Supramolecular solvents with restricted-access properties (SUPRAS-RAMs) are proposed as a new approach for integrating extraction and sample cleanup in the quantification of benzodiazepines (BDZs) in urine by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The SUPRAS-RAM was synthesized in situ in the urine by the addition of 1-hexanol (154 µL) and tetrahydrofuran (THF) (600 µL). BDZ extraction was driven by both hydrogen bonds and dispersion interactions. Removal of proteins and polar macromolecules was performed by the action of the SUPRAS through chemical and physical mechanisms. Phospholipids were removed by precipitation during SUPRAS extract evaporation. A multivariate method was used for the optimization of the extraction process by applying Box-Behnken response surface design. The proposed method was validated according to the guiding principles of the European Commission Decision (2002/657/EC). Method detection and quantification limits for the target BDZs were in the intervals 0.21-0.85 and 0.67-2.79 ng/mL, respectively. The repeatability and reproducibility (expressed as relative standard deviations) were in the range 2-6% and 3-8%, respectively. The method enabled the simultaneous extraction of BDZs (recoveries in the range 84-105%) and the removal of matrix effects. The method was applied to the analysis of 13 urine samples using external calibration. Five out of 13 samples tested positive in alprazolam and lorazepam at concentrations in the range 5.4-74 ng/mL. The method allows simple and quick sample treatment with minimal solvent consumption while delivering accurate and precise data.
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