Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS |
| |
Authors: | Radim Simerský Ondřej Novák David A Morris Vladimír Pouzar Miroslav Strnad |
| |
Institution: | (1) Laboratory of Growth Regulators, Faculty of Science, Palacky University & Institute of Experimental Botany AS CR, Šlechtitelů 11, 78371 Olomouc, Czech Republic;(2) Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nám. 2, 16610 Prague 6, Czech Republic |
| |
Abstract: | We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones
and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined
with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ4-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked
immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10−10 mol L−1 and cross-reactivity with most Δ4-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts
prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry UPLC-ESI(+)-MS/MS]. In
multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to
10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples
spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting
and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species,
that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations
comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone,
were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant
concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea. |
| |
Keywords: | Ultra-performance liquid chromatography (UPLC) Tandem mass spectrometry (MS/MS) Immunoaffinity purification Steroids Plant extracts Digitalis purpurea Nicotiana tabacum Inula helenium |
本文献已被 SpringerLink 等数据库收录! |
|