[Determination of diazepam and its three metabolites in fish by pass-through solid phase extraction purification...

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.

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.

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.

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