Analysis of folates using combined affinity and ion-pair chromatography

Abstract

Publisher Summary This chapter presents the analysis of folates, using combined affinity and ion-pair chromatography. Folate is extracted from tissues under conditions that protect against oxidative degradation and γ-Glu-X carboxypeptidase degradation. Although fresh tissues are preferable, tissues that have been frozen in liquid nitrogen immediately after harvesting and stored at –70° can be used. It is important that frozen tissues not be allowed to thaw, as extensive hydrolysis of the folate polyglutamates will occur. The method described in the chapter allows simultaneous measurement of intact one-carbon and polyglutamyl derivatives of folate from tissues or food using a two-step procedure. The first step is folate purification by affinity chromatography, using immobilized bovine milk folate-binding protein (FBP). The second step uses ion-pair high-performance liquid chromatography (HPLC) to separate the folate derivatives with diode array detection to identify and quantify the folate derivatives.