Quantification of Adenine Nucleotides and Adenosine

The concentration of adenine nucleotides (ATP, ADP, AMP) and adenosine (Ado) released into the culture medium were determined using HPLC. The medium (400 µl) was transferred into a cold Eppendorf tube containing 50 µl of 0.1 m perchloric acid and 10 μm theophylline (as an internal standard). The medium was centrifuged (at 3510 × g for 10 min at 0°C-4°C), and the supernatant was kept at −20°C until analysis. Online solid phase extraction coupled to the column-switching technique was applied for quantification of nucleotide content in the samples. HPLC separation was performed by a Shimadzu LC-20 AD Analytical System using UV (Agilent 1100 VW, set at 253 nm) detection. A phenyl-hexyl packed (7.5 × 2.1 mm) column was used for online sample enrichment, and separation was completed by coupling to an analytical C-18 (150 × 2.1 mm) column. The flow rates of the mobile phases [Phase A: 10 mm potassium phosphate buffer with 0.25 mm EDTA; Phase B: additional components, such as 0.45 mm octane sulfonyl acid sodium salt, 8% acetonitrile (v/v), 2% methanol (v/v), pH 5.55] were 350 and 450 µl/min, and they were applied in a step gradient (Baranyi et al., 2006 (link)). The sample enrichment flow rate of buffer A was 300 µl/min for 4 min, and the total run time was 55 min. Concentrations were calculated by an internal standard 2 point calibration curve method [(A_i × f × B)/(C × D_i), where A_i is the area of nucleotide components, B is the sample volume, C is the injection volume, D_i is the response factor for 1 pmol nucleotide standard, and f is the international standard factor (IS area in the calibration/IS area in the actual sample)]. The data are expressed as pmol per ml.