Identification of Monohydroxylated Products of 3-Keto-4-ene Steroid Hormones Based on Relative Ion Intensity Feature
In this study,the mass fragmentation routes responsible for the generation of m/z 97.06,109.06,121.06,and 123.08 fragment ions from testosterone in positive ion mode were proposed in depth.Then the impacts of hydroxyl substitution site on relative ion intensity(RII)properties of those featured fragment ions were summarized through incorporating the fragmentation pattern of testosterone and the findings described in the previous articles.RII ratio between m/z 109.06 and 97.06 signals exhibited tight correlation with D-ring hydroxylation.Greater RII of m/z 121.06 and 97.06 usually corresponded to hydroxylation at C-2 and C-7 sites,respectively.RII of m/z 121.06 displayed the lowest value in MS2 spectrum when C-6 site was hydroxylated.The monohydroxylated products of five steroid hormones,namely progesterone,testosterone,androstenedione,17α-hydroxyprogesterone and deoxycorticosterone,were characterized using liquid chromatography-hybrid triple quadrupole-tandem time of flight-mass spectrometry(LC-QTOF-MS)followingin vitroincubation with human liver microsomes in the presence of the whole NADPH-regeneration system.As a result,a total of 37 kinds of metabolites were detected and structurally deciphered based on the high-resolution m/z values of both precursor ions and fragment ions.Particularly,31 ones,including 8,6,5,8,and 4 kinds of monohydroxylated metabolites for progesterone,testosterone,androstenedione,17α-hydroxyprogesterone,and deoxycorticosterone,respectively,were confirmatively identified through fortifying the correlations between RII patterns and hydroxylation site to the high-resolution mass profiles.Moreover,the structures of two metabolites,such as 21-hydroxyprogesterone and 17α-hydroxyprogesterone,were justified by matching with authentic compounds.Above all,the identification confidences of hydroxylated products of 3-keto-4-ene steroid hormones were dramatically elevated through taking both RII features and high-resolution mass values into consideration.These findings offered a robust approach for the structural elucidation of steroid hormone metabolites,providing valuable insights into the metabolic pathways of steroid hormones and their roles in diseases,and laid the foundation for a comprehensive characterization of the steroid hormone-targeted submetabolome.
Steroid hormonesLiquid chromatography-hybrid triple quadrupole-tandem time of flight-mass spectrometryIsomersRelative ion intensity
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