A fluorimetric assay for cortisol.
A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 muM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at lambda (ex): 460 nm; lambda (em): 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.
Appel
Daniel
D
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Schmid
Rolf D
RD
Dragan
Calin-Aurel
CA
Bureik
Matthias
M
Urlacher
Vlada B
VB
D001711:Biotransformation; D003350:Cortodoxone / Q000737:chemistry; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006854:Hydrocortisone / Q000032:analysis*; D006900:Hydroxylation; D008956:Models, Chemical; D010422:Pentanones / Q000737:chemistry; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013256:Steroids / Q000032:analysis / Q000737:chemistry
D005456:Fluorescent Dyes; D010422:Pentanones; D012997:Solvents; D013256:Steroids; D003350:Cortodoxone; D006854:Hydrocortisone