Quantitative analysis of a bacteria-derived antibiotic in nematode-infected insects using HPLC–UV and TLC–UV methods

Abstract 3,5-Dihydroxy-4-isopropylstilbene (ST), an antibiotic produced by the bacterial symbiont Photorhabdus luminescens of the nematodes of the genus Heterorhabditis was determined quantitatively in nematode bacterium-infected insects using HPLC or TLC for separation and UV for quantification. Comparable and reproducible results were obtained with both HPLC–UV and TLC–UV methods. Several factors, including solvents for extraction of the antibiotic from the infected insects, eluents for TLC development and programs for HPLC operation, were investigated. Of the four solvents used, namely acetone, methanol, ethyl acetate and diethyl ether, acetone had the highest extraction efficacy, and the ST recovery rate was about 95%. ST can be easily separated from all other bacterial metabolites on a TLC plate using a mixture of chloroform–methanol (98.5:1.5) or by HPLC using acetonitrile and water as the mobile phase.