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.

Keywords: Drosophila melanogaster; Girard reagent; ecdysone.

Fig. 1.

Derivatization of ecdysteroids by Girard reagents. Ecdysteroids - ecdysone: R1 = R6 = OH; R2 = R3 = R4 = R5 = H; 20-hydroxyecdysone: R1 = R4 = R6 = OH, R2 = R3 = R5 = H; makisterone A: R1 = R4 = R6 = OH, R2 = R3 = H, R5 = CH3; 2-deoxy-20-hydroxyecdysone: R4 = R6 = OH, R1 = R2 = R3 = R5 = H; polypodine B: R1 = R2 = R4 = R6 = OH, R3 = R5 = H; ponasterone A: R1 = R4 = OH, R2 = R3 = R5 = R6 = H. Girard reagents - Girard P: R = pyridine; Girard T: R = trimethylamine; Girard C: R = COOH. Structure of the end product of ecdysone derivatization (including the localization of the concomitantly produced double bond) was established by tandem mass spectrometry and NMR. Note that the reaction yields two E/Z isomers of each hydrazone.

Fig. 2.

Derivatization of ecdysone with Girard P. A: Mass spectrum acquired by direct infusion of the reaction mixture (4 h at 85°C). B: Relative abundance of dh-G(p)E (shown in arbitrary units) at different reaction temperatures (50°C, 70°C, and 85°C) and incubation times. The yield was determined by LC-MS/MS in MRM mode using neutral loss of pyridine as a monitored transition. The abundances of chromatographic peaks of its E/Z isomers were combined and normalized to the abundance of internal standard (muristerone A, 2.5 pM) spiked into the reaction mixture prior injection. Starting concentrations of E and G(p) were 50 μM and 0.3 mM, respectively. Error bars represent SD of two independent measurements.

Fig. 3.

Major fragmentation pathways of ecdysone. Intact [M+H]⁺ ion undergoes up to four successive water losses; in parallel, neutral loss of the hydrocarbon side chain occurs via the cleavage of C20-C22 bond followed by water loss.

Fig. 4.

Tandem mass spectra acquired from the dehydrated hydrazones of ecdysone and various Girard reagents. A: Native E. B: dh-G(p)E. C: dh-G(t)E. D: dh-G(c)E. MS/MS spectra in A–C were acquired in positive ion mode and in D in negative ion mode. Fragments designated with “-Py”, “-TMA,” and “-CO₂” are products of neutral loss of, respectively, pyridine, TMA, and carbon dioxide from corresponding molecular cations of dehydrated Girard hydrazones. Collision energies were 23, 46, 46, and 39 eV, respectively.

Fig. 5.

Fragmentation pathways of dh-G(p)E in positive mode. Experimental m/z are from the spectrum in Fig. 4D.

Fig. 6.

Fragmentation pathways of dh-G(c)E in negative mode. Experimental m/z are from the spectrum in Fig. 4C.

Fig. 7.

LC-MS/MS detection of three major endogenous ecdysteroids. XIC traces of (A, B) ecdysone, (C, D) 20-hydroxyecdysone, and (E, F) makisterone A in Drosophila pupa extract in MRM mode on a triple quadrupole mass spectrometer; individual transitions are indicated at each panel. Each ecdysteroid was monitored by two transitions: one transition (A, C, E) was specific for TMA neutral loss from dh-G(t) precursors; the second complementary transition (B, D, F) accounted for further C17-C20 cleavage followed by neutral loss of the hydrocarbon chain. Fragmentation pathways of dh-G(t) derivatives are exemplified in Fig. 5. E/Z isomers (see Fig. 1 for details) were detected by the same transitions and their peaks were chromatographically resolved. E, F: Peaks with the retention times of 32.2 min and 33.29 min correspond to E/Z isomers of makisterone A.