Multiresponse Optimisation Applied to the Development of a TLC Autography for the Detection of Tyrosinase Inhibitors.

INTRODUCTION: Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. OBJECTIVES: To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. METHODS: Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. RESULTS: The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. CONCLUSION: The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way.