Determination of Four Nitrofuran Metabolites in Gelatin Chinese Medicine using Dispersive Solid Phase Extraction - Solid Phase Extraction - Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ), semicarbazide hydrochloride (SEM) and 1-aminohydantoin (AHD) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of (A) acetonitrile and (B) 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ using LC-MS were found to be 0.2 µg/L, 0.3 µg/L, 0.2 µg/L and 0.2 µg/L, whereas their limit of quantification were 0.6 µg/L, 0.8 µg/L, 0.6 µg/L and 0.5 µg/L. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was extracted through dispersive solid phase extraction using 1,000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent), and then purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the extraction procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in 95: 5 (v/v water: acetonitrile). The recoveries of these nitrofuran metabolites under the 2 µg/kg, 5 µg/kg and 20 µg/kg spiking levels were between 85.6% and 95.6%. These metabolites after the extraction were stable at 4°C for 24 hours. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).