一阶导数光谱法快速检测蛋黄中斑蝥黄含量的研究

Rapid Determination of Canthaxanthin in Egg Yolk by First Order Derivative Spectroscopy

土鸡蛋供不应求,不法商贩为谋取暴利将斑蝥黄添加到饲料中制造假土鸡蛋,严重损害了消费者权益。斑蝥黄和蛋黄本身的色素(叶黄素、玉米黄素、胡萝卜素)均为线性多烯分子,相似的结构和相近的分子量使得它们被同时萃取、难以分离。斑蝥黄和蛋黄色素提取液的紫外-可见吸收光谱严重重叠,无法采用传统光谱法检测。将紫外-可见分光光度法与一阶导数光谱法有机结合,经简单萃取而未分离的情况下,建立了蛋黄中斑蝥黄的快速检测方法,有效排除了蛋黄中天然色素对斑蝥黄检测的干扰。首先用乙醇和氯仿混合溶剂提取蛋黄中天然色素,用紫外-可见分光光度计对蛋黄提取液和标准斑蝥黄样品进行光谱扫描,然后用Origin软件对光谱做一阶导数处理,并用Adjacent-Averaging方法对一阶导数光谱进行平滑去除噪声处理。根据蛋黄提取液、标准斑蝥黄样品及两者混合物的一阶导数光谱特征和蛋黄提取液一阶导数光谱零交叉点位置,确定448, 467及520~579 nm可作为斑蝥黄的检测波长。用蛋黄提取液和标准斑蝥黄样品混合物的一阶导数光谱值对斑蝥黄浓度作图得工作曲线,考察了448, 467, 520及535 nm,工作曲线的线性关系及检测限,最终确定520 nm为斑蝥黄的最佳检测波长:该波长下工作曲线的线性回归方程为Y=0.001 01C+0.000 180 9,R2=0.992 9,线性范围为0~17.68μg·mL^-1,检测限为0.58μg·mL^-1。为了验证该方法在实际样品检测中的效果,取来自不同养殖场的三种鸡蛋样本进行添加回收实验,结果表明:当斑蝥黄添加量为1~5μg·mL^-1时,样品的平均回收率在96.4%~102.8%之间,相对标准偏差在2.53%~5.67%之间。该方法无需复杂的样品前处理步骤、无需大型仪器、操作简单、结果准确,成本低,能用于检测蛋黄中斑蝥黄含量。

In order to make huge profits,the illegal traders add canthaxanthin to chicken feed to make fake native eggs,which seriously damages the rights and interests of consumers.Both canthaxanthin and yolk coloring(lutein,zeaxanthin and carotene)are linear polyene molecules with similar structures and similar molecular weights;they are extracted at the same time and are difficult to separate.The ultraviolet-visible spectra of canthaxanthin and yolk extract overlap seriously,so it is impossible to detect canthaxanthin by traditional spectroscopy.In this paper,ultraviolet-visible spectrophotometry and first derivative spectrophotometry were combined to establish a rapid method for the determination of canthaxanthin in egg yolk without separation after simple extraction,the interferences of lutein,zeaxanthin and carotene on the detection of canthaxanthin were effectively eliminated.Firstly,the yellow compounds in the yolk were extracted with a mixed solvent of ethanol and chloroform.The spectra of yolk extract and canthaxanthin standard solution were scanned by ultraviolet-visible spectrophotometer.Then the spectra were processed for first-order differential processing by Origin software and were smoothed to eliminate noise by Adjacent-Averaging method.According to the first derivative spectra’characteristics of yolk extract,standard canthaxanthin sample and their mixture and the zero-cross point position of the first derivative spectrum of yolk extract,448,467 and 520~579 nm can be used as detection wavelength of canthaxanthin.The first derivative spectrum value of the mixture of yolk extract and the standard canthaxanthin solutions was plotted against the concentration of canthaxanthin to obtain the straight working line.The linear relationship and detection limit of the working line at 448,467,520 and 535 nm wavelengths were investigated.The results showed that the optimal detection wavelength of canthaxanthin was 520 nm.The linear regression equation of the working line at 520 nm was Y=0.00101C+0.0001809 and R 2=0.9929.The linear range was 0~17.68μg·mL^-1,and the detection limit was 0.58μg·mL^-1.In order to verify the effectiveness of this method in actual sample measurement,three egg samples from different producing areas were added and recovered.The results showed that the average recovery of the samples ranged from 96.4%to 102.8%,and the relative standard deviation ranged from 2.53%to 5.67%.The method does not need complicated sample pretreatment steps and large-scale instruments;it is simple,accurate and low cost and can be used to detect canthaxanthin in egg yolk.