Journal of Chromatography B, 878 (2010) 925–932 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography–tandem mass spectrometry Catherine Blais a,∗ , Thierry Blasco b , Annick Maria a , Chantal Dauphin-Villemant a , René Lafont c a UPMC Univ Paris 06, UMR CNRS 7622, Equipe Biogenèse des signaux hormonaux, Case 29, 7 Quai Saint Bernard, F-75005 Paris, France b UPMC Univ Paris 06, Plate-forme de Spectrométrie de Masse et Protéomique, IFR 83, Case 41, 4 Place Jussieu, F-75005Paris, France c UPMC Univ Paris 06, Laboratoire BIOSIPE ER3, Case 29, 7 Quai Saint Bernard, F-75005 Paris, France article info Article history: Received 20 August 2009 Accepted 14 February 2010 Available online 23 February 2010 Keywords: Drosophila Development Ecdysone Mass spectrometry Phytosterol Sterol abstract Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20- Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophila melanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography–mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Ecdysteroid hormones control major developmental events and reproduction of insects. The chemical diversity of these polyhy- droxylated steroids has been underlined by extensive studies using advanced analytical methods [1]. Even if 20-hydroxyecdysone (20E) is classically considered as the major steroid hormone in the fruit fly Drosophila melanogaster, the reality is more complex. As other insects, Drosophila depends exclusively on dietary sterols to produce its steroids. During larval growth of higher Diptera, ecdysteroid biosynthesis takes place in specialized cells of a com- plex endocrine structure, the ring-gland. Secreted ecdysteroids lack the hydroxy group at C-20, as the enzyme catalyzing 20- hydroxylation, ecdysone 20-monooxygenase, is not expressed in the ring-gland itself, but in various peripheral tissues including fat body [2,3]. Owing to both their inability to dealkylate 24-alkyl sterols to produce cholesterol and their capability to use different Abbreviations: 20E, 20-hydroxyecdysone; MaA, makisterone A; 20dMaA, 20- deoxymakisterone A; 24epiMaA, 24-epi-makisterone A; CE, collision energy; DP, desolvating potential; EcR, ecdysteroid receptor; EIA, enzyme immunoassay; TIC, total ion chromatogram. ∗ Corresponding author. Tel.: +33 1 44 27 65 84; fax: +33 1 44 27 65 09. E-mail address: Catherine.Blais@snv.jussieu.fr (C. Blais). sterols as substrates for their steroid hormones, Drosophila larvae consequently contain both 27C- and 28C-ecdysteroids, previously identified as 20E and makisterone A (MaA) [4,5]. The relative pro- portions of these ecdysteroids depend on the sterol composition of the larval diet [4,5]. Earlier studies demonstrated that Drosophila ring-glands produce both ecdysone and 20-deoxymakisterone A (20dMaA) (Fig. 1), but a third less polar unidentified ecdysteroid was also detected [4,6]. Drosophila larvae feed partly on yeasts, which are known to contain particular 28C-sterols (the major one being ergosterol), differing from those of higher plants by the stere- ochemistry of their 24-alkyl substituent. Whenever used without dealkylation by insects to produce ecdysteroids, such fungal sterols would not give rise to MaA, but instead to its 24-epimer, as was found to be the case for a leaf-cutting ant [7]. The presence of 24-epi-makisterone A (24epiMaA) in Drosophila was therefore an attractive hypothesis, thus prompting us to rein- vestigate the nature of Drosophila ecdysteroids. High-performance liquid chromatography coupled to enzyme immunoassay (HPLC- EIA) was used as a first step of purification and pre-identification of ecdysteroids. This very sensitive and specific technique is how- ever limited to ecdysteroids which are recognized by our currently used antisera. Recently, liquid chromatography coupled to electro- spray tandem mass spectrometry (LC–MS/MS) has been used to identify ecdysteroids in biological samples from plants [8,9] or ani- mals [10,11]. This specific technique is sensitive enough to quantify 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.02.018