[Determination of diazepam and its three metabolites in fish by pass-through solid phase extraction purification combined with ultra-performance liquid chromatography-tandem mass spectrometry].

Abstract

Objective: A stable isotope dilution-through solid phase extraction(SPE)-ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method was established for rapid determination of diazepam and its three metabolites(nordiazepam, temazepam and oxazepam) in fish.

Methods: Sample was extracted with acetonitrile, then salted out with sodium chloride and degreased by freezing centrifugation. The acetonitrile layer diluted to a ratio of 90 percents with pure water, and through C_(18)/PSA SPE column for purification. Purification solution was collected with the first 1 mL discarded, and nitrogen blow to near dry.1.0 mL initial mobile phase was used for reconstitution, and then separated by BEH C_(18)(100 mm × 2.1 mm, 1.7 μm) chromatography column using 0.1% formic acid aqueous and 0.1% formic acid methanol at gradient elution, and detected with triple quadrupole mass spectrometry.

Results: One-step purification with C_(18)/PSA SPE column would effectively reduce matrix interference, simplify operation and improve work efficiency. The linear ranges of 4 target compounds were 0.2 to 10.0 ng/mL and the correlation coefficients were greater than 0.999. The recoveries were in the range of 92.0%-120.0% with the relative standard deviations ranging from 4.5%-10.7%(n = 6). The method limits of detection were 0.2 μg/kg. Then the established method was used for the determination of 10 positive fish samples, and the content of diazepam and nordiazepam were 68.4%-100% in fish samples, and it indicated that diazepam and nordiazepam were the main biomarker.

Conclusion: The established method is simple, rapid, sensitive and selective, which is suitable for the simultaneous determination of diazepam and its three metabolites in fish.