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

Kyung Mi Joo, Seolyeong Kim, Ye Ji Koo, Miri Lee, Su Hyun Lee, Dalwoong Choi, Kyung Min Lim

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

WST-1 [Water Soluble Tetrazolium-1; 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-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 μm, 2.1 × 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 μg/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.

Original languageEnglish
Pages (from-to)412-419
Number of pages8
JournalToxicology in Vitro
Volume60
DOIs
StatePublished - Oct 2019

Bibliographical note

Funding Information:
This work was supported by the grants from National Research Foundation (NRF) No. 2018R1D1A1B07042919, 2018R1A5A2025286 and Biosolution Co.

Funding Information:
This work was supported by the grants from National Research Foundation (NRF) No. 2018R1D1A1B07042919 , 2018R1A5A2025286 and Biosolution Co .

Publisher Copyright:
© 2019 Elsevier Ltd

Keywords

  • Color interference
  • Cytotoxicity assay
  • Method validation
  • UPLC
  • WST-1 assay

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