Tcc 3000

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).

This analysis was developed following the methodology described by Polanía, Londoño, Ramírez, and Bolívar (2022) [30 (link)]. Briefly, the samples analyzed were prepared by dissolving the sample in a mixture of methanol: 0.2% water in formic acid (1:1), mixing (5 min), sonication (5 min), filtering, and subsequent injection into the chromatographic equipment. An ultra-high efficiency liquid chromatograph (UHPLC), Dionex Ultimate 3000 (Thermo Scientific, Sunnyvale, CA, USA), equipped with a binary gradient pump (HP G3400RS), an automatic sample injector (WPS 300TRS) and a thermostated column unit (TCC 3000), was used.

A high-performance liquid chromatography (HPLC) system consisting of an LPG-3400 pump (Thermo Scientific, Tokyo, Japan) with flow control valve, auto sampler (WPS-3000), and temperature controller compartment for the column (TCC-3000) was utilized in this study. Separation was performed in gradient mode with a flow rate of 0.5 ml min⁻¹ over a 5-min run time through a reversed phase C18 column. A CAPCELL PAK C18MG II (250 mm × 4.6 mm i.d., Shiseido, Tokyo, Japan) HPLC column was used. The temperature for the auto sampler and column oven was maintained at 10°C and 30°C, respectively. The chromatographic run was performed with a gradient, and the mobile phase consisted of solvent A (HPLC grade water + 0.05% formic acid) and solvent B (HPLC grade acetonitrile + 0.05% formic acid) with a “T” switch tube only 200 μl/min of total flow (0.5 ml/min) introduced into the mass spectrometry (MS) detector.