Determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (LPME) and gas chromatography-mass spectrometry (GC-MS)

In this study, it is shown a method for the determination of benzodiazepines and their main metabolites in urine samples by hollow-fiber liquid-phase microextraction (LPME) in the three-phase mode. Initially, the hydrolysis step was performed using 100 μL of sodium acetate 2.0 mol/L buffer solution (pH 4.5), 25 μL of β-glucuronidase enzyme and incubation for 90 min at 55 °C. In parallel with hydrolysis, the LPME fiber (9 cm) was prepared. Its pores were filled with a mixture of dihexyl ether: 1-nonanol (9:1). Afterwards, a solution of 3.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). After hydrolysis, the fiber was submersed in the alkalinized urine (pH 10) containing 10% NaCl. Samples were then submitted to orbital shaking (2400 rpm) for 90 min. The acceptor phase was later withdrawn from the fiber, dried and the residue derivatized with trifluoroacetic anhydride (TFAA) for 10 min at 60 °C with further addition of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide containing 1% tert-butyldimethylchlorosilane (MTBSTFA) for 45 min at 90 °C followed by determination by gas chromatography-mass spectrometry (GC-MS). The calibration curves obtained showed linearity over the specified range, with a similar sensitivity to traditional techniques and a higher detection capability compared to most of the miniaturized methods described in the literature. The method has been developed and successfully validated and applied to urine samples from real cases of benzodiazepines intake.