Endocrine Abstracts

Valuable in endocrine studies, reliable quantification of multiple steroids remains an analytical challenge. The steroidogenic pathway contains many similar and same mass steroids and without separation prior to detection then this can affect the number of steroids that can be confidently measured in a sample. We developed an LC-MS/MS approach that relies upon isotopically labelled internal standards to track steroids and confirm retention times. A calibration curve covering 0.0025 – 100 ng/mL was prepared. The method allows simultaneous profiling of up to16 steroids including progestogens, androgens, glucocorticoids, mineralocorticoids and estrogens and applied it to rodent samples of both plasma and tissue in rodents of different ages and sex. A Waters I-Class Acquity UPLC system fitted with a Kinetex C18 (150 x 2.1 mm; 2.6 um) column with a mobile phase of water and methanol with 50 uM ammonium fluoride to encourage negative ion formation at 0.3 mL/min was used to separate steroids, with positive and negative ionisation through polarity switching on a Sciex QTrap 6500+ mass spectrometer. Plasma samples were extracted using supported liquid extraction in 96-well format on an SLE200 extraction plate. We assessed varying volumes, with reliable quantitation in 10 uL up to 100 uL, with corticosterone detectable in as little as 10 uL but 100 uL profiling the majority of steroids in the method, depending on the age and sex of the rodents. The method was validated in plasma according to bioanalytical guidelines, and applied to over 100 mouse and 100 rat plasma samples. This LC-MS/MS method was also used to analyse homogenates of rodent tissue (10-50 mg), extracted through phosopholipid depletion plates. The method succesfully detected up to 14 steroids in the tissue homogenates and allowed profiling of steroids both circulating and in tissue in individual rodents.