Measurement of steroid levels by GC/MS/MS v1
Authors: Ghoumari Abdel 
Published: June 2017

Steroids are determined by GC/MS/MS according to the purification protocol described by Meffre et al. (Meffre, Endocrinology, 2007) with minor modifications (Gonzalez Deniselle, Endocrinology, 2016). Briefly, plasmas (50-100 µl) and individual brains (60-100 mg) from mice are taken up for steroids quantification. -Extraction is performed by adding 10 volumes of methanol and internal standards are introduced for steroid quantification: 1 ng of 13C3-testosterone (Isoscience, PA, USA) for testosterone; 1 ng of epietiocholanolone for 5a-dihydrotestosterone, androstenediol, pregnenolone, 20a-dihydropregnenolone, 3b,5a-tetrahydroprogesterone, 3a,5a-tetrahydrodeoxycorticosterone; 1 ng of 13C3-androstenedione for androstenedione; 13C3-estradiol for estradiol; 1 ng of 13C3-progesterone for progesterone, 20a-dihydroprogesterone; 1 ng of 13C3-deoxycorticosterone for deoxycorticosterone; 1 ng of 2H6-5a-dihydroprogesterone (CDN Isotope) for 5a-dihydroprogesterone and 5a-androstanedione; 10 ng of 2H8-corticosterone for corticosterone. -Samples are purified and fractionated by solid-phase extraction with the recycling procedure (Liere et al, 2004 J Lipid Res). -The unconjugated steroids-containing fraction is filtered and further purified and fractionated by HPLC. The HPLC system is composed of a WPS-3000SL analytical autosampler and a LPG-3400SD quaternary pump gradient coupled with a SR-3000 fraction collector (Thermoscientific, USA). -The HPLC separation is achieved with a Lichrosorb Diol column (25 cm, 4.6 mm, 5 µm) in a thermostated block at 30°C. The column is equilibrated in a solvent system of 90% hexane and 10% of a mixture composed of hexane/isopropanol (85/15). -Elution is performed at a flow-rate of 1 ml/min, first 90% hexane and 10% of hexane/isopropanol (85/15) for 8 min, then with a linear gradient to 100% of hexane/isopropanol (85/15) in 2 min. -This mobile phase is kept constant for 10 min and a linear gradient to 100% MeOH is applied. -The column is washed with MeOH for 15 min. Three fractions are collected from the HPLC system: 5a-dihydroprogesterone and 5a-androstanedione were eluted in the first HPLC fraction (3-13 min) and next silylated with 50 µl of a mixture N-methyl-N-trimethylsilyltrifluoroacetamide/ammonium iodide/dithioerythritol (1000:2:5 vol/wt/wt) for 15 min at 70°C. The second fraction (13-25 min) containing testosterone, 5a-dihydrotestosterone, androstenediol, pregnenolone, 20a-dihydropregnenolone, 3b,5a-tetrahydroprogesterone, 3a,5a-tetrahydrodeoxycorticosterone, androstenedione, estradiol, progesterone, 20a-dihydroprogesterone and deoxycorticosterone is derivatized with 25 µl heptafluorobutyric anhydride (HFBA) and 25 µl anhydrous acetone for 1h at 20°C. Corticosterone is eluted in the third fraction (25-33 min) and derivatized with 25 µl HFBA and 25 µl anhydrous acetone for 1h at 80°C. -All the fractions are dried under a stream of N2 and resuspended in hexane for GC-MS/MS analysis. -Brain and plasma extracts are analyzed by GC/MS/MS with an AI 1310 autosampler (Thermo Fisher Scientific, USA). The Trace 1310 gas chromatograph is coupled with a TSQ 8000 mass spectrometer (Thermo Fisher Scientific, USA). - The mass spectrometer is used in tandem mode using Argon as collision gas. Injection is performed in the splitless mode at 250°C (1 min of splitless time) and the temperature of the gas chromatograph oven is initially maintained at 50°C for 1 min and ramped between 50 to 200°C at 20°C/min, then ramped to 300°C at 10°C/min and finally ramped to 350°C at 30°C/min. The helium carrier gas flow is maintained constant at 1 ml/min during the analysis. -The transfer line and ionization chamber temperatures are 330°C and 200°C, respectively. -Electron impact ionization is used for mass spectrometry with ionization energy of 70 eV. -GC/MS/MS signals are evaluated using a computer workstation by means of the software Excalibur®, release 3.0 (Thermoscientific, USA). - Identification of steroids is supported by their retention time and according to two or three transitions (Lamy, Theriogenology, 2016). -Quantification is performed according to the more abundant transition for the calibration solutions and for the biological extracts. -The precision was in the 95-105% range, respectively, and the limits of quantification of 0.002 ng/g. -The inter-assay coefficient of variation estimated at roughly 5-10% for both steroids. The coefficient of correlation is measured from extracts corresponding to 10, 25, 50 and 100 mg of rat brain and is in the range 0.9942-0.9975.