Kinetex rp18

The HPLC gradient elution analysis was performed on Merck-Hitachi LaChrom Elite System (Merck, Darmstadt, Germany) with diode array detector L-2455, thermostat L-2300, pump L-2130 and autosampler L-2200 with the Kinetex RP18 (5 μm, 150 × 4.6 mm) chromatographic column (Phenomenex, Torrance, CA, USA) as stationary phase. The mobile phase was consisted of methanol-water-0.1% formic acid (gradient 5–100% (v/v) of methanol) at 30 °C and the run time was 60 min. The analysis was performed with the detection wavelength 280 nm, the sample injection volume was 10 μL and the flow rate was set to 1.0 mL min⁻¹. Methanol Chromasolv for HPLC was purchased from Sigma-Aldrich (St. Louis, MO, USA) and formic acid from Polish Reagents (Polish Reagents, Gliwice, Poland); double-distilled water was used.

The concentration of the solutions of mABA-labeled standard and of the binary mixtures was determined by HPLC-UV-MS using the UV signal at 254 nm. Separation was performed on a RP-C₁₈-column (Phenomenex Kinetex RP18, 100 × 2.1 mm, 2.6 μm) at 40 °C: flow rate, 0.2 mL min⁻¹; eluent, H₂O + 1% HOAc (A) and ACN + 1% HOAc (B); t = 0, 90% A and 10% B; linear gradient to 20% A and 80% B in 30 min; and injection volume, 3 μL (COS standards) and 8 μL (standard binary mixtures), n = 6. Quantification was based on an external calibration with mABA-labeled 2,3,6-tri-O-methyl glucose standard (0.38–3.82 ∙ 10⁻⁴ M). For MS parameter, see chapter Instrumentation.