关键词: 超高效液相色谱-四极杆-飞行时间质谱, 非法添加药物, 渔药, 快速筛查

Abstract:

With the rapid expansion of fisheries, one of the most significant limitations to the sustainable development of fisheries in China is the quality and safety of fishery products owing to the abuse of fishery drugs and the use of illegal and/or restricted chemicals in fishery drugs. A range of chemicals that are potential hazards to fishery drugs were selected for screening in this study. A comprehensive analytical method was developed, based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), for the rapid screening of 86 types of illegally added chemicals in fishery drugs. The fishery drug samples were extracted with 80% (v/v) acetonitrile aqueous solution and diluted to reduce matrix effects. The 86 target compounds were separated on an ACQUITY PREMIER HSS T3 column (100 mm×2.1 mm, 1.8 μm), with methanol and 0.1% formic acid as mobile phases, via gradient elution. The extract was directly analyzed by UPLC-Q-TOF-MS using electrospray ionization in the positive mode. The external standard method was used for quantification. In this study, the extraction reagent and purification procedure were selected to develop a simple and effective pre-treatment protocol. The effects of the chromatographic column, mobile phase, and fragmentation voltage on the separation and sensitivity of the 86 substances were evaluated to determine the optimum instrument conditions. An accurate mass database and fragment ion library were created for the rapid qualitative and quantitative analysis of the 86 illegally added chemicals in fishery drugs. The retention time, isotopic abundance and spacing, and precise mass of the principal diagnostic ion for each analyte were used for identification. The information on the fragment ions obtained from the target MS/MS profiles was compared with that from a database to ensure the accuracy of the qualitative results. The chromatographic peak area of each target analyte was used for quantification. The analytical detection was based on the retention time deviation of ±0.35 min, accurate mass deviation of ±10×10^-6, and major adduct forms, including [M+H]⁺, [M+Na]⁺, and [M+NH₄]⁺. To evaluate the matrix effects of the 86 target chemicals at varied dilution ratios, two types of antibiotics and four types of Chinese herbal medicines were selected as typical samples. Considering the instrument tolerance as well as sensitivity and accuracy of the procedure, the recommended dilution ratios for antibiotics and Chinese herbal medicines were 50 times and 10 times, respectively. Two different types of calibration curves were prepared; one was the solvent calibration curve for antibiotics and the other was the matrix calibration curve for Chinese herbal medicines. For a given concentration, the calibration curves of the 86 target chemicals were linear with correlation coefficients of at least 0.99. The recoveries ranged from 76.8% to 112.1% with relative standard deviations (RSDs) (n=3) of less than 11.7%. The limit of quantification (LOQ) ranges of the compounds in Chinese herbal medicines and antibiotics were 1-15 mg/kg and 5-75 mg/kg, respectively. To evaluate the screening detection limits (SDLs) of each compound, a mixed standard solution was added to a fishery drug sample at varied concentrations. The SDL ranges of the compounds in Chinese herbal medicines and antibiotics were 1-15 and 5-50 mg/kg, respectively. This approach resulted in SDLs that satisfy the actual screening requirements. Because of its rapid nature, simplicity, accuracy, and sensitivity, the method may be used in the high-throughput screening and identification of illegally added chemicals in many types of fishery drugs. This method was applied to a monitoring project for the quality and safety of fishery inputs in Zhejiang Province. Sixty fishery drug samples were evaluated, among which eight Chinese herbal medicine samples were found to contain unspecified ingredients and one antibiotic sample was found to be free of any active substances. Thus, an effective technical method to monitor the quality and safety of fishery drugs was developed.

Key words: ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS), illegally added chemicals, fishery drugs, rapid screening