Simultaneous analysis of circulating 25‐hydroxy‐vitamin D3, 25‐hydroxy‐vitamin D2, carotenoids, retinoids, tocopherols, and oxidized and reduced coenzyme Q10 by HPLC with photo diode‐array detection using C18 and C30 columns alone or in combination

Abstract

Modern studies require high‐throughput assays that provide many biomarkers from a single specimen. Several circulating lipid‐soluble agents have been shown to serve as useful biomarkers for disease prevention. We aimed to develop a simple, fast, and accurate HPLC‐diode array detection assay to simultaneously measure such agents. A C18 column alone, a C30 column alone, or these connected in series measured 25‐(OH) vitamin D2 and D3, retinol, α, β‐, γ‐ and δ‐tocopherol, all major carotenoids and their isomers, and ubiquinol‐10 and ubiquinone‐10 in a single chromatographic run. The C18 column alone resolved all analytes except the D vitamers and lycopene isomers and co‐eluted lutein with zeaxanthin and β‐ with γ‐tocopherol. The C30 column alone resolved all carotenoids and many of its isomers, β‐ and γ‐ tocopherol, and D vitamers but not the Q10 compounds. Connecting the C18 and C30 column in series with a DAD both between and after the columns resolved all analytes but the run time was unacceptably long (90 mins). This serial connection of columns but with only one DAD and a 6‐port valve between the columns resolved all analytes in 45 mins. Shortening run times in any system resulted in the resolution of fewer analytes. Our proposed methods can be custom designed to meet the specific needs of projects depending on analyte requirements (www.uhcancercenter.org/analyticallab). Supported by NCI Grant P30 CA 717890.