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High‐performance liquid chromatography/mass spectrometry with thermospray lonization of free ecdysteroids
Author(s) -
Evershed Richard P.,
Prescott Mark C.,
Kabbouh Mohamed,
Rees Huw H.
Publication year - 1993
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1290070615
Subject(s) - chemistry , chromatography , thermospray , high performance liquid chromatography , mass spectrometry , ecdysone , hydroxylation , fragmentation (computing) , detection limit , mass spectrum , ion , ammonium acetate , tandem mass spectrometry , organic chemistry , selected reaction monitoring , hormone , biochemistry , computer science , enzyme , operating system
High‐performance liquid chromatography/mass spectrometry (HPLC/MS) with thermospray ionization has been applied to the analysis of free ecdysteroids (polyhydroxylated steroids that serve as arthropod moulting hormones). Of a range of analytical conditions that were tested, optimum results were obtained by the use of re versed‐phase HPLC using a methanol + water mixture containing 0.1 M ammonium acetate. The positive‐ion spectra of ecdysteroids showed pseudo‐molecular ions, [M + H] + , and fragment ions corresponding to the sequential losses of hydroxyl groups. The [M + H − H 2 O] + and [M + H − H 2 O] + ions were the most abundant for all the compounds tested. Ecdysteroids bearing a C‐20 hydroxyl group (e.g. 20‐hydroxyecdysone) showed ions characteristic of cleavage of the side‐chain from the steroid nucleus. This mode of fragmentation was much less obvious in the case of ecdysteroids lacking C‐20 hydroxylation (e.g. ecdysone). Full‐scan mass spectra were readily obtained from 1 μg of free ecdysteroids, while a limit of detection of 5 ng (signal‐to‐noise ratio 3) was attained in the selected‐ion monitoring mode. The application of HPLC/MS to the study of ecdysteroids in biological materials is demonstrated by the analysis of extracts of newly laid eggs of the desert locust ( Schistocerca gregaria ) and pupae of the cotton leafworm ( Spodoptera litoralis ).