Study On The Standard Detection Technology Of Conjugated Linoleic Acid Glycerides

Objective: This study was designed to prepare high purity conjugated linoleic acid triglycerides,1,3-conjugated linoleic acid diglycerides,1,2-conjugated linoleic acid diglycerides and conjugated linoleic acid monoglycerides which things can provide standards for the detection of conjugated linoleic acid glycerides.Established aqualitative and quantitative method for the determination of conjugated linoleic acid glyceride using high performance liquid chromatography-Evaporative light-scattering detector(HPLCELSD).Methods: With safflower oil as a raw material,Conjugated linoleic acid was prepared by alkali isomerization method,and the conjugated linoleic acid was purified by urea inclusion method.The conjugated linoleic acid glycerides was prepared by esterification reaction with conjugated linoleic acid and glycerol under the solvent-free system using lipase Novozyme435 as catalyst.The conjugated linoleic acid glycerides were separated and purified by column chromatography,thin layer chromatography and liquid phase chromatography.The high purity conjugated linoleic acid triglycerides,1,3-conjugated linoleic acid diglycerides,1,2-conjugated linoleic acid diglycerides and conjugated linoleic acid monoglycerides were prepared as reference substance.The structure and purity of four conjugated linoleic acid glycerides were identified by infrared spectroscopy,gas chromatography and liquid chromatography.HPLC-ELSD method was established to analyse conjugated linoleic acid glycerides by optimizing the conditions such as column,mobile phase and detector parameters.Results: The conjugated linoleic acid samples with high content of up to 95% were prepared by alkali isomerization method and purified by urea inclusion method.The conjugated linoleic acid triglycerides,1,3-conjugated linoleic acid diglycerides,1,2-conjugated linoleic acid diglycerides and conjugated linoleic acid monoglycerides were prepared by enzyme synthesis,and they can be used as reference substances after separated and purified.The structures of the four conjugated linoleic acid glycerides were detected by infrared spectroscopy and found contain carbonyl groups,C = C-C = C,cis CHR = CHR’,trans CHR = CHR’ and other groups.The content of CLA was more than 99% in the four conjugated linoleic acid glycerides which determined by gas chromatography.The purity of the four conjugated linoleic acid glycerides which determined by liquid chromatography reached 99%.The HPLC-ELSD method of conjugated linoleic acid glycerides shows good linearity and short detection time.The detectable limits for four conjugated linoleic acid glycerides were 0.06、0.20、0.20、0.25 mg/kg and their quantitative determination limits were 0.6、1.2、1.2、1.2 mg/kg.The relative standard deviation were between 1.46%-4.71%.Conclusion: High purity conjugated linoleic acid triglycerides,1,3-conjugated linoleic acid diglycerides,1,2-conjugated linoleic acid diglycerides,conjugated linoleic acid monoglycerides which can used as reference substances were successfully prepared.The structure of four conjugated linoleic acid glycerides was confirmed by infrared spectroscopy.The purity of four conjugated linoleic acid glycerides was confirmed by gas chromatography and liquid chromatography.The HPLC-ELSD method for the determination of conjugated linoleic acid glycerides was established.This method has the advantages of easy operation,good linear correlation with double logarithm,good detection precision and high sensitivity.Conjugated linoleic acid glycerides products can be detected rapidly and this method has a guiding significance for production of glycerol ester products.