LC‐MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

Abstract

Roles of steroid hormones, and compounds that can influence their levels in cells, are of increasing interest in e.g. cancer research, partly because resistance to hormone therapies often complicates treatment. To elucidate the processes involved, the hormones and related compounds need to be accurately measured. Reversed-phase liquid chromatography with dynamic multiple reaction monitoring mass spectrometric detection in electrospray mode is capable of providing such measurements. Therefore, LC-MS/MS was developed for sensitive, selective analysis of 11 steroid hormones, cholesterol and two prostaglandins. The effects of the tissue matrix, and solid-phase extraction (SPE) sample clean-up, on the LC-MS/MS signals of the hormones were also investigated. The results show that the developed LC-MS/MS method, following SPE clean-up to reduce matrix interference, can detect selected steroids in extracts of mouse tissues. The method provides linear measurements of the steroids at concentrations up to few ng/μL, and limits of detection in the range 0.03-0.2 pg/μL (for some compounds lower than those of previously reported methods).

Analysis of ketone‐based neurosteroids by reactive low‐temperature plasma mass spectrometry

“…In parallel‐reaction monitoring mode, the most abundant product ions were m / z 97.065 for P4, T, and 17P4, m / z 121.065 for CORT, m / z 255.211 for DHT, m / z 253.195 for DHEA, and m / z 100.084 for P4‐D9. The detected fragment ions were consistent with previously reported fragment ion spectra . Detection with HPLC/MS/MS gave coefficients of determination R 2 >0.98 (Table ).…”

Mammalian Cells Engineered To Produce New Steroids

“…The substrate concentration was chosen because of the low solubility of the less hydroxylated steroids in the medium . Lipids were extracted from the spent cells and medium and measured by LC‐MS . The yield per milliliter of spent medium was determined for each product when a standard was available.…”

“…Lipids were analyzed with an Agilent 1200 series HPLC system and 6530 qTOF mass spectrometer. Steroids were separated with a Thermo Scientific Hypersil GOLD C18 column (1.9 μm, 50×2.1 mm) in an acetonitrile gradient in water with formic acid (0.1 %, v / v ) . Known products were compared with standards for quantification.…”

Mammalian Cells Engineered to Produce Novel Steroids

“…The substrate concentration was chosen because of the low solubility of the less-hydroxylated steroids in media 13 . Lipids were extracted from the spent cells and media and measured by LC-MS 14,15 . The yield per milliliter of spent media was determined for each product when a standard was available.…”

“…Steroids were separated with a Thermo Scientific Hypersil GOLD C18 column (1.9 μm, 50 x 2.1 mm) in an acetonitrile gradient in water with 0.1% formic acid v/v 15 . Known products were compared to standards for quantification.…”