| Blueberry is one of the fruits with high economic value,which is popular among people.It is not only because of its unique flavor,but also because it contains many active substances.The antioxidant effect of phenolic acids in the fruit of the blueberry is second to that of anthocyanin.Chlorogenic acid,as the highest content of phenolic acid in the fruit of the blueberry,has many biological activities and application value.At present,there are many methods for the detection of chlorogenic acids,but few researches on the chlorogenic acids in the fruits of the blueberry are found,and there are no methods for the extraction,separation and identification of chlorogenic acids in the fruits of the blueberry.In this paper,the extraction technology,separation and purification technology and detection and analysis conditions of chlorogenic acid,including chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A and isochlorogenic acid B,were systematically optimized in this paper,which laid a foundation for the further study of chlorogenic acids in the fruit of yueorange,It can also provide reference for the future study on the determination methods of chlorogenic acids and other phenolic acids in the fruits of blueberry.The main results are as follows:1.The best extraction technology of five chlorogenic acids in blueberry was determined in this experiment:the extraction method was liquid nitrogen freezing grinding,the extraction solvent was 90%methanol,the extraction method was ultrasonic extraction method,the extraction time was 10 min.2.The best purification process of five chlorogenic acids in blueberry was determined in this experiment:the HLB adsorbent was used as the column filling material of solid-phase extraction,5 ml methanol was added for activation,then 5 ml of water was added to activate,and 5 ml water was added to the rotating flask for three times,and then 2.3 ml methanol was added to the solid-phase extraction column for collection and volume was determined to 5 ml.3.The optimum conditions for the determination of five chlorogenic acids in the fruits of blueberry were determined by this experiment:the wavelength is 320 nm,the chromatographic column is Waters CORTECS~?UPLC~?Shield RP18(2.1 mm×150 mm,1.6?m),the same protective column is used,the column temperature is 40?,the flow rate is 0.3 m L/min,and the injection volume is 1?L.The mobile phase A is 0.5%formic acid solution,the mobile phase B is acetonitrile,the elution procedure is 100%a solution(0 min)?90%A solution(1 min)?80%A solution(10 min)?75%A solution(16 min)?60%A solution(18 min)?10%A solution(19 min)?10%A solution(20 min),21 min returned to the initial state,equilibrium for 8 min.4.In this study,the ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)is established to determine chlorogenic acids in blueberry fruits.The linear correlation coefficient is above 0.99,the detection limit(0.011?g/m L?0.033?g/m L)and the limit of quantification(0.035?g/m L?0.108?g/m L)are good.The recovery is over 86%.The method has high precision and good stability,and the matrix effect is weak.5.This method can detect five chlorogenic acids in blueberry fruits:this method is used to detect five chlorogenic acids in six blueberry varieties,the results show that the total content of the five chlorogenic acids in the fruits of"Northland"is the highest among the six varieties of bilberry.It is 204.23 mg/100g.The total content of the five chlorogenic acids in the"O'Neill"fruit is the lowest among the six bilberry fruits,at 67.75 mg/100g.Among the five chlorogenic acids,the content of chlorogenic acid is the highest,accounting for 97.65%to 99.45%. |
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