专一性酶解-高效液相色谱-荧光检测法测定3种食用油中4种多环芳烃

Determination of 4 Polycyclic Aromatic Hydrocarbons in 3 Edible Oils by HPLC with Fluorescence Detection After Sample Pretreatment by Specific Enzymolysis

称取食用油(动物油、植物油或经用于煎炸的食用油)2.0g与pH 8.0磷酸盐缓冲溶液5mL和脂肪酶0.2g混匀后,于(37±2)℃振荡酶解2h。加入碳酸钾1.0g皂化样品中的脂肪,以及乙醇5mL使在后续的提取中降低溶液的乳化现象。于此溶液中加入水5mL及正己烷提取4种多环芳烃{PAHs,即苯并[a]蒽(BaA)、苯并[b]荧蒽(BbF)、苯并[k]荧蒽(BkF)和苯并[a]芘(BaP)}2次,每次用正己烷15mL,振荡5min,离心5min,移取上层提取液,合并后于40℃旋蒸至干。用乙腈溶解残渣并定容至1.0mL,此溶液经0.22μm滤膜过滤,取滤液供在仪器工作条件下进行高效液相色谱(HPLC)分离后测定。选择Dikma Plus C18色谱柱为固定相,以不同体积比的(A)水和(B)乙腈的混合液为流动相进行梯度洗脱。上述4种PAHs的保留时间依次为9.3,12.0,12.6,13.9min。用荧光检测器分别测定其发射荧光的强度。在此4种PAHs中,BaA的荧光发射波长(λem)为380nm,BbF、BkF和BaP的λem在406nm波长处。分别取上述3种食用油的空白样品作为基体,加入加4种PAH的混合标准溶液系列制作标准曲线,测得其线性范围在相同的区间(0.1~5.0μg·L^-1),其检出限(3S/N)为0.03μg·kg^-1。分别以3种食用油样品作为基体,按标准加入法进行回收试验,测得其回收率均在90.0%以上,测定值的相对标准偏差(n=5)均小于9.0%。

A portion(2.0g)of edible oil sample(animal oil,vegetable oil or oil after use in frying)was mixed with 5mL of pH 8.0phosphate buffer solution and 0.2g of lipase,and enzymolyzed for 2hat(37±2)℃under stirring.1.0g of K2CO3and 5mL of ethanol were added and mixed thoroughly to saponify the fat and to reduce emulsification of the solution during the following extraction.Four PAHs(BaA,BbF,BkF and BaP)in the solution after adding 5mL of water were then extracted twice with n-hexane(15mL for each extraction)by shaking for 5min,and after centrifugation for 5min,and the extracts of the upper phase were collected,combined and evaporated to dryness at 40℃.The residue was dissolved and diluted to 1.0mL with acetonitrile.The solution was filtered through 0.22μm filtering membrane and the filtrate was used for HPLC analysis under the intrumental working condition.Dikma Plus C18chromatographic column was used as the stationary phase,and mixtures of(A)water and(B)acetonitrile in various ratios were used as mobile phases in the gradient elution.Retentiom times of 9.3,12.0,12.6,13.9min were found for the 4PHAs respectively.Fluorescence detection(FLD)was made in the HPLC determination,and values of intensity of the emission fluorescence of the 4analytes were measured at theλem of 380nm for BaA and at theλemof 406nm for BbF,BkF and BaP.Standard curves for the 4analytes were prepared with the mixed standard solution of the 4PHAs added separately to the 3oil samples as matrixes,giving same linearity range between 0.1and 5.0μg·L^-1.Values of detection limits(3S/N)found were same of 0.03μg·kg^-1 for the 4PAHs.Tests for recovery were made by standard addition method using the 3oil samples as matrixes,and values of recovery found were all over 90.0%,and values of RSDs(n=5)found were all below 9.0%.