Study On The Qualitative And Quantitative Analysis Methods Of Components And Plasticizer DBP In The Production Of DHA Glycerides Produced By Schizochytrium Sp.

Docosahexaenoic acid(DHA)is an essential fatty acid of the human body,mainly in the form of triglycerides(TAGs)in marine fish and marine micro-organisms.DHA has many functions such as preventing and treating cardiovascular diseases,promoting brain development,anti-inflammatory,anti-cancer,and improving eyesight,etc.It has an important influence on human health.The human body cannot supplement enough DHA through diet.Therefore,the industrial production of DHA supplements that can be taken is of great significance.Schizochytrium sp.is one of the raw materials for the industrial production of DHA glycerides.Establish a qualitative and quantitative analysis method of DHA glyceride components produced by Schizochytrium sp.,the detection method of the plasticizer din-butyl phthalate(DBP)content in DHA glyceride,and explore the factors of DBP migration,have important guiding significance for the industrial production of highcontent and high-quality DHA glycerides.The research content of this paper is as follows:A gas chromatography(GC)analysis method for DHA glycerides was established.DHA glyceride and KOH-methanol solution undergo esterification reaction to generate DHA methyl ester.The chromatographic conditions are as follows: chromatographic column is Agilent HP-5(30 m×0.32 mm,0.25 μm),column temperature adopts program temperature increase,sampler temperature is 250℃,FID detector temperature is 270℃,nitrogen flow rate is 1m L/min.The area normalization method is used for quantification,which is fast,efficient and accurate,and is suitable for the qualitative and quantitative analysis of DHA glycerides in industrial production.The results of GC analysis showed that the contents of docosapentaenoic acid(DPA)and DHA in DHA glycerides were 14%and 75%,respectively.A reversed-phase high performance liquid chromatography(RP-HPLC)analytical method for simultaneous determination of fatty acids(FAs),monoglycerides(MAGs),diglycerides(DAGs)and triglycerides(TAGs)in DHA glycerides was established.The chromatographic conditions are as follows: chromatographic column is Shimadzu Shimpack GIST C18(150 mm×2.1 mm,5 μm),mobile phase is acetonitrile-water solution,using multi-stage gradient elution,flow rate is 0.3 m L/min,column temperature is 30℃,detection wavelength is 210 nm.The area normalization method is used for quantification.This method does not require sample pretreatment,has good reproducibility,and accurate qualitative and quantitative results.A reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry(RP-HPLC-ESI-MS)analysis method for DHA glycerides was established.The chromatographic conditions were as follows: chromatographic column is Shimadzu Shim-pack GIST C18(150 mm×2.1 mm,5 μm),mobile phase is acetonitrilewater solution,10 mmol ammonium acetate is added to acetonitrile,using multi-stage gradient elution,flow rate is 0.3 m L/min,column temperature is 30℃,detection wavelength is 210 nm.Mass spectrometry conditions were as follows: positive ion full scan mode,interface voltage 4 k V;negative ion full scan mode,interface voltage-3 k V;positive ion product ion scan mode,collision energy 15 e V～35 e V.This method has good reproducibility and high accuracy,and does not require standard samples and sample pretreatment.At the same time,using the positive and negative ion full scan mode combined with the positive ion product ion scan mode,direct sample injection for qualitative and quantitative analysis of FAs,DAGs,TAGs and FAs in the sn-1,2,3positions.The qualitative and quantitative analysis method of reversed-phase high performance liquid chromatography-mass spectrometry(RP-HPLC-MS/MS)for the plasticizer di-nbutyl phthalate(DBP)in DHA glycerides was established.The sample was extracted with acetonitrile.Chromatographic conditions: chromatographic column is Shimadzu GIST C18(150 mm×2.1 mm,5 μm),mobile phase is acetonitrile-water solution,gradient elution,column temperature is 40℃,flow rate is 0.3m L/min.Mass spectrometry conditions: positive ion multiple reaction monitoring scan(MRM)detection,external standard method for quantification.The linear correlation coefficient was 0.9994,the qualitative limit was 0.18 μg/L,the standard spike recovery was 86.31～114.08%,and the relative standard deviation(RSD)was 1.70～6.10%.The analysis found that the main reason for the increase in DBP of DHA glyceride products was the migration of DBP in the plastic hose and esterification enzyme during the production.Avoid using plastic hoses.and esterification enzyme is treated with a special solvent to remove DBP.The DBP migration rate is reduced from 80.42 mg/kg to 8.37 mg/kg.The DBP content in the final DHA glyceride product is less than 0.11 mg/kg.