Controversial caffeine caught by clever LC-MS/MS method

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  • Published: Jan 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Controversial caffeine caught by clever LC-MS/MS method

Caffeine can be deadly

Most of us like the odd cup of tea or coffee, but a very high caffeine intake from oral sprays, e-cigarettes or powdered caffeine can have serious health consequences, particularly when combined with high alcohol consumption.

Whether it’s our morning coffee or afternoon tea, we tend to think of caffeine as a harmless, mild stimulant. However, as the 16th century physician Paracelsus observed, ‘All things are poison and nothing is without poison. It is only the dosage that makes a thing not a poison.’ The human LD50 of caffeine has been estimated as 150 to 200 mg/kg body weight, giving a dose of 10.5 to 14 g for a person weighing 70 kg. However, those suffering from liver disease are at greater risk since they may not be able to metabolise caffeine properly. People with heart conditions are also more likely to suffer fatal consequences. The US FDA warns of the dangers of taking large amounts of caffeine, noting that it has been responsible for at least two deaths. New sources of caffeine, such as high energy drinks, e-cigarettes, oral sprays or caffeine powder (either swallowed or inhaled), increase the likelihood of harm. High amounts of caffeine can be particularly dangerous when taken alongside large quantities of alcohol as the caffeine seems to make ‘binge drinking’ more likely.

The problem of analysing caffeine in blood

Although there are a number of analytical methods for measuring plasma caffeine levels, most are either long-winded or fail to detect caffeine metabolites. The liver metabolises caffeine to the related compounds paraxanthine, theothylline and theobromine. These have their own pharmacological effects, so pharmacokinetic studies require accurate measurement of the levels of metabolites, in addition to caffeine itself.

University of Tennessee researchers, writing in the journal Biomedical Chromatography, detail their attempts to find a better method for plasma caffeine analysis. They wanted to avoid lengthy extraction methods, so that rapid results could be obtained from plasma samples. The answer was found by using a simple protein precipitation technique to clean up the plasma. Addition of methanol containing 125 mM formic acid, followed by centrifugation, gave a solution that could be directly injected into the HPLC system. Too much background noise was noted if the formic acid was not used.

The authors found aqueous methanol acidified with formic acid to be a suitable eluting solvent. A quick 6-minute gradient system was devised. The amount of formic acid in the eluting solvent was optimised, with 25 mM proving best. It was found that a high temperature (35 °C) and flow rate (700 μl/min) were needed for complete peak separation. The mass spectrometer used a positive ion electrospray tandem system. A splitter (1:1 ratio) was used to prevent the high flow rate overwhelming the mass spectrometer. Deuterated caffeine and paraxanthine were used as internal standards, being added with the precipitating methanol solution. These gave their own distinctive peaks in the mass spectrum. The use of deuterated standards reduced errors to a minimum, since any losses of the substances under investigation during the process would be mirrored by their deuterated analogues.

The method was tested on healthy human volunteers who had consumed caffeine. Blood samples were taken, giving concentration versus time plots for caffeine and its metabolites.

New method improves analysis of caffeine and its metabolites

The new LC-MS/MS method allows for rapid analysis of caffeine and its three main metabolites. The novel use of deuterated internal standards helps to minimize sources of error. The new method will enable the pharmacokinetics of caffeine absorbed by novel methods, such as e-cigarettes or nasal sprays, to be readily determined.

Related Links

Biomedical Chromatography, 2016, Early View paper. Chen et al. Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application.

FDA consumer advice on pure powdered caffeine

Drug and alcohol dependence, 2016, 99, 1-10. Reissig et al. Caffeinated energy drinks – A growing problem.

Wikipedia, Caffeine

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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