溶剂萃取分离-高效液相色谱-串联质谱法测定对虾中12种抗生素的残留量

HPLC-MS/MS Determination of Residual Amounts of 12 Antibiotics in Prawn with Separation by Solvent Extraction

将对虾先经冷冻干燥,再用球磨机碾碎制成粉末状分析样品。取此样品1.00g,用体积比为1∶4的加有0.1mol·L^-1 Na2EDTA的Mcllvaine缓冲溶液(pH 4.00±0.05)-乙腈混合液每次10mL,超声提取3次,将样品中的12种抗生素(包括5种磺胺类、2种喹诺酮类、2种大环内脂类、3种四环素类)溶入提取液中。将3次提取液合并,并加入经乙腈饱和的正己烷20mL,充分振荡使可能带入提取液中的脂类杂质溶于正己烷中,离心分层后将上层正己烷取出并弃去。将下层溶液在40℃时蒸发至其体积不变,加水将溶液稀释至200mL,此时溶液中所含有机溶剂不应超过5%(体积分数),以免其在后续固相萃取(SPE)分离中穿透SPE柱而导致被测物流失。将上述200mL溶液以1.0~1.5mL·min^-1的流量通过经活化的CNWBOND LC-C18小柱进行净化,待溶液全部通过,用5%(体积分数)甲醇溶液5mL淋洗小柱,弃去全部流出液。将SPE小柱负压抽干,用甲醇6mL将被测物从小柱上洗脱,所得洗脱液用吹氮蒸至近干,用甲醇溶解残渣并定容至1.0mL。此溶液经0.22μm针式过滤器过滤,取滤液按仪器工作条件进行分析。选择ZORBAX Eclipse XDB-C18色谱柱为分离柱,用不同体积的(A)1%(体积分数)乙酸溶液和(B)甲醇组成的混合液作为流动相进行梯度洗脱。采用电喷雾(ESI)离子源并在多反应监测(MRM)模式下按不同的保留时间测得了所测定的12种药物的峰面积。此12种药物标准曲线的线性范围均为1~100μg·L^-1,其检出限(3S/N)为0.52~3.02ng·g^-1。加标回收试验给出的回收率为70.2%~102%,测定值的相对标准偏差(n=5)均小于14%。

The prawn sample was freeze-dried and ball-milled to powdery form,and 1.00g of the powdered sample was taken for analysis.The sample was extracted thrice with a mixture of Mcllvaine buffer solution containing 0.1mol·L^-1 Na2EDTA(pH 4.00±0.05)and acetoritrile at a volume ratio of 1∶4by ultrasonication and 10mL of the mixed extraction solvent were used for each extraction,to extract the 12drugs(including 5sulphonamides,2quinolones,2macrolides and 3tetracyclines)from the sample into the solvent.The 3extracts were combined and shaken thoroughly with 20 mL of n-hexane saturated with acetontrile to dissolve the fatty impurities,that might be carried over in the extract,into the n-hexane.After centrifuging,the upper layer of n-hexane was discarded and the lower layer was evaporated at 40℃until the volume of the solution was kept unchanged.The water was added to make the volume of the solution to 200mL.It should be examined at this instant if the total volume of organic solvent in the 200mL solution exceeded 5%by volume.Because when the organic solvent in the solution exceeded 5%,some of the analytes in the solution might pass through the SPE column in the follow-up SPE purification,thus leading to missing errors.The diluted 200mL solution was then purified by passing through the activated CNWBOND LC-C18 micro-column at a flow-rate of 1.0-1.5mL·min^-1.When all of the solution was passed through the micro-column,5mL of 5%(volume fraction)methanol solution were used to rinse the column.All the effluent was discarded and the SPE micro-column was dried under reduced pressure.The analytes on the column were then eluted with 6mL of methanol,and the eluate was evaporated to near-dryness by N2-blowing.The residue was taken up and its volume was made up to 1.0mL with methanol.The solution was filtered through 0.22μm needle-filter and the filtrate was used for analysis under the instrumental working conditions.ZORBAX Eclipse XDB-C18chromatographic column was selected as stationary phase in HPLC separation,and mixtures of(A)0.1%(volume fraction)acetic acid solution and(B)methanol in various ratios were used as mobile phases in the gradient elution.Peak areas of the 12drugs were measured by MS/MS at their respective retention times,under the MS/MS conditions of ESI ionization source and mode of MRM.Linearity ranges of the standard curves of the 12drugs were found same between 1-100μg·L^-1,with detection limits(3S/N)in the range from 0.52to 3.02ng·g^-1.Recovery was tested by standard addition method,giving results of recovery ranged from 70.2%to 102%.Values of RSDs(n=5)found were all less than 14%.