Cortisol d4

Serum samples were collected by cardiac bleeds to assess systemic metabolism between groups; 200 µL of serum was spiked with 0.2 ng of internal standard (corticosterone-d8 and cortisol-d4; purchased from Sigma-Aldrich, UK). Steroids were extracted via liquid-liquid extraction with 2 mL of tert-methyl butyl ether (MTBE). MTBE was evaporated to dryness under nitrogen at 55°C. Samples were reconstituted in 125 µL of 50/50 methanol/water for liquid chromatography tandem mass spectrometry analysis.15 16 (link) Samples were measured on a Waters Xevo-XS mass spectrometer coupled to an Acquity uPLC with an electrospray ionisation source in positive ionisation mode. Steroids were identified by comparison to authentic reference standards, (Sigma-Aldrich), with matching retention time and identical mass transitions and quantified relative to a calibration series. Concentrations were calculated relative to internal standard corticosterone to corticosterone-d8 and 11-DHC and its isomer metabolite to cortisol-d4.

The steroid hormone standards were separately dissolved in methanol individually (10 µg/mL). Stock solutions were combined and then serially diluted with commercially available blank plasma to produce standard solutions for a calibration curve (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0 ng/mL for each compound). Quality control (QC) samples were prepared at three concentration levels, higher, middle, and lower limit of quantification, and abbreviated as HQC, MQC, and LQC, respectively, based on the dynamic ranges of the analytes (Table 2). Cortisol-d₄ (Sigma-Aldrich) was used as the internal standard and dissolved in methanol to produce a 50 ng/mL IS solution. All stock solutions were sealed and stored at −20 °C until use. Before use, each solution was thawed at room temperature for 30 min.

Standards of prednisolone, prednisone, cortisone, cortisol, dihydrocortisone, aldosterone, allotetrahydrocortisol, urocortisol, tetrahydrocortisone, corticosterone, deoxycorticosterone, α-cortolone and 6β-hydroxycortisol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Internal standards were prednisolone-d₈ (TRC, Canada), cortisol-d₄ and prednisolonde-d₄ (Sigma-Aldrich). Reagents were of analytical grade (Merck, Darmstadt, Germany) when used for extraction purposes and of liquid chromatography-mass spectrometry (LC-MS) Optima grade (Fisher Scientific, Loughborough, UK) for ultra-high performance LC (UHPLC) tandem MS (MS/MS) and high-resolution MS (HRMS) applications. Ultrapure water was obtained by usage of a purified-water system (Sartorius AG, Goettingen, Germany). Stock solutions were prepared in ethanol at a concentration of 200 μg mL⁻¹ and stored in dark glass bottles at −20 °C.

HPLC-grade methanol, isopropanol, and methyl tert-butyl ether (MTBE) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Acetone was acquired from Labsynth (Diadema,SP, Brazil), and all ultrapurified water was generated on-site using a Milli-Q 3 Ultrapure Water System (Merck-Millipore, Darmstadt, Germany). The steroid standards were acquired from Sigma-Aldrich (St. Louis, MO, USA). The internal standards cortisol-d₄, testosterone-¹³C₃, and estradiol-d₂ were purchased from Sigma Aldrich (St. Louis, MO, USA) and progesterone-d₉ was purchased from Steraloids Inc (Newport, RI, USA).
Standards’ stock solutions for all of the steroids and their respective internal standards were gravimetrically prepared for calibration. A mass of 1.0 to 3.0 mg of each hormone was accurately weighed and dissolved in 15.0 mL of methanol to prepare solutions ranging from 24.63 to 70.42 μg g^-1. The working and calibration solutions were prepared accordingly by dilutions with methanol. Six different concentration levels were used for the calibration curves, and three recovery levels were prepared daily and analyzed during three sequential days to evaluate the method recovery and performance.

Cortisol (hydrocortisone) and cortisone were purchased from Acros Organics, NJ, USA. Cortisol-d4 was obtained from Sigma-Aldrich, MO, USA. Ammonium acetate, potassium phosphate (monobasic), sodium phosphate (dibasic), potassium chloride, sodium chloride, and acetonitrile (HPLC grade) were all provided by Fisher Scientific, NJ, USA. Milli-Q water was prepared by passing distilled water through the Milli-Q System (Millipore, Bedford, MA, USA). We prepared phosphate-buffered saline (PBS) by combining 0.01 M sodium phosphate, 0.0018 M potassium phosphate, 0.137 M sodium chloride, and 0.0027 M potassium chloride (pH = 7.4, adjusted with HCl). The study was approved by the Research Ethics Committee of King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, under RAC No. 2191297.

Ethyl ether and HPLC-grade water were supplied by CARLO ERBA (Italy). HPLC-grade methanol (MeOH) was purchased from Thermo Fischer Scientific.
6 PLUS1 Multilevel Serum Calibrator Set MassChrom Steroids Panel 1&2 allowing the simultaneous quantification of 7 steroids (progesterone, 11-deoxycorticosterone, corticosterone, 17OHP, 11-deoxycortisol, cortisol and androstenedione) among the proposed panel of 15 steroids was purchased from Chromsystems (Germany), as well as internal quality controls (QC) MassCheck Steroid Panel 1&2 Serum Control.
Internal standards: corticosterone-D8 and 11-deoxycorticosterone-D8 were purchased from LGC (Teddington, UK) and separately dissolved in MeOH to get stock solutions at 1 mg/mL, stored at −20°C. Internal standard solutions of cortisol-D4, 11-deoxycortisol-D5, androstenedione-¹³C3, 17OHP-D8 and progesterone-D9 were purchased from Sigma-Aldrich. A mix of the 7 internal standard solutions was further prepared in MeOH.