Rapid and Simple Measurement of Retinol in Human Dried Whole Blood Spots

Abstract

We describe an improved method for the measurement of retinol in dried blood spots (DBS) on filter paper. Retinol in human DBS on filter paper was analyzed by normal phase HPLC after a simple extraction method. Retinol associated with its binding protein was eluted from the paper into aqueous solution facilitated by ultrasonic agitation. Retinol associated with retinol binding protein was denatured with acetonitrile, and then retinol was isolated in a single hexane extract and analyzed directly by HPLC. When analyzing DBS, the individual plasma volume of the spots was calculated by measuring the sodium content or by weighing the blood spots. The described method yielded low intra- and interassay variability (<6%), with sufficient sensitivity (detection limit, 0.1 micromol/L) and good recovery (97% spike). Compared with matching plasma samples, DBS retinol consistently decreased 18-23% during the 1st wk of storage. After 1 wk, retinol remained stable in the blood spots at 23 degrees C for >3 mo. In conclusion, the analysis of retinol in DBS by HPLC is comparable to retinol analysis in serum. The variability of the method was reduced by using sodium concentration to estimate sample volume. Collection of DBS for retinol analysis is appropriate under field conditions, where it is difficult to centrifuge or freeze blood samples.