Simultaneous quantification of individual intermediate steroids in silkworm ecdysone biosynthesis by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring

The concentration changes of endogenous ecdysteroids are closely related to the regulation of insect growth and development. Although they are frequently measured by immunoassays with anti-steroid antibodies, the separate estimations of the individual concentrations of ecdysone and other ecdysteroids with similar chemical structures are quite difficult to accomplish. In this study, an efficient method for the simultaneous, individual quantification of intermediate steroids in ecdysone biosynthesis was developed, using LC–MS/MS. By employing multiple reaction monitoring (MRM) in the MS detection, the selectivity and sensitivity of the method were greatly enhanced, allowing the estimation of trace amounts of steroids in biological samples from silkworm prothoracic glands and hemolymph.

► A quantification method for intermediates in ecdysone biosynthesis was developed.
► The method employs the LC–MS/MS with multiple reaction monitoring.
► All of the intermediate ecdysteroids can be quantified simultaneously.
► The method's sensitivity is comparable to those of conventional RIA and ELISA.
► The method was applied to biological samples from silkworms during their life cycle.