Supramolecular Solvents Applied to Liquid–Liquid Microextraction for LC–HRMS Detection of Doping Drugs in Urine


Eighty drugs prohibited in athletic competition were analyzed, with seven samples returning adverse findings compared to previous methods.

New research from Universidad de Córdoba in Córdoba, Spain, and the Doping Control Laboratory at the Carlos III Health Institute in Madrid, Spain, explores the capability of supramolecular solvents (SUPRAS) in making comprehensive liquid–liquid microextraction (LLME) for multiclass screening of certain drugs found in urine using liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS) (1).

Doping for athletes. Golds, silver and bronze medal and doping syringe and pills with capsules on a gray background | Image Credit: © adragan -

Doping for athletes. Golds, silver and bronze medal and doping syringe and pills with capsules on a gray background | Image Credit: © adragan -

LLME is a technique used for sample preparation in analytical chemistry. It involves the extraction of target analytes from a liquid sample into a small volume of an immiscible organic solvent. LLME allows for the enrichment and purification of analytes from complex matrices such as biological fluids. It is particularly useful for multiclass screening of compounds, including drugs, in various samples. By using specific solvents and extraction conditions, LLME enables efficient extraction of analytes across a wide range of polarities.

Specifically, the study published in the Journal of Chromatography A selected 80 substances, or their metabolites, from a list of compounds belonging to any of 10 categories on the World Anti-Doping Agency (WADA) Prohibited List covering a range of polarities, physiochemical properties, and threshold concentrations.

Anti-doping laboratories are examples of such environments in which screening of multiclass substances is relevant and highly valuable where maximum permitted levels have been set and positive and negative samples must be quickly determined. LC–HRMS has been the technique of choice for targeted and untargeted analysis of thousands of compounds in this way, with solid-phase extraction (SPE) the traditionally preferred sample preparation technique. However, SPE does not provide the green, cost-effective analysis usually desired for high-throughput multiclass screening methods.

Up to now LLE, and organic solvent-based extraction more broadly, have not been considered viable alternatives to SPE in this application because extraction would be conditioned by the similarity of the polarities between analytes and solvents. But the different polarity microenvironments in the amphiphilic structures of SUPRASs suggest they can extract analytes across a wide range of polarity. SUPRASs are naturally non-selective extractants but can and have been tailored to remove major matrix macrocomponents such as carbohydrates, proteins, and humic acids. The researchers said that to their knowledge, SUPRASs had not previously been applied to wide screening analysis based on LC–HRMS.

This study employed a SUPRAS made of 1,2-hexanediol, sodium sulfate, and water synthesized directly in urine, with the applicability of the method demonstrated by the screening of 36 blinded and anonymized samples previously analyzed by other chromatographic methods. Some of the selected substances under analysis were alcohol, amide, amine, carboxyl, ester, ether, ketone, and sulfonyl. Interfering peaks were not observed for any of the 80 total substances measured. Between 84% and 93% of the drugs in question were sufficiently extracted, and 83% to 94% did not show matrix effects across 10 tested urines.

Moreover, in seven of the samples, there were adverse analytical findings versus results obtained by the previous and more conventional methods. Because of this enhanced specificity, as well as high recovery rate (70% to 120% in the approximately 90% of drugs successfully extracted), the researchers said using SUPRASs for LLME in preparation for LC–HRMS analysis was a valuable approach that would not necessitate any complicated changes to skills, practices, or equipment already in common use.


(1) González-Rubio, S.; Caballero-Casero, N.; Ballesteros-Gómez, A.; et al. Supramolecular solvents for making comprehensive liquid–liquid microextraction in multiclass screening methods for drugs of abuse in urine based on liquid chromatography–high-resolution mass spectrometry. J. Chromatogr. A 2023, 1701, 464061. DOI: 10.1016/j.chroma.2023.464061

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