[8] Separation and Quantitation of Retinyl Esters and Retinol by High-Performance Liquid Chromatography

This chapter describes the separation and quantitation of retinyl esters and retinol by high- performance liquid chromatography (HPLC). Quantitation is based on the intrinsic absorbancy of retinol at a wavelength where few other lipids interfere, and use of internal standards as a convenient means of accurate quantitation is presented. Retinyl ester separation is achieved using a two-step discontinuous gradient consisting of (1) acetonitrile–water, 88:12 (v/v) followed by (2) acetonitrile–water, 98:2. Distilled and deionized water is filtered through a Millipore filter (HAWP, 0.45/μm and mixed with an appropriate volume of HPLC-grade acetonitrile under light vacuum for approximately 0.5 hr to remove dissolved air. For retinol analysis, a single isocratic elution is used. The concentration of retinyl ester standards can be determined by spectrophotometry at 324–326 nm. Retinol is purified by chromatography on aluminum oxide before use. Accurate quantitation requires that internal standard and endogenous retinyl esters behave identically with regard to solubility in solvents used to extract or redissolve lipids. When a single internal standard, retinyl pentadecanoate, is added one found that the recovery of medium-chain esters of retinol could be overestimated under conditions where their solubility exceeded that of retinyl pentadecanoate. The chapter also discusses the preparation of samples for total vitamin A (retinol) analysis such as HPLC analysis of retinol concentration (total vitamin A) in rat plasma (A).