Analysis Of Triacylglycerols In Edible Oil By Pre-column Derivatization Of Bromine Addition And Reversed-phase High Performance Liquid Chromatography

Edible oils and fats are an important part of the human diet,because they can supply energy and many essential fatty acids,for example,linoleic and linolenic acids.Triacylglycerols are the main constituents of edible oils,which have a serious impact on physicochemical and nutritional properties of edible oils and fat.The profiling anlysis of TAGs is a challenging task because of the presence of numerous TAGs species with similar structure and physicochemical properties.High Performance Liquid Chromatography?HPLC?is the most widely used method in analysis of edible oils and fats.There are two main HPLC methods for the separation of TAGs species:non-aqueous reversed-phase HPLC?NARP-HPLC?and silver-ion HPLC?Ag-HPLC?.In NARP-HPLC mode,the retention of TAGs depends on equivalent carbon numbers?ECNs?.Whereas,TAGs are separated mainly according to the number and position of double bonds?DBs?in Ag-HPLC mode.Both these two methods are difficult to separate TAGs with similar structures.Therefore,the efficient separation method of TAGs is still an important issue in the analysis of edible oil.In this study,we established a pre-column derivatization method using bromine addition reaction for improving the separation selectivity of TAGs by NARP-HPLC.The derivative reaction conditions and HPLC conditions were investigated.Oil sample was dissolved in n-hexane to 5mg/mL,and then was reacted with Br₂-CCl₄?1:1,v/v?solution at room temperature in dark place.The derivatization reaction time is 3 to 4 hours.The mixture was chromatographically separated using a C18 column at 25?and isocratic elution with methanol:n-hexane?91.5:8.5;v/v?.Elution was performed at a solvent flow rate of 1mL/min.Detection was accomplished with a variable wavelength UV detector?VWD?at 230nm.The¹H-NMR experiment result of the reacted mixture indicates that the derivatization was well done and the C=C bonds were completely reacted.The analytical result of triolein using HPLC shows that the derivatized product is mainly a single product.The analysis by FT-MS shows almost no odd number of bromine in the derived TAG compounds.The separation chromatograms of sunflower oil display that the chromatographic peaks increase from original about 4 to about 9 after derivatization,which means the separation selectivity of TAGs has been significantly improved.Simultaneously,five peaks of derived TAGs and possible numbers of bromine addition were identified from the mass spectra considering m/z values and characteristic fragment ions.The method was applied to the profiling of TAGs of22 kinds of vegetable oils,and three kinds of animal oil?lamb fat,chicken fat,lard?.The results show that the same kind of edible oil from different manufacturers is similar in TAG profile,whereas there are significant differences in TAGs composition of different kinds of edible oil.Therefore,HPLC separation can be improved by pre-column bromine addition derivation and used for recognizing different edible oils and fats.The bromine addition pre-column derivatization method presents simple,rapid,reproducible and complete reaction with TAG.Combing HPLC,the method offers a useful technique for profiling of TAG and recognizing different edible oils and fats.