Sébastien Gagné and Éric Langlois have made a breakthrough in toxicology: an innovative and very efficient method for urinary ortho-cresol analysis, a plus for the clientele of the IRSST’s laboratories.

Their article on the subject, Ultrahigh-performance liquid chromatography–tandem mass spectrometry quantitation of o-cresol in hydrolyzed human urine to assess toluene exposure, has been published in the scientific journal Rapid Communications in Mass Spectrometry.

The Genesis of a Solution

“We went off the beaten track to develop this method,” remarks Gagné. “I’ve been working at the IRSST for 13 years, and this is the most impressive work I’ve ever done.”

End-of-shift (and end-of-week) urinary o-cresol levels reflect same-day esposure and correlate with toluene exposure intensity.

“o-Cresol is a molecule we routinely measure as a marker of toluene exposure in workers. However, the sensitivity of this parameter is so-so, and only made it possible to assess exposure where the TWAEV was 50 ppm. The method was also often inconclusive due to interference. In addition, detection limits were not low enough. The lowering of exposure values in the Regulation respecting occupational health and safety (ROHS) led to a review of the biological reference value, called the biological exposure index (BEI), which obliged us to revise the method. We took this opportunity to take a close look at all the problems inherent in this method

We thus took a molecule that was incompatible with the type of instrument we were using and converted it to make it compatible. We derivatize it with dansyl chloride, which makes it compatible with a liquid chromatography-mass spectrography system.”

o-Cresol is a volatile molecule that cannot be analyzed by liquid chromatography. The scientists at the IRSST converted it in the urine sample preparation process to make it non-volatile and easily ionizable in the instrument.

“This transformation enabled us to look for very low detection limits and eliminate the interferences that we previously had. The older method had several peaks and it was very difficult to find o-cresol in the ‘forest of peaks.’ With the new method, we have two easily identifiable peaks and can selectively search for o-cresol.”

“We are the first in the world to have done this!” exclaims Gagné.

Usefulness

“In practical terms, this discovery enables us to measure the intensity of exposure levels in workers. Biological monitoring measures the total body burden of a substance in all possible absorption routes: dermal, respiratory and digestive. The level of o-cresol gives the individual’s level of exposure to toluene. The more toluene a worker is exposed to, or has absorbed, the higher the level of o-cresol in the urine. This level will never be zero,” cautions Gagné, “because there is always a proportion that comes from atmospheric pollution, cigarette smoke and diet, but at least it will be below the reference value.”

“Usually, volatile molecules are analyzed with an instrument using gas chromotography. Non-volatile or semi-volatile molecules are analyzed with liquid chromatography. We’ve gone off the beaten track by taking a volatile molecule and making it non-volatile, in order to analyze it with another technique.”

Future Avenues of Research

“We are continuing our explorations and working on using this method for other molecules, such as hydroxypyrene, the biomarker for polycyclic aromatic hydrocarbons (PAHs). This is another volatile molecule that we want to convert into a non-volatile one for analysis by liquid chromatography. We’ve opened up a new avenue for the analysis of volatile compounds,” explains Gagné.

Collaboration and Co-construction

The scientific literature mentions that research groups derivatize their molecules using agents such as dansyl chloride, which appeared to IRSST scientists to be the most suitable for o-cresol.

“We tried it out, and the result met our expectations. For a chemist, this discovery is an incredible source of pride! I didn’t work alone, but in close collaboration with my colleague, Eric Langlois.”

Sébastien Gagné is the author and co-author of some forty articles.

What Is Toluene?

Toluene is a volatile organic compound used in domestic and industrial applications. The main routes of exposure to toluene in the workplace are inhalation and dermal contact. Exposure to theis substance can cause severe damage to the nervous system. Measuring exposure to toluene is therefore crucial to preventing occupational diseases.

Toluene is metabolized mainly as hippuric acid, S-benzylmercapturic acid and epoxides. These are rapidly converted to o-/p-cresol, which is then excreted in the urine as glucoronides and conjugated sulphates. o-Cresol and its conjugates can be chemically hydrolized to form free o-cresol, which can then be used as a urinary biomarker of toluene exposure. As the IRSST chemist explained, however, current analytical methods for quantifying o-cresol in hydrolyzed urine are weakened by interferences and are not suffiently sensitive. It was therefore necessary to develop a liquid chromatography with tandem mass spectrometry (LC-MS-MS) method for assessing toluene exposure. This was achieved at the IRSST!