1200 series hplc system

An Agilent 1200 Series HPLC system coupled to an AB Sciex API4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer was used to monitor two product ions, one for quantitation and one for identity confirmation, using scheduled multiple reaction monitoring for each steroid and internal standard. Chromatography was conducted on a Restek (Bellefonte, PA, USA) Ultra Biphenyl column (250 mm x 4.6 mm, 5.0 μm particle size) with acetonitrile and methanol both containing 0.1 % formic acid (volume fraction) for reproductive hormones. A flow of 500 μL/min was used for a solvent gradient of 20 % acetonitrile increased to 45 % over 30 min., then increased to 80 % over 1 min and held for 4 min, then washed with 100 % acetonitrile for 5 min and re-equilibrated at 20 % for 10 min. Corticosteroids were separated on an Agilent (Santa Clara, CA, USA) Eclipse Plus C18 column (21 mm X 150 mm, 5.0 μm particle size) with methanol and water both containing 0.1 % acetic acid (volume fraction). Column conditions were as follows: flow rate of 250 μL/min, and isocratic method of 46 % methanol for 20 min, a wash of 100 % methanol for 13 min, and re-equilibrated for 10 min. Additional information on compound and instrument parameters can be found in Boggs et al. (Boggs et al., 2017 (link)).

Metabolite extracts were analyzed for targeted metabolites listed in Supplemental Table 4 using an LC-MS/MS method adapted from (Young et al., 2011 (link)). A Phenomenex 150 mm x 2 mm Synergi Hydro-RP column was used on an Agilent 1200 Series HPLC system coupled to an AB Sciex 5500 QTRAP system. LC was performed with an injection volume of 20 μL, using gradient elution of 10 mM tributylamine and 15 mM acetic acid (aqueous phase) with acetonitrile (organic phase) at a constant flow of 0.3 mL/min. The gradient profile of the organic phase is as follows: 0% B (0 min), 8% B (10 min), 16% B (15 min), 30% B (16.5 min), 30% B (19 min), 90% B (21.5 min), 90% B (26.5 min), 0% B (26.6 min), and 0% B (30.5 min). MS analysis was performed in negative mode using a multiple reaction monitoring (MRM) acquisition method. Data acquisition was performed on the ABSciex Analyst 1.7 software. Absolute quantification of intracellular metabolites was performed using the Analyst MultiQuant 3.0.3 Software. For isotope labeling profiles, MSConvert was used to convert LC-MS/MS data files (ABSciex .wiff and .wiff.scan) to an open-source format (Holman et al., 2014 ). A combination of the pyOpenMS (Rost et al., 2014 (link)) and SciPy (Virtanen et al., 2020 (link)) packages in Python, was used for the process of peak finding, window sizing, and noise assessment across multiple isotopes.