Does the Saponification‐GC Method Underestimate Total Cholesterol Content in Samples Having Considerable Cholesterol Esters?

Abstract Cholesterol, in free or esterified form, is a sterol that is found in animal products. Free cholesterol is one of the unsaponifiable matters, so most quantification methods involve saponification of the acyl lipids under heating and alkaline conditions to obtain the unsaponifiable fraction for further GC quantification. We examined the stability of cholesterol in the harsh saponification environment and the completeness of the hydrolysis reaction of cholesterol ester. Cholesterol was found to be stable in a model saponification system. However, only about 50 % of the cholesterol ester was hydrolyzed with a long saponification time (up to 18 h). This incomplete reaction will lead to an underestimation of the total cholesterol if a sample has a significant amount of sterol esters. In this study, 5.1 % of the total sterol in egg yolk was found to be esterified cholesterol, but yolk's total cholesterol content quantified by using standard methods was lower than that determined using a commercial kit that measures total cholesterol. More research is needed to further understand the stability and reactivity of both free and esterified cholesterol in foods in order to use appropriate methodology for cholesterol quantification.