Ascentis express c18

HPLC analysis was performed on a Thermo Scientific Dionex Ultimate 3000 HPLC system (Thermo Scientific, Bremen, Germany) equipped with an LPG-3400SD pump, TCC-3000 column oven, and UV VWD-3100 detector.
According to the United States Pharmacopeia’s (USP) pending monograph for azacitidine [34 ] and certificate of analysis (CoA) of azacitidine reference material supplied by Merck Life Science S.r.l. (Milan, Italy), the HPLC analysis using a reversed-phase high-performance liquid chromatography (RP-HPLC; Ascentis Express C18, 150 mm × 4.6 mm, 2.7 μm; Merck Life Science S.r.l. (Milan, Italy) with a linear A-B gradient (0–4.8 min 0% B, 4.8–12 min 0% to 15% B, 12–15 min 15% B, 15–18 min 15% to 30% B, 18–24 min 30% to 50% B, 24–27 min 50% to 0% B, 27–33 min 0% B) at a flow rate of 0.8 mL/min and a total run time of 33 min was performed. Solvent A consisted of 1.54 g/mL ammonium acetate in water (0.02 M, pH 6.9 ± 0.1) and solvent B consisted of solvent A:methanol:acetonitrile (50:30:20).
UV absorbance was measured at 210 nm. The column temperature was kept at 30 °C. The injection volume was 20 μL.
The Chromeleon data system software (Version 7.2.8) was used for data acquisition and mathematical calculations.
The extensive validation of the analytical method was carried out according to ICH Q2(R1) guidelines [27 ] (see Supplementary Materials).

Degradation experiments were performed at 30 °C in mineral salts medium (MSM) with the following composition: g L⁻¹: 3.78 Na₂HPO_{4 ×} 12 H₂O; 0.5 KH₂PO₄; 5.0 NH₄Cl; 0.2 MgSO₄ × 7 H₂O and 0.01 yeast extract under (1) mono-substrate conditions with 1 mg L⁻¹ of DCF and initial OD₆₀₀ set at 0.750, (2) co-metabolic conditions with glucose at a concentration of 0.1% (v/v), 1 mg L⁻¹ of DCF and initial OD₆₀₀ set at 0.05, (3) co-metabolic conditions with glucose (0.1%, v/v) and sodium acetate at the concentration of 5.6 mM, 1 mg L⁻¹ of DCF and initial OD₆₀₀ set at 0.05. The degradation rate was determined with the High-Performance Liquid Chromatography (HPLC) technique using Merck Hitachi reversed-phase chromatograph (Mannheim, Germany), equipped with a column Ascentis Express^® C18, pre-column Opti-Solv^® EXP and UV/Vis DAD detector. The mobile phase consisted of acetonitrile, methanol and 1% acetic acid (50:30:20, v/v, flow-rate 1 mL min⁻¹). The detection wavelength was set at 276 nm. DCF in the supernatant was identified by comparing the HPLC retention time and UV spectra with that of the external standard [23 (link)].

Analyses were carried out on a Ascentis Express C18, 15 cm × 4.6 mm internal diameter (i.d.), with particle size of 2.7 µm (Merck Life Science, Merck KGaA, Darmstadt, Germany). The injection volume was 5 µL, and the mobile phase consisted of water/formic acid (99.9:0.1, v/v) (solvent A) and ACN/formic acid (99.9:0.1, v/v) (solvent B); the linear gradient profile was as follows: 0 min, 0% B; 15 min, 5% B; 65 min, 20% B; 95 min, 35% B; 100 min, 100% B; 101 min, 0% B. The flow rate for separation and detection was 1 mL/min, and it was split to 0.2 mL/min prior to MS detection.