Synergi 2.5 μm

For MDA content determination samples of mammary tumours were subjected to gentle alkaline saponification and derivatisation with 2,4-dinitrophenylhydrazine (DNPH) to form MDA-DNPH adducts [32 (link)]. A liquid chromatograph (UFLCXR system; SHIMADZU, Tokyo, Japan) equipped with C18-column (Synergi 2.5 μm, Hydro-RP, 100 Å, 100 × 2 mm, Phenomenex) and a DAD operated in the UV range of 195–420 nm was applied for MDA chromatographic analyses. As mobile phase linear binary gradient of acetonitrile in water was used. Solvent A consisted of water–acetonitrile (95:5, v/v) and solvent B of 100% acetonitrile. Identification of MDA-DNPH adducts peaks was based on retention time and absorption UV spectra of analytical standard—1,5-pentanedialdehyde (Sigma, St. Louis, MO, USA) solution (λ_max = 306 nm).

The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an Interchim Uptisphere120-3HDO C18 or a Synergi, 2.5 μm Fusion-RP C₁₈, 100 Å, 100 × 2 mm (Phenomenex®, Torrance, California, USA). Mobile phase A was 2 mM ammonium acetate and mobile phase B was 80% acetonitrile containing 2 mM ammonium acetate. Gradient elution started with 0% B and increased to 12% B after 10 min and to 80% after 12 min. After 4 min elution at 80% B and subsequently regeneration of starting conditions to 100% A after 5 min, the column was equilibrated at 100% A for 8 min. The flow rate was 0.2 mL/min and the column temperature 30 °C. High-resolution mass spectra of precursor and product ions were recorded by a ThermoFinnigan LTQ Orbitrap XL. The parameters of the mass spectrometer were tuned with a freshly mixed solution of adenosine (5 μM). The parameters were sheath gas flow rate, 16 arb; auxiliary gas flow rate, 11 arb; sweep gas flow rate, 4 arb; spray voltage, 5.0 kV; capillary temperature, 200 °C; capillary voltage, 20 V, tube lens 65 V.