Agilent 1200 infinity system

Sample preparation: an aliquot of the culture liquid (0.25 mL) was diluted 10 times with a mixture of acetonitrile:isopropanol (50:45, v/v), mixed and centrifuged within 8 min at 12,100 × g. The supernatant was used for analysis. HPLC analyses were performed on an Agilent Infinity 1200 system (Agilent Technologies, Germany) with a Symmetry C18 column (4.6 × 250 mm, 5 μm) with a Symmetry C18 precolumn (3.9 × 20 mm, 5 μm,) (Waters, Milford, MA, USA) at 50 °C and a flow rate of 1 mL/min. Steroid assay was performed using two mobile phases: I—acetonitrile:2-propanol:water (50:45:5 (v/v/v)) and II—acetonitrile:water:TFA (60:40:0.02 (v/v/v)) with gradient elution as described [43 ], UV-detection at 200 (I) and 240 nm (II). Calibrations were performed by the external standard method based on peak areas. The results were processed using the ChemStation Rev. software. B.04.03 (Agilent Technologies, USA). Retention times (Rt) for mobile phase I: cholesterol, Rt 10.3; phytosterols, Rt 9.5–10; pregnenolone, Rt 3.16; for mobile phase II: progesterone, Rt 10.56. Rt of 3-methoxymethyl derivatives are given in Table 1.

HPLC analyses were performed using reversed-phase HPLC on an Agilent Infinity 1200 system (Agilent Technologies, Germany SA) with a Symmetry column (250 × 4.6 mm, 5 μm) with a Symmetry C18 precolumn (5 μm, 3.9 × 20 mm) (Waters, Milford, MA, USA) at 50 °C and a flow rate of 1 mL/min. Steroid assays were performed using two mobile phases (acetonitrile:water:acetic acid (60:40:0.01, v/v/v) and acetonitrile:2-propanol:water (50:45:5, v/v/v)) with UV-detection at 200 nm (for compounds with the 3β-ol-5-ene configuration) and 240 nm (for compounds with the 3-oxo-4-ene configuration).

Analyses were performed with an Agilent 1200 infinity system (Agilent Technologies) using ELSD Sedex 100 and DAD (280 nm) detectors, an Agilent Zorbax Eclipse Plus C18 RRHD (2.1 × 50 mm, 1.8 μm) as the stationary phase, with a mobile phase composed of A = H₂O and Na₂HPO₄ (10 mM), Na₂B₄O₇ (10 mM), pH = 8.2 and B = MeOH:acetonitrile:H₂O, 45:55:10 pH = 8.2 with a gradient from 98:2 to 0:100, over 13 min, 0.2 mL/min, column temperature: 40 °C, sample volume injection: 1 μL.