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The glucuronide metabolites are important both for toxicology and for pharmacokinetics of many drugs and xenobiotics. Even though glucuronides oftenlack the pharmacological activity, after de-conjugation, the free aglycone can regain that activity and may present an environmental burden. Furthermore, glucuronides may be used as markers of the past substance exposure, like for example the ethyl-glucuronide in body hair for alcohol abuse or the cannabinoid-glucuronides for cannabis abuse. Therefore, quantification of glucuronide metabolites in biological matrices may be very important. However, due to their highly hydrophilic nature, the separation of glucuronides from the matrix (body fluids, hair, and environmental samples) may be difficult. For that purpose, many sample preparation techniques can be used, including the liquid extraction, solid phase extraction, dried blood spots, protein precipitation, direct injection, on-line solid phase extraction and others. Furthermore, some glucuronides, such as acyl- and N-glucuronides can be highly unstable. Glucuronides can be measured either by a direct or an indirect approach. With the latter, the glucuronides are cleaved back to their parent aglycone by enzymes known as the ŽŽ- glucuronidases, afterwards the freed aglycone is quantified according to its calibration curve. With direct measurement, unchanged glucuronides are quantified by their MRMs, where some significant benefits can be expected, namely, quicker sample preparation, better accuracy and precision, and selectivity towards glucuronide isomers. Tandem mass spectrometry is the corner stone for direct glucuronide quantification because it offers both exceptional selectivity and sensitivity. However, this approach requires the use of authentic glucuronide standards. This necessity can be avoided with thesemi-quantitative approach, where the response factor for glucuronide is used to quantify it against the calibration curve for the parent. The recent advancement of mass spectrometersŽʼ speed and sensitivity coupled with higher mass accuracy promises an even easier measurement of glucuronides in complex biological matrices, allowing a faster method development, robust response, and quicker sample preparation. Notwithstanding the described improvements, the glucuronide identification and quantification in difficult matrices will still remain a challenge for the analyst.

Quantification of Glucuronide Metabolites in Biological Matrices by LC-MS/MS