Determination of 10 Benzodiazepines in Urine of Domestic Animals by HPLC-MS/MS with MSPE Using Magnetic Graphene as Adsorbent

An aliquot (10.0 mL) of the urine sample of domestic animal was taken and mixed with 50 mg of the self-prepared Fe₃O₄-G; the acidity of the mixture was adjusted to pH 8.0 by adding 10 mmol·L^-1 NaOH solution and the mixture was then extracted ultrasonically for 20 min. The solid and liquid phases were separated with magnetic iron, the liquid phase was discarded, and the solid phase was then extracted thrice with CH₃CN (2 mL were used for each extraction of 5 min). The 3 extracts were combined and evaporated to near-dryness by N₂-blowing in a water bath at 40℃. 1.0 mL of a mixture of acetonitrile and 0.1% (φ) formic acid solution (1+9) was added to dissolve the residue. The solution was filtered through 0.22 μm filtering membrane, and the filtrate was used for HPLC separation of the 10 benzodiazepines (BZDs) and their determination by MS/MS, under precribed instrumental conditions. Acquity UPLC HSS T3 column was used as stationary phase, and mixtures of (A) 0.1% (φ) formic acid solution and (B) acetonitrile containing 0.1% (φ) formic acid solution mixed were used as mobile phase in the separation by programmed gradient elution. The modes of ESI, positive-ion scanning and MRM were adopted in MS/MS analysis. As shown by the results, linear relationships between values of peak areas of the 10 BZDs and their mass concentrations were obtained in the same range of 1.0-100.0 μg·L^-1, with values of lower limits of determination (10S/N) in the range of 0.09-0.40 μg·L^-1. Using blank pig's urine sample as matrix, recovery was tested by standard addition method, giving results of recovery in the range of 74.6%-95.2%, values of RSDs (n=6) were found ranged from 2.5% to 9.4%.