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A novel approach for the simultaneous quantification of eight benzodiazepines (BZDs) using dispersive liquid-liquid microextraction (DLLME) and field-amplified sample stacking (FASS) combined with micellar electrokinetic chromatography (MEKC) was investigated and evaluated in the context of precision, accuracy, sensitivity, linearity, detection and limits of quantification (LOQ). The absolute recovery rates of BZDs were above 90.65%. The limits of detection (LOD) were 20 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 30 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam, while the LOQ was set at 50 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 100 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam. Linearity was confirmed in the range of 50-2,000 ng/mL for chlordiazepoxide, estazolam, temazepam and midazolam, and 100-2,000 ng/mL for clonazepam, lorazepam, lormetazepam and medazepam, with a correlation coefficient greater than 0.9987 for all analytes. The elaborated procedure meets all the requirements of analytical methods. During the extraction procedure, a mixture of 1 mL of ethanol and 500 µL of dichloromethane, used as the disperser and extraction solvent, respectively, was rapidly injected into 3 mL of a urine sample. A significant improvement in sensitivity was achieved when DLLME was used to extract BZDs from the urine sample and FASS as an on-line preconcentration technique was developed. For the best separation of analytes, the running buffer was composed of 30 mM SDS, 10 mM sodium tetraborate and 15% methanol (pH 8.8), whereas a sample buffer was composed of 10 mM SDS and 2 mM sodium tetraborate. Moreover, a fused-silica capillary [inner diameter (i.d.) of 75 µm and length of 50 cm], photodiode array detection, pneumatic injection for 15 s and a voltage of 23 kV were applied. The applicability of the method has been confirmed for the analysis of BZD in urine samples collected from patients who were treated with or abused these drugs.

Simultaneous Separation of Eight Benzodiazepines in Human Urine Using Field-Amplified Sample Stacking Micellar Electrokinetic Chromatography