Study And Application On Determination Methods Of Cholesterol In Eggs

Because cholesterol content in eggs is very high, consumers have much concerns. Although the controversy about the metabolism of cholesterol in human is unkown, it is greatly necessary to determinate cholesterol contents in eggs. At present, there is no standard method to determine egg cholesterol in China. The technological conditions of ultrasonic assisted extraction which was used on extraction of egg cholesterol was optimized in this research. A rapid and easy sample preparation method that involved no-heating saponification and dSPE (dispersive solid phase extraction) clean-up was developed to determine the level of cholesterol in egg yolk. The developed method utilized high performance liquid chromatography with an ultraviolet detector (HPLC-UVD) as a separation instrument. The two methods were compared. The cholesterol contents in different kinds of eggs and egg products were determinated. Change of cholesterol contents in egg yolks during the incubation period were studied. Main results for this study are as follows:The ultrasonic extraction technology was optimized by single factor experiments, the orthogonal design. The optimal process conditions was obtained:extraction temperature,70℃; time,30min; ultrasonic power,500W. The optimal chromatographic conditions of RP-HPLC to determine cholesterol contents in egg yolk was obtained. The sample can be separated in12min. inear range of0.106-1.06mg/mL, linear range is0.106～1.06mg/mL, the correlation coefficient is greater than0.99, the method validation results show that:the RSD of method precision (RSD3.27%), reproducibility (RSD3.40%) and recovery (81.9%～90.4%, RSD4.35%) are small, the detection limit is33.3mg/kg, show that the method has good precision, good reproducibility, the results are accurate and reliable, and the sample solution to be detected at15h is relatively stable (RSD2.11%).The total cholesterol contents of duck egg, browns shell egg, white leghorn eggs, Silkie eggs, green shell eggs, Soil eggs, low-cholesterol eggs and quail eggs were determinated by this method. The cholesterol contents of Soil eggs is highest (241.85mg) and quail eggs is lowest(45.32mg)(P<0.05). However, we found that there were deficiencies such as:large quantity of organic solvent, time-consuming, low recovery, so it is not suitable for rapid determination. The dSPE extraction technology was optimized by single factor experiments, the orthogonal design. The optimal process conditions was obtained:quantity of concentrated ammonia,4g; quantity of isopropyl alcohol,1:20; quantity of (NH4)SO44g; NH2; The optimal chromatographic conditions of RP-HPLC to determine cholesterol contents in egg yolk was obtained. The sample can be separated in15min. linear range of0.1036～1.036mg/mL, the correlation coefficient is greater than0.99, the method validation results show that:the RSD of method precision (3.06%), reproducibility3.03%) and recovery (93.8%～102.5%, RSD3.56%) are small, the detection limit is30.63mg/kg, show that the method has good precision, good reproducibility, the results are accurate and reliable, and the sample solution to be detected at24h is relatively stable (RSD2.09%).Extraction purification and bound cholesterol hydrolysis can be simultaneousl by d-SPE method. Less quantity of organic solvent, not necessary to evaporation before analysis, not concentrated Saponification is completed at room temperature, can effectively avoid the oxidation of cholesterol. Finally, the two methods are compared. The results are basically the same, but the d-SPE method can significantly shorten the preparation time, effectively improve the efficiency of the analysis.The total cholesterol contents of fertilized eggs and fertilized decreased with increasing incubation time, but there is difference between the change rule of egg cholesterol. The total cholesterol of fertilized egg yolk decreased slowly firstly, but decreased fast with increasing incubation time. The total cholesterol of egg yolk is fewer changes, decline rate during13-21d was faster than4～13d (P<0.05), total cholesterol dropped to93.32mg lastly, only42.88%of original。As for infertilized eggs, the decline rate during0-13d was very small, decline rate increased gradually after13d, Total cholesterol contents dropped to134.13mg at21d. Experiments show the change of total cholesterol of the fertilized egg has negative correlation to DNA content, embryo weight and weight less; positive correlation to total fat contents.. It reveals the differences between the fertilized eggs and fertilized eggs in total cholesterol changes during incubation. It preliminary confirmed the correlation of egg yolk total cholesterol and chicken embryos growth condition and fat metabolic rate.Cholesterol content in egg products are affected by different processing ways. Compared to fresh duck eggs, cholesterol content of salted duck eggs and alkaline-preserved duck eggs increased by13.39%,14.80%(P<0.05); Compared with fresh eggs, cholesterol content of boiled eggs do not changed. Cholesterol content of tea eggs decreased significantly by22.86%(P<0.05).