Development and validation of UPLC method for WST-1 cell viability assay and its application to MCTT HCE (TM) eye irritation test for colorful substances

Abstract

WST-1 [Water Soluble Tetrazolium-1; 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenye-2H-tetrazolium, monosodium salt)] is widely used in the cell viability assays replacing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A water-soluble formazan dye (4-[1-(4-Iodophenyl)-5-(4-nitrophenyl)formaz-3-yl]-1,3-benzene disulfonate, disodium salt) is produced from the reduction of WST-1 tetrazolium, of which optical density at 450 nm is measured to evaluate cell viability. Colorful substances may interfere with spectrometric measurement, and a method to specifically detect WST-1 formazan is required. Here, a simple, rapid, sensitive, and specific ultra-performance liquid chromatography coupled to UV detector (UPLC-UV) was developed and validated for the WST-1 formazan. For the application to cell viability assay, the supernatant from WST-1 assay was injected without sample preparation procedure and a single run was completed within 5 min. Chromatographic separation was achieved on BEH C18 column (1.7 mu m, 2.1 x 50 mm) using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 2.5-120 mu g/mL WST-1 formazan, which encompasses WST-1 formazan concentrations from 2% cell viability to 2 fold of 100% cell viability. The intra- and inter-day precisions were measured to be below 5% and accuracies were within the range of 91.8-104.9%. The validated method was successfully applied to the test of colorful substances in vitro eye irritation test with a human cornea-like epithelium, and in vitro cytotoxicity in HaCaT, human keratinocyte cell line.