Determination of Retinol by Aqueous Reverse Phase Open Column System

The AOAC method and other open column techniques which are used to determine retinol content, are time consuming, require technical skills, and do not always result in a complete separation of retinol from other fluorescent components. In some cases cis-trans isomers are formed due to the long term exposure of retinol to oxygen, light, adsorbents and solvents. The colorimetric method hes disadvantages which include a rapidly fading blue color, sensitivity of the reagent to moisture, and interference from carotenoids. The more recently developed HPLC method is rapid, reproducible, nondestructive, quantitative and gives high resolution of closely related compounds. The instrumentation, however, is expensive to purchase and maintain, uses expensive solvents and is not available to nutritional scientists in many parts of the world. Therefore there is clearly a need for a method that has the simplicity of the AOAC method coupled with the accuracy and speed of the HPLC method for the determination of retinol in food products and serum. It was found that packing the HPLC adsorbent (50 um C18) in an open column and eluting it with an isocratic, aqueous solvent system consisting of methanol and water as the mobile phase gave good separation of retinol from the extracted sample. The method was evaluated by measuring the retinal concentration in milk, infant formula, margarine, egg yolk and liver and was compared with both AOAC and HPLC methods. The method was used to measure the retinal concentration in serum as compared to the HPLC method. The retinal values of milk, infant formula, egg yolk, margarine and liver obtained by RP-C18 open column were very similar to that of AOAC and HPLC methods, and there was no significant differences among these methods when compared over a set of samples. A correlation coefficient between RP-C18 and HPLC estimates and RP-C18 and AOAC estimates were 0.993 and 0.999, respectively, indicating that a highly significant correlation exists between these methods for the determination of retinal from the different samples. The study showed that the RP-C18 method is comparable with both the HPLC and the AOAC methods for retinal analysis from these food products. The recovery study for retinal from the analyzed samples was found to be 97%, 98% and 96% for RP-C18, HPLC and AOAC respectively, indicating that a good recovery was obtained by this method. Also the study showed that the retinal values of serum obtained by RP-C18 method were very similar to that of HPLC and indicated no significant differences among the two methods when compared over a different set of samples. A correlation coefficient between the RP-C18 and HPLC estimates was 0.963 for retinal analysis, indicating a highly significant correlation between these two methods. Also the study showed that the RP-C18 method gave comparable values with the HPLC method for retinol from serum. The retinal separation from the extracted sample could be achieved in 10-15 minutes by the reverse phase open column method. The low cost and ease of operation make this method suitable for routine assays of retinal content in serum and food products. This method can be an alternative technique for clinical laboratories that cannot afford to have HPLC and to nutritional scientists in many parts of the world