液相色谱-串联质谱法测定葡萄中链霉素和双氢链霉素

Determination of streptomycin and dihydrostreptomycin in grapes by liquid chromatography-tandem mass spectrometry

研究系统地优化了样品前处理过程及仪器分析中影响链霉素和双氢链霉素残留分析准确度与响应灵敏度的各主要因素,建立了葡萄中链霉素和双氢链霉素残留的快速精准定量分析方法。葡萄样品经磷酸溶液(pH=2)超声提取、Oasis HLB单固相萃取柱富集净化后,采用Waters HSS T3色谱柱(100 mm×2.1 mm,1.8μm)分离,柱温35℃,进样量2μL,以0.1%甲酸水溶液-甲醇溶液(60∶40,v/v)为流动相进行等度洗脱,在正离子、电喷雾电离源多反应监测模式下测定,外标法定量。链霉素和双氢链霉素在2~400μg/L质量浓度范围内线性关系良好,相关系数(R2)分别为0.9991和0.9997;在5、10、20和40μg/kg 4个添加水平下的平均回收率为76.8%~91.9%,相对标准偏差为0.4%~10.2%;链霉素和双氢链霉素的检出限(LOD)为1μg/L,定量限(LOQ)为5μg/kg。为验证该方法的适用性,将方法适用于无籽红提、新郁葡萄、夏黑葡萄等实际样品中进行添加回收实验,链霉素和双氢链霉素的平均回收率分别为77.2%~83.9%和70.8%~78.9%,RSD为3.0%~15.6%。该法的准确度和精密度均符合葡萄中链霉素和双氢链霉素分析要求,且操作简便、准确,灵敏度高,适用于葡萄中链霉素和双氢链霉素残留量的检测分析。

Streptomycin(STR)and dihydrostreptomycin(DSTR)are two of the most common aminoglycoside antibiotics used in veterinary medicine.STR is produced by some streptomyces griseus strains,and DSTR is a derivative of STR.In recent years,STR has been widely used in grapes to induce denuclearization.However,high levels of STR may have adverse effects like serious ototoxicity and nephrotoxicity.Therefore,to ensure the quality of grapes and the health of consumers,the regulation of STR and DSTR levels in grapes is required.An analytical method was developed for the identification and quantification of STR and DSTR in grapes by liquid chromatography-tandem mass spectrometry(LC-MS/MS).STR and DSTR are highly polar compounds due to the presence of various amino and hydroxyl groups in their structure.The determination of STR and DSTR poses a considerable analytical challenge,both during sample preparation and instrument analysis.In this study,the main factors governing the response,recovery,and sensitivity of these compounds,such as the type of chromatographic column,the type and proportion of the mobile phase and extraction solvent,the dosage of sodium 1-hexane sulfonate solution,and elution solvent and its volume,were investigated during sample pretreatment and instrument analysis.The STR and DSTR residues in the grape sample were extracted by ultrasonication with a phosphoric acid solution(pH 2),and cleanup and enrichment was performed using an Oasis HLB solid phase column.The analysis was performed using a UPLC Waters HSS T3 column(100 mm×2.1 mm,1.8μm)at the column temperature of 35℃.The injection volume was 2μL.The mobile phase consisted of 0.1%formic acid aqueous solution and methanol with a volume ratio of 60∶40.ESI-MS/MS was operated in multiple reaction monitoring(MRM)mode.External standard calibration curves were used for quantification.Based on the optimized method,both analytes displayed good linearity between 2 and 400μg/L.The correlation coefficients were 0.9991-0.9997.Recoveries in spiked blank grape samples(5,10,20,and 40μg/kg)ranged from 76.8%to 91.9%,with the relative standard deviations(RSDs)less than 10.2%,in compliance with the current legislation.The limits of detection and the limits of quantification of both analytes were 1μg/L and 5μg/kg,respectively.To assess the feasibility and potential of the proposed approach for routine analyses of STR and DSTR in other kinds of grape samples,the developed method was applied to the analysis of these compounds in red grapes,xinyu grapes,and xiahei grapes.The recoveries of STR and DSTR in the three kinds of blank grape samples were 77.2%-83.9%and 70.8%-78.9%,respectively,and the RSDs ranged from 3.0%to 15.6%.The results showed that the optimized methods can yield satisfactory recoveries for the analytes in grapes.In this method,the combination of Waters HSS T3 column to overcome the difficulties of the retention and separation of these highly polar compounds in the reverse phase,avoids the use of an ion-pair additive in the mobile phase to increase their retention,which is known to cause severe contamination of the column and serious ion suppression with electrospray ionization detection.In addition,the ideal enrichment and purification effect can be achieved by adding a sodium 1-hexane sulfonate solution to the superstratum extract with the use of only Oasis HLB for sample treatment.The method described herein has the advantages of easy operation,accuracy,and selectivity,making it feasible for the identification and quantification of STR and DSTR residues in grapes.