Sudan dyes in seconds: Paper spray mass spectrometry for chilli powder tests

Sudan dyes in chilli

No-one had heard of Sudan dyes until 2003. Until then, these red compounds had been added safely to consumer products like plastics, leather, solvents, oils, waxes, and polishes, as well as cosmetics and drugs for external application. Then an official lab in France found that Sudan I had been added illegally to chilli powder and chilli products, presumably to improve their colour. Sudan dyes cause cancer in animals, so the EU authorities regard them as unsafe for human consumption.

The next year, in 2004, hundreds of different types of food product, including soups, ready meals and curry powders, were taken off the shelves in the UK when they too were found to be contaminated with Sudan dyes. The source was an imported batch of Worcestershire sauce that had been added to many different foods during their preparation. Since then, other types of spice, such as curcuma and sumac, and virgin palm oil have also been found to be tainted with these dyes.

The revelations spurred scientists into action and a raft of methods was developed to detect the dyes in food products, with HPLC prominent among them. HPLC with detection by mass spectrometry, chemiluminescence and UV-visible detection has been successfully applied, as well as capillary electrochromatography with amperometric detection.

These methods have been accompanied by a variety of extraction procedures. Molecularly imprinted SPE, ionic liquid-based liquid-liquid microextraction, cloud point extraction, stir bar microextraction, hollow fibre liquid phase microextraction and pressurised liquid extraction are just some of those that have been involved.

These two-step methods, with extraction followed by analysis, have now been challenged by a rapid procedure involving the minimum of sample preparation. The key to the success of the new method is paper spray mass spectrometry, which is one form of ambient mass spectrometry. The technique was announced in the US in 2010 by the Graham Cooks lab at Purdue University, from where many ambient ionisation techniques have originated, including one of the first: desorption ionisation mass spectrometry.

The point of paper

In paper spray, a tiny volume of solution, typically 10-20 µL, is spotted onto a triangular piece of paper. The apex of the triangle is positioned in front of the mass spectrometer inlet. When a high voltage is applied to the wetted paper, charged droplets form at the apex before desolvating to form dry ions, as in electrospray ionisation. Unlike some other forms of ambient ionisation, no sheath gas or sheath liquid is required.

Giovanni Sindona and colleagues from the University of Calabria, Italy, have now adapted paper spray mass spectrometry to measure Sudan dyes in chilli pepper. They described their application in Journal of Mass Spectrometry, where they applied it to the simultaneous measurement of Sudan I, II, III, IV and Para Red, which are all related azo dyes.

The sample preparation was simple. A small amount of powdered chilli pepper was mixed with a solution containing the deuterated analogues of each dye and vortexed for just one minute with an acetonitrile-chloroform solution. A drop of the supernatant was spotted onto the paper triangle with dimension 5 x 15 mm. After drying, a small drop of methanol was added to the paper and it was positioned in front of the mass spectrometer source.

When a voltage of 5 kV was applied to the paper, the ions were produced at the tip and drawn into the source of a triple quadrupole mass spectrometer for collision-activated dissociation. In precursor-ion scanning mode, the common ion at m/z 156 was monitored, corresponding to the β-naphthol fragment (or m/z 162 for the deuterated standards) of each dye molecule. This scan revealed the parent ions for all five dyes, which were the corresponding protonated molecules.

Simultaneous analysis in seconds

Each individual dye was confirmed by tandem mass spectrometry experiments and measured from the ratios of the abundances of the ions for the dye and its labelled standard. The smallest amount detectable, while maintaining a signal-to-noise ratio of more than three, was 1 ppm. Above this value, the calibration curves expressed good linearity up to 15 ppm.

For spiked samples of powdered chilli pepper, containing 1, 5 or 10 ppm of each Sudan dye and Para Red, the accuracies of the method were in the range 94-118% and the relative standard deviations were acceptable at up to 12.7%.

The key advantages of this method are its ability to measure all five dyes in one run, in less than 60 seconds, without resorting to chromatography. Apart from the minute volumes of solvent used to spot the paper triangle, the method is also green. Using a triple quadrupole mass spectrometer has the bonus that the presence of each dye can be confirmed from its fragmentation pattern in tandem mode.

It would be easy to extend this method to the analysis of the Sudan dyes in other types of foods, providing a rapid, high-throughput way to detect deliberate or accidental contamination.