HPLC quantification of AMP and ATP

Intracellular ATP and AMP contents were measured by HPLC⁷⁴. In brief, FL83B cells grown in the presence or absence of CO-EtOAc were washed twice with cold phosphate-buffered saline and immediately centrifuged for 2 min at 1000 × g (4 °C). Cell pellets were lysed in 150 μl of perchloric acid (4% v/v) and the lysates were then neutralized with 2 M KOH/0.3 M 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, St Louis, MO, USA). After centrifuging at 13,000 × g for 10 min, the supernatant was analyzed by HPLC (HPLC-Pro Star from Varian, Walnut Creek, CA) with a Spherisorb column (ODS II, 5 mm, 0.46 × 25 cm, Z22.697-1, Sigma-Aldrich, St Louis, MO, USA) at a flow rate of 1.0 ml/min. The mobile phase used was an isocratic mixture of 20 mM KH₂PO₄ and 3.5 mM K₂HPO₄-3H₂O at pH 6.1. The adenine nucleotides were analyzed spectrophotometrically at 254 nm. Each elution peak was compared with AMP, ADP, and ATP standards (Sigma-Aldrich, St Louis, MO, USA) to confirm its identity. The order of eluted nucleotides was ATP, ADP, and AMP.