Chromeleon software 7

A UHPLC analysis of adenosine and cordycepin was performed using a Vanquish™ Flex UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a VF-D11-A Diode Array Detector (DAD) set at 254 nm (Thermo Fisher Scientific, Waltham, MA, USA). The system consisted of a VF-P10-A binary pump, a VF-10A-A thermostated autosampler (4 °C), and a VC-C10-A thermostated column compartment (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved through a Hypersil Gold Vanquish C18 column (150 mm × 2.1 mm, 1.9 μm) equipped with a Hypersil Gold security guard and thermostated at 30 °C (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of water/methanol (92:8, v/v). The flow rate was set to 200 μL/min, and the injection volume was 2.5 μL. The data were evaluated using Chromeleon Software 7.2.10 (Thermo Fisher Scientific, Waltham, MA, USA). The calibration curves for the quantification were obtained by using authentic standards of adenosine and cordycepin. The analysis was performed in triplicate.

The HPLC analyses were performed using a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Germering, Germany) equipped with an Ultimate 3000 pump, an Ultimate autosampler, an Ultimate column compartment, and a fluorescence detector (FLD). The Chromeleon software 7.2.10 (Thermo Scientific) was used to control the HPLC system and data acquisition.
The chromatographic separations were achieved using a Dikma C18 reverse-phase column (100 mm × 4.6 mm i.d., 5 μm particle size), with a guard column packed with the same material. The column was maintained at 35 °C throughout the analysis. The mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B), delivered at a flow rate of 1.0 mL/min. The gradient elution procedure was set as follows: 2.0 min, 20% B; 4.0 min, 90% B; 10.0 min, 90% B; 11.0 min, 20% B; 16.5 min, 20% B. The injection volume was 10 μL. Moreover, the HPLC-FLD fingerprints (data matrices for samples) were acquired at 310 nm for excitation wavelengths and 350 nm to 450 nm for emission wavelengths. The temperature of the flow cell was 45 °C, while the sensitivity of the FLD detector was set to 5.