Dispersive liquid–liquid microextraction with back extraction using an immiscible organic solvent for determination of benzodiazepines in water, urine, and plasma samples

Dispersive liquid–liquid microextraction (DLLME) as an attractive miniaturized extraction procedure needs to be modified in order to improve its performance for extraction from complex matrices. To achieve this goal, in this study a new DLLME method was introduced coupled with a back extraction step based on using an immiscible organic solvent. By applying this methodology, during DLLME procedure the analytes were transferred into the extraction solvents, then they were separated from aqueous solution and mixed with an immiscible organic solvent (acetonitrile) as an acceptor phase for preconcentration of the target analytes. Four benzodiazepines (BZDs) were selected as model compounds for developing and evaluating the performance of the method. Gas chromatography with a microelectron capture detector (GC-μECD) was applied for separation and determination of the target drugs. Several parameters affecting the extraction efficiency including pH, type and volume of extraction and acceptor solvent, ionic strength, and extraction time were investigated and optimized. Accordingly, the enrichment factors were obtained in the range of 16–497. Linearity of the method was determined to be in the range of 0.01–200.0 μg L−1 for diazepam, 0.05–200.0 μg L−1 for midazolam, 0.02–200.0 μg L−1, and 1.0–200.0 μg L−1 for flurazepam and alprazolam in water, urine, and plasma samples, respectively. The limits of detection and quantification for the target benzodiazepines were in the range of 0.005–1.0 μg L−1 and 0.01–5.0 μg L−1, respectively. Also, intra and inter-day precisions were obtained in the range of 4.0–13.4% and 6.0–14.5%, respectively. The presented method was successfully applied for extraction and highly sensitive determination of BDZs in water, plasma, and urine samples.