title: Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila.
abstract: Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; and.
authors.forename: Oksana
authors.initials: O
authors.lastname: Lavrynenko
authors.affiliation: Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Nedielkov
authors.affiliation: Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Heiko M
authors.initials: HM
authors.lastname: Möller
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; and. Electronic address: shevchenko@mpi-cbg.de.
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
mesh_terms: D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004330:Drosophila; D026461:Ecdysteroids / Q000032:analysis*; D053719:Tandem Mass Spectrometry
chemical_list: D026461:Ecdysteroids; D001622:Betaine; C012332:Girard's reagent T
keywords: Drosophila melanogaster; Girard reagent; ecdysone