Chocolate threats: Carcinogenic PAHs

Hidden dangers in chocolate

While children around the world are digging into their cache of Easter eggs, scientists are concerned about the amounts of carcinogenic polycyclic aromatic hydrocarbons that the treats might be harbouring. It is well known that cocoa butter contains higher levels of PAHs than other types of oils and fats as a result of poorer refining during processing and inadequate drying practices. They find their way into foods and sweets containing chocolate and contribute to the overall exposure of an individual. At particular risk are children, who tend to eat more chocolate than adults and have a lower safe threshold.

The European Food Safety Authority recently recognised that its criteria for detecting PAHs in food were deficient. They relied on the occurrence of a single PAH, benzo[a]pyrene, as a marker of PAHs but they have recently agreed to expand this to a panel of four PAHs from April 2013 in order to protect consumers.

Benzo[a]pyrene remains one of the four biomarkers with a maximum allowable limit of 5.0 µg/kg fat. The other three are benz[a]anthracene, benzo[b]fluoranthene and chrysene and the maximum allowable limit for all four together will be 35.0 µg/kg fat from April 2013 and 30.0 µg/kg fat from April 2015. The separate value for benzo[a]pyrene alone is retained to ensure that previous and future data can be compared.

As these limits come into force, scientists must ensure that they can be met by appropriate analytical procedures. In 2012, Devendra Kumar Patel and colleagues from CSIR-Indian Institute of Toxicology Research in Lucknow developed an HPLC method for PAHs in candies with ng/g detection limits. However, it involved time consuming sample preparation procedures and organic solvents, so they have come up with an alternative method involving SPME and GC/MS.

Chocolate testing

Their method was aimed at detecting 16 PAHs in chocolate, including the four biomarkers selected by the EFSA. Using chocolate purchased from a local market in Lucknow, small portions were spiked with known amounts of the PAHs during method development while the SPME procedures were optimised.

The researchers began with a poly(dimethylsiloxane) fibre because it is known to have a large capacity for non-polar compounds like PAHs. The best results were achieved by homogenising the chocolate with an aqueous 10% sodium chloride solution at pH 6.5 by heating to 65°C. At that stage, the SPME fibre was introduced into the solution through the septum to trap the PAHs.

Direct immersion was better than headspace analysis, which failed to extract the higher-molecular-weight PAHs with four or more rings that tend to be more carcinogenic and mutagenic than their low-ring counterparts.

The fibre was removed from the solution and inserted into the injection port of a GC/MS system for electron ionisation and detection by selected ion monitoring. One ion was selected for quantification and two further ions were selected for confirmation of the individual compounds. All 16 PAHs were separated on a total ion chromatogram allowing their contents to be determined.

PAHs present

The optimised method gave detection limits of 0.004-0.438 ng/g in SIM mode. These were 0-100 times more sensitive than GC/MS in full-scan mode which were, in turn 8-10 times more sensitive than GC-FID. Average recoveries of 85-105% showed that other substances in the chocolate did not have much effect on the extraction.

The method was applied to try and detect PAHs in ten sweet chocolate bars. Two of the bars were found to contain fluoranthene (0.024 and 0.041 ng/g) and pyrene (0.082 and 0.079 ng/g). These have been labelled "not classifiable as to carcinogenicity to humans" by the EPA but are carcinogenic in animals.

Each of the bars also contained one further PAH, either benzo[a]pyrene (0.320 ng/g), which is a human carcinogen, or benzo[k]fluoranthene (0.230 ng/g), which is a possible human carcinogen. A further chocolate bar was found to contain six PAHs at concentrations in the range 0.027-0.312 ng/g.

Although the sensitivity greatly exceeds that obtained using GC-FID, Patel recognised that some organisations cannot afford the GC/MS instrumentation or the skilled personnel to run it. As a back up, the SPME technique can be used with GC-FID, albeit at poorer sensitivities of 0.09-3.31 ng/g, for detecting and measuring PAHs in chocolate and related food products.