Steroids were extracted from 200 µL of serum by liquid/liquid extraction using MTBE (tert-methyl butyl ether) as described previously (11 (link)). DHEA sulfate (DHEAS) was extracted from 20 µL of serum after protein precipitation as described by Chadwick et al. (12 (link)).
A Waters Xevo Mass Spectrometer with an electrospray ionization source (in positive ionization mode) and an attached Acquity liquid chromatography system was used to identify and quantify the steroids. Steroids were eluted using a HSS T3, 1.8 µm, 1.2 × 50 mm column using an optimized methanol/water 0.1% formic acid gradient system. After initial analysis, samples were evaporated and derivatized to form oxime derivatives to improve the sensitivity to DHEA.
For accurate quantitation using liquid chromatography–tandem mass spectrometry, two mass transitions for each steroid analyte and its isotopically labeled internal standard were defined, followed by quantification facilitated by referring to a calibration series spanning the expected concentration range for unconjugated steroids, 0.25–500 ng/mL, and for DHEA sulfate (DHEAS), 0.25–10 µg/mL.
A Waters Xevo Mass Spectrometer with an electrospray ionization source (in positive ionization mode) and an attached Acquity liquid chromatography system was used to identify and quantify the steroids. Steroids were eluted using a HSS T3, 1.8 µm, 1.2 × 50 mm column using an optimized methanol/water 0.1% formic acid gradient system. After initial analysis, samples were evaporated and derivatized to form oxime derivatives to improve the sensitivity to DHEA.
For accurate quantitation using liquid chromatography–tandem mass spectrometry, two mass transitions for each steroid analyte and its isotopically labeled internal standard were defined, followed by quantification facilitated by referring to a calibration series spanning the expected concentration range for unconjugated steroids, 0.25–500 ng/mL, and for DHEA sulfate (DHEAS), 0.25–10 µg/mL.
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