Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Delta 4- and Delta 7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Delta 4- and Delta 7-DA have important biological activities and are produced by two different biological pathways in C elegans. Here we have described a fast separation method for these two isomers using a 13 mu m core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Delta 4- and Delta 7-DA in C elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.