A RAPID METHOD FOR THE DETERMINATION OF VITAMIN-E FORMS IN TISSUES AND DIET BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING A NORMAL-PHASE DIOL COLUMN

This paper describes a simple method for the analysis of tocopherols i n tissues by which frozen tissues -70 degrees C were pulverized at dry ice temperatures (-70 degrees C) and immediately extracted with hexan e. There was no need to remove the coeluting lipids from tissues by sa ponification, since at that level of neutral lipids in the sample, the re was no reduction in fluorescence response. For the analysis of oil, in which large amounts of neutral lipids were coextracted, a 20% redu ction of fluorescence response was observed, but the response was equa l for all tocopherol forms, and was appropriately corrected. Saponific ation was used only when tocopherol esters were present and only after an initial hexane extraction to remove the free tocopherols in order to avoid their loss by saponification, particularly non alpha-tocopher ol and tocotrienols. All the tocopherols and tocotrienols were separat ed on a normal-phase diol (epoxide) column that gave consistent and re producible results, without the disadvantages of nonreproducibility wi th silica columns, or the lack of separation with reversed-phase colum ns. The tocopherols were quantitated by using a tocopherol form not pr esent in the sample as an internal tocopherol standard, or using an ex ternal tocopherol standard if all forms were present, or when the samp le was saponified. Piglet heart and liver samples showed the presence of mainly a tocopherol, with minor amounts of beta- and gamma-tocopher ol and a tocotrienol, but no delta-tocopherol. Only small amounts of t ocopherol esters were present in the liver but not in the heart.