Retinol analysis in dried blood spots by HPLC

There are many advantages to measuring vitamin A in dried blood spots (DBS) from a finger prick as compared to plasma collected by venipuncture, The a dvantages include easier collection, transport and storage; accessibility t o younger and more remote populations; and decreased risk of disease transm ission. We describe a method for the extraction of retinol from DBS for ana lysis by HPLC and initial comparison to plasma retinol, The effects of vari ous buffers, detergents, antioxidants and chelators were evaluated to estab lish the most effective approach to elute the retinol, retinol binding prot ein (holo-RBP) complex from the blood collection cards. The process involve s ultrasonic agitation to elute holo-RBP into a phosphate buffer containing an antioxidant and metal chelator. The holo-RBP complex was denatured by t he addition of ethanol containing additional antioxidants permitting the ex traction of free retinol into hexane, Following solvent evaporation, the ex tract was dissolved in methanol for HPLC analysis. The initial measured ret inol levels in freshly collected DBS declined for 6 -10 d whether stored at 25, 4 or -20 degrees C, but remained consistent thereafter (homeostatic). By incorporating a "recovery/volume adjustment" factor, measured retinol va lues in homeostatic DBS were adjusted to the equivalent of plasma retinol. For 17 normal adults, the correlation coefficient was 0.90 between plasma r etinol and adjusted DBS retinol in samples that had been stored at -70 degr ees C for < 9 mo. The use of this new sample matrix for vitamin A assessmen t will allow access to previously unavailable populations.