Ecdysteroids represent a large class of polyhydroxylated steroids which, due to their anabolic properties, are marketed as dietary supplements. Some ecdysteroids also act as important hormones in arthropods, where they regulate molting, development, and reproduction and many of these insects are miniature organisms that contain submicroliter levels of circulating biofluids. Analysis of ecdysteroids is further complicated by their very low abundance, large fluctuations during development, and difficult access to a pooled sample, which is important for quantitative measurements. In this work, we propose a new method that overcomes the described difficulties and allows validated quantification of four ecdysteroids in minimal amounts of biological material. After methanolic extraction, detectability of the ecdysteroids is increased 16- to 20-fold by conversion to their 14,15-anhydrooximes. These are further purified by pipette tip solid-phase extraction on a three-layer sorbent and subjected to HPLC-MS/MS analysis. Full validation was achieved using hemolymph from larvae of the firebug Pyrrhocoris apterus as a blank matrix and by the determination of ecdysteroids in a single Drosophila larva. The lower limit of quantifications for the four target ecdysteroids (20-hydroxyecdysone, ecdysone, makisterone A, and 2-deoxyecdysone) were 0.01; 0.1; 0.05; and 0.025 pg·ml-1 (20; 200; 100; 50 fmol ml-1), respectively, with very good accuracy, precision (expressed as relative standard deviation <15%) and recoveries (96%-119.9%). The application potential of the new method was demonstrated by quantification of ecdysteroids in various biological materials including human serum.

Laboratory of Analytical Biochemistry and Metabolomics Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Analytical Biochemistry and Metabolomics Biology Centre Czech Academy of Sciences České Budějovice Czech Republic; Department of Chemistry of Natural Compounds Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Prague Czech Republic

Laboratory of Analytical Biochemistry and Metabolomics Biology Centre Czech Academy of Sciences České Budějovice Czech Republic; Faculty of Health and Social Sciences Institute of Laboratory Diagnostics and Public Health University of South Bohemia České Budějovice Czech Republic

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Slama K., Lafont R. Insect hormones - ecdysteroids - their presence and actions in vertebrates. Eur. J. Entomol. 1995;92:355–377.

Parr M.K., Botrè F., Naß A., Hengevoss J., Diel P., Wolber G. Ecdysteroids: a novel class of anabolic agents? Biol. Sport. 2015;32:169–173. PubMed PMC

Parr M.K., Zhao P., Haupt O., Ngueu S.T., Hengevoss J., Fritzemeier K.H., et al. Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone. Mol. Nutr. Food Res. 2014;58:1861–1872. PubMed

James L., Nation S. Insect Physiology and Biochemistry. 3rd ed. CRC Press; Boca Raton, FL: 2015.

Isenmann E., Ambrosio G., Joseph J.F., Mazzarino M., de la Torre X., Zimmer P., et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Arch. Toxicol. 2019;93:1807–1816. PubMed

Karatt T.K., Sathiq M.A., Laya S., Ajeebsanu M.P.M., Karakka Kal A.K., Subhahar M.B., et al. An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry. Rapid Commun. Mass Sp. 2023;37 PubMed

Budesinsky M., Vokac K., Harmatha J., Cvacka J. Additional minor ecdysteroid components of Leuzea carthamoides. Steroids. 2008;73:502–514. PubMed

Hunyadi A., Herke I., Lengyel K., Bathori M., Kele Z., Simon A., et al. Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting. Sci. Rep. 2016;6 PubMed PMC

Thiem B., Kikowska M., Maliński M.P., Kruszka D., Napierała M., Florek E. Ecdysteroids: production in plant in vitro cultures. Phytochemistry Rev. 2017;16:603–622. PubMed PMC

Petryk A., Warren J.T., Marqués G., Jarcho M.P., Gilbert L.I., Kahler J., et al. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proc. Natl. Acad. Sci. U. S. A. 2003;100:13773–13778. PubMed PMC

Lavrynenko O., Rodenfels J., Carvalho M., Dye N.A., Lafont R., Eaton S., et al. The ecdysteroidome of Drosophila: influence of diet and development. Development. 2015;142:3758–3768. PubMed

Niwa R., Niwa Y.S. Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond. Biosci. Biotechnol. Biochem. 2014;78:1283–1292. PubMed

Blais C., Blasco T., Maria A., Dauphin-Villemant C., Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2010;878:925–932. PubMed

Cardinal-Aucoin M., Rapp N., Steel C.G. Circadian regulation of hemolymph and ovarian ecdysteroids during egg development in the insect Rhodnius prolixus (Hemiptera) Comp. Biochem. Physiol. A, Mol. Integr. Physiol. 2013;166:503–509. PubMed

FDA . U.S. Department of Health and Human Services Food and Drug Administration; Silver Spring, MD: 2018. Bioanalytical Method Validation Guidance for Industry; pp. 1–44.

EMA . European Medicines Agency; United Kingdom: 2011. Guideline on bioanalytical method validation; pp. 1–23.

Ványolós A., Bathori M. New perspectives in the analysis of ecdysteroids: a promising group of biologically active compounds. Curr. Pharm. Anal. 2008;4:162–175.

Yamanaka N., Rewitz K.F., O'Connor M.B. Ecdysone control of developmental transitions: lessons from Drosophila research. Annu. Rev. Entomol. 2013;58:497–516. PubMed PMC

Hodgetts R.B., Sage B., O'Connor J.D. Ecdysone titers during postembryonic development of Drosophila melanogaster. Dev. Biol. 1977;60:310–317. PubMed

Martin-Creuzburg D., Westerlund S.A., Hoffmann K.H. Ecdysteroid levels in Daphnia magna during a molt cycle: determination by radioimmunoassay (RIA) and liquid chromatography-mass spectrometry (LC-MS) Gen. Comp. Endocrinol. 2007;151:66–71. PubMed

Le Bizec B., Antignac J.-P., Monteau F., Andre F. Ecdysteroids: one potential new anabolic family in breeding animals. Analytica Chim. Acta. 2002;473:89–97.

Bielby C.R., Morgan E.D., Wilson I.D. Gas chromatography of ecdysteroids as their trimethylsilyl ethers. J. Chromatogr. A. 1986;351:57–64.

Destrez B., Pinel G., Monteau F., Lafont R., Le Bizec B. Detection and identification of 20-hydroxyecdysone metabolites in calf urine by liquid chromatography-high resolution or tandem mass spectrometry measurements and establishment of their kinetics of elimination after 20-hydroxyecdysone administration. Anal. Chim. Acta. 2009;637:178–184. PubMed

Hikiba J., Ogihara M.H., Iga M., Saito K., Fujimoto Y., Suzuki M., et al. Simultaneous quantification of individual intermediate steroids in silkworm ecdysone biosynthesis by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2013;915-916:52–56. PubMed

Venne P., Yargeau V., Segura P.A. Quantification of ecdysteroids and retinoic acids in whole daphnids by liquid chromatography-triple quadrupole mass spectrometry. J. Chromatogr. A. 2016;1438:57–64. PubMed

Dinan L., Balducci C., Guibout L., Foucault A.S., Bakrim A., Kumpun S., et al. Ecdysteroid metabolism in mammals: the fate of ingested 20-hydroxyecdysone in mice and rats. J. Steroid Biochem. Mol. Biol. 2021;212 PubMed

Ambrosio G., Joseph J.F., Wuest B., Mazzarino M., de la Torre X., Diel P., et al. Detection and quantitation of ecdysterone in human serum by liquid chromatography coupled to tandem mass spectrometry. Steroids. 2020;157 PubMed

Lavrynenko O., Nedielkov R., Moller H.M., Shevchenko A. Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila. J. Lipid Res. 2013;54:2265–2272. PubMed PMC

Liu S., Sjövall J., Griffiths W.J. Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes. Rapid Commun. Mass Sp. 2000;14:390–400. PubMed

Galyautdinov I.V., Ves’kina N.A., Afon’kina S.R., Khalilov L.M., Odinokov V.N. Synthesis of 20-hydroxyecdysone oxime, its diacetonide, and their 14,15-anhydro derivatives. Russ. J. Org. Chem. 2006;42:1333–1339.

Rappsilber J., Mann M., Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007;2:1896–1906. PubMed

Pesek J.J., Matyska M.T. Ammonium fluoride as a mobile phase additive in aqueous normal phase chromatography. J. Chromatogr. A. 2015;1401:69–74. PubMed

Takkis K., Aro R., Kõrgvee L.-T., Varendi H., Lass J., Herodes K., et al. Signal Enhancement in the HPLC-ESI-MS/MS analysis of spironolactone and its metabolites using HFIP and NH4F as eluent additives. Anal. Bioanal. Chem. 2017;409:3145–3151. PubMed PMC

Gaudl A., Kratzsch J., Bae Y.J., Kiess W., Thiery J., Ceglarek U. Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair. J. Chromatogr. A. 2016;1464:64–71. PubMed

Kamba M., Mamiya Y., Sonobe H., Fujimoto Y. 22-deoxy-20-hydroxyecdysone and its phosphoric ester from ovaries of the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 1994;24:395–402.

Guerriero A., Pietra F. Isolation, in large amounts, of the rare plant ecdysteroid Ajugasterone-C from the mediterranean zoanthid Gerardia savaglia. Comp. Biochem. Physiol. B: Comp. Biochem. 1985;80:277–278.

Parvy J.P., Blais C., Bernard F., Warren J.T., Petryk A., Gilbert L.I., et al. A role for βFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster. Dev. Biol. 2005;282:84–94. PubMed

McKinney D.A., Strand M.R., Brown M.R. Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids. Gen. Comp. Endocrinol. 2017;253:60–69. PubMed PMC

Wainwright G., Prescott M.C., Lomas L.O., Webster S.G., Rees H.H. Development of a new high-performance liquid chromatography-mass spectrometric method for the analysis of ecdysteroids in biological extracts. Arch. Insect Biochem. Physiol. 1997;35:21–31.

Ovarian transcriptome analyses indicate that weak juvenile hormone signaling underlies the molecular basis of oogenesis deficiencies in mosquitoes