This follows their critical 2011 review of the US EPA’s draft assessment of formaldehyde. Although the EPA document said that the evidence is “sufficient to conclude a causal association” between formaldehyde exposure and a variety of cancers, the NAS review said that there were “recurring methodologic problems” in this study.

The new document from NAS is their own independent assessment of the literature through to November 2013. Here they conclude that there is “sufficient evidence of carcinogenicity” in humans for nasopharyngeal cancer, sinonasal cancer and myeloid leukemia, and “convincing relevant information” that formaldehyde induces mechanistic events associated with the development of cancer.

These and other aspects lead the committee to conclude that “formaldehyde should be listed in the RoC [Report on Carcinogens] as “known to be a human carcinogen”.”.

Picked up by:

• AllGov

Is carbon tetrachloride still being emitted despite global ban?

Studies carried out by a team led by Qing Liang at the NASA Goddard Space Flight Center, Maryland, USA, have suggested that the observed slow decline of the ozone-depleting carbon tetrachloride (tetrachloromethane) can only be explained if it is still being emitted (see also this press release). This stands in contrast to the near-zero emissions estimate based on production and feedstock usage, a result of the regulations initiated by the 1987 Montreal Protocol.
Liang’s research, which is based on computer modelling of the concentration gradient between the northern and southern hemispheres, estimates that current unknown emissions are still about 30% of pre-treaty peak emissions. He says “it is now apparent there are either unidentified industrial leakages, large emissions from contaminated sites, or unknown CCl₄ sources”.

Picked up by:

• Science World Report

Plant volatiles – An area that keeps on growing

From time to time, I scour the scientific literature for interesting examples of where people have used thermal desorption in scientific research – which is great for highlighting some of the latest trends in how people are harnessing the power of modern analytical technology.

As an organic chemist (and former Publishing Editor of the journal Natural Product Reports), plant volatiles particularly interest me, and I’m always keen to see what’s been happening in this small but rapidly growing field. So here’s my choice of the most interesting papers from the last few years where our TD equipment has helped researchers to study plant volatiles.

David Barden

Insects know the best time for ripe pickings

It’s been known for some time that volatiles released by plants are used by insects to home in on their favourite food plants – see for example this BBC article on how the attractiveness of flowers to moths is affected by pollution. Now it’s also been shown that they can use this information to switch between different food sources according to the time of year.

Work by Adriana Najar-Rodriguez and colleagues at the Institute of Agricultural Sciences, Zurich, Switzerland, found that the Oriental fruit moth could tell when both peach and pear trees were in season, on the basis of the changing blends of volatiles emitted over the course of the year.

Using Markes’ UNITY-ULTRA automated thermal desorption system with GC–MS analysis, they found that a set of five aldehydes – hexanal, (E)-hex-2-enal, heptanal, benzaldehyde and octanal – were present in the volatile blends at the stages when the peach and pear trees were most attractive to the moths. The authors suggest that this information should be used when designing chemical attractants for female fruit moths.

Birds use volatile emissions to find lunch

In a fascinating piece of research, carried out by a team led by Luisa Amo at the Netherlands Institute of Ecology, Wageningen, Great Tits were found to be more attracted to caterpillar-infested apple trees than uninfested trees, even when they could not see the larvae or the leaf damage caused by them.

This is an example of a ‘tritrophic’ system – that is, one involving three levels of the food chain, and in essence an extension of the plant–herbivore system described above.Such systems are well-studied,* but this one is remarkable because it involves caterpillars being predated by birds, not other insects.

Despite this fundamental difference, the system studied appears to operate in the same way as normal – namely, the apple trees release a distinct set of volatiles when the leaves are attacked by the caterpillars, and these volatiles are used by the birds to locate the trees where a meal can be found.

To help confirm that the birds were using volatile cues, the authors enclosed tree branches with a polymer bag, and flushed the collected vapours onto sorbent tubes, with analysis by Markes’ UNITY thermal desorber and GC–MS. This analysis showed that the infested trees emitted more alpha-farnesene and dodecanal than uninfested trees, but less 1,2,4-trimethylbenzene, oct-1-en-3-ol, methoxy phenyl oxime, non-1-ene and and octan-3-ol.

The authors say that their findings are in line with previous studies on the use of smell by birds to detect prey, and indeed indicate that it may be more important than previously thought. They add that this area of research is exciting because birds eat far more caterpillars than other insects – so the potential benefits for pest control obtainable by breeding plants with enhanced volatile emissions may be correspondingly greater.

* The insect predators don’t always eat their prey straight away. Many studies of tritrophic systems look at parasitic wasps, which inject their eggs into living caterpillars. These caterpillars eventually suffer a grisly death by being eaten alive by the wasp larvae. See this BBC article for a rare example of research into this area being picked up by the mainstream media.

Volatiles help plants distinguish relatives from strangers

Research led by Richard Karban at the University of California, Davis, USA, has shed light on how plants might communicate with each other, by identifying specific compounds that correlate with the ability of neighbouring plants to repel insect herbivores.

In the study, which also involved scientists from Japan and Finland, portable dynamic headspace equipment was used to sample volatiles emitted from branches of 59 sagebrush plants in Sagehen Natural Reserve, California, USA. Volatiles were collected onto sorbent tubes from Markes and then analysed with Markes’ TD-100 automated thermal desorber, in conjunction with GC–MS.

They found that although volatile profiles differed greatly, individual profiles were generally dominated by either alpha-thujone or camphor, and that just two additional compounds (beta-thujone and cis-salvene) were sufficient to classify these two ‘chemotypes’ of sagebrush. Importantly, a separate experiment involving analysis of compounds from seed-raised plants showed that the chemotypes were highly heritable, 80% of ‘children’ having the same chemotype as their ‘mother’.

They then investigated the effectiveness of plant communication by collecting headspace from one plant and incubating it with another plant for 24 hours, before exposing the plant to the open air and so to insect herbivores. They found that the transferred volatiles were more effective at deterring herbivory in the recipient when the plants were the same chemotype. The authors suggest that this knowledge, together with the fact of chemotype heritability, suggests that plant volatiles could help plants to distinguish ‘relatives’ from ‘strangers’, so increasing the value of the information encoded by plant volatiles.

For more on this research, see this article on the phys.org website.

Raised CO₂ makes Brussels sprout plants less attractive to aphids

Another piece of research by Adriana Najar-Rodriguez and colleagues at the Institute of Agricultural Sciences, Zurich, Switzerland, shows for the first time that plant adaptations to rising carbon dioxide (CO₂) levels reduce the colonisation of plants by herbivorous insects.

In the work, Brussels sprout plants were reared inside climate-controlled chambers, and headspace volatiles collected passively over 6 hours onto Radiello sorbent samplers. The sorbent cartridges were then desorbed using Markes’ UNITY-ULTRA, with analysis by GC–MS.

They found that 10 weeks’ exposure to doubled CO₂ resulted in a number of changes to the volatile profile – namely the appearance of (Z)-hex-3-enyl acetate, dodecane and tridecane, the disappearance of decanal and undecanal, and statistically significant reductions in 10 terpenoids, as well as a 50% fall in dimethyl disulfide.

At the same time, they used wind-tunnel choice experiments to investigate the preferences of winged cabbage aphids for the different plants, and found that the colonisation of plants reared under doubled CO₂ was 26% lower than regular plants.

The authors also found that the rate at which carbon dioxide or water entered or left the leaf through the stomata (‘stomatal conductance’) was significantly reduced, and suggest that this, together with possible changes in leaf enzyme activity, could explain the reduction in volatile emissions. They then suggest that this in turn (perhaps in concert with additional cues such as changes in plant size) reduces the attractiveness of the plants to the aphids.

Whatever the underlying mechanism, the authors say that this is one of the very few cases where changes in atmospheric CO₂ have been shown to influence herbivory. They also point out that the changes in volatile emissions could have important ecological implications – such as the ability to attract predators of the herbivores (as I’ve described in Luisa Amo’s research above).

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David Barden received his Ph.D. in Organic Chemistry from Cambridge University in 2004, and during his time as an editor at the RSC wrote news pieces for Chemistry World on various scientific topics. He is now Technical Copywriter at Markes International, where he draws on the expertise of his colleagues to explain how new thermal desorption and mass spectrometry technologies can be applied to analyse volatile organic compounds in a wide variety of situations.

Up close and personal – The chemicals used in personal care products

Recent research published in Occupational & Environmental Medicine carried out by Swedish scientists has again highlighted the exposure of hairdressers to potentially hazardous chemicals, this time of carcinogenic and sensitising toluidines in hair dyes and hair-waving (‘perming’) products.

This is just the latest in a long line of news stories about chemicals in personal care products – including methylisothiazolinone in suncream, triclosan in antibacterial products, and phthalates in nail polish,

The latest research, led by Gabriella Johansson at Skåne University Hospital in Lund, Sweden, has been picked up by a number of outlets, including Reuters, ChemicalWatch, SpectroscopyNOW, Medical Xpress, Nature World News and MedicalResearch.com. What the researchers found was that m-toluidine was present above the limit of detection in the blood of >97% of a cohort of hairdressers, personal dye users and controls (a total of 387 people), with the figure for  o-toluidine being ~50%. The chemicals were detected indirectly by GC–MS analysis of their adducts with haemoglobin, which is a good way of assessing average exposure because of haemoglobin’s relatively long lifetime.

This of course isn’t the first time that chemicals originating from hair dyes have raised alarm bells – p-phenylenediamine gained attention in late 2011 following a death from a suspected allergic reaction. The issue this time is that toluidines, unlike p-phenylenediamine, were banned from cosmetics in the EU in the late 1970s (as stipulated on page 18 of the original 1976 EU directive), so it is a surprise to find them in the blood at all.

Two facts uncovered in Johansson’s study seem to suggest that these haircare products are the source of the toluidines. Firstly, they found that o-toluidine concentrations increased with the number of hair-waving treatments, and that m-toluidine increased with the number of hair-dyeing treatments. Secondly, and more tellingly perhaps, they analysed a commerical (multi-component) hair-waving product and found both o-toluidine (up to 0.23 ng/g) and m-toluidine (0.15 ng/g).

However, the matter is not as clear-cut as it might seem, because, as the authors say “We evaluated the exposure … [for] hairdressers, consumers and controls, and found no overall significant difference.” Likewise, research last year led by Marie Thi Dao Tran at Copenhagen University Hospital found that “the prevalence and the severity of fragrance-related symptoms [of chemical intolerance] were similar in hairdressers and the general population”.

Such research points to a complex picture, with possibly multiple routes of exposure subject to factors not accounted for in the current study. Clearly, there is a need to devote more attention to where the compounds are coming from, as the authors themselves suggest: “A study measuring both exposure to aromatic amines and product analysis … would strengthen the conclusions about hairdressers’ exposure to carcinogenic aromatic amines, and is encouraged”.

Indeed, such analysis should really go hand-in-hand in this sort of study, and is essential in order to draw any meaningful conclusions from the work. Analysing all the haircare products used by several hundred people is not going to be a small task, but perhaps is illustrative of the amount of effort that needs to be made to link cause and effect in such a complex area.

The good news is that this sort of analysis – once a highly labour-intensive endeavour – is becoming increasingly quick and straightforward, because of advances in sample preparation and automation. Advanced analytical techniques like GC×GC–TOF MS are also playing their part, by making it possible to get reliable, quantitative information out of such highly complex samples – as we’ve illustrated ourselves in Application Note 522 for the case of allergens in cosmetics.

David Barden received his Ph.D. in Organic Chemistry from Cambridge University in 2004, and during his time as an editor at the RSC wrote news pieces for Chemistry World on various scientific topics. He is now Technical Copywriter at Markes International, where he draws on the expertise of his colleagues to explain how new thermal desorption and mass spectrometry technologies can be applied to analyse volatile organic compounds in a wide variety of situations.