Ods 2 hypersil column

The samples were prepared using the protocol of Cardoso et al. and Masubuchi et al. with some modification [24 (link),25 (link)]. The minced tissues were homogenized, and then 1 M HClO₄ (w/v) was transferred into the homogenates in a volume equal to five times their weights. They were centrifuged for 15 min at 5000 rpm. The supernatants were neutralized with 1 M K₂CO₃ and then centrifuged again. The clear supernatants were injected into HPLC for determining the levels of cytosolic adenine nucleotides.
A Thermo ODS-2 Hypersil column (250 × 4.6 mm) was used to obtain adenine nucleotide levels. The analysis was conducted under the following conditions: mobile phase, 50 mM aqueous triethylamine (TEA) buffer (adjusted with phosphoric acid to pH 6.5; A) and acetonitrile (B). Gradient elution was performed from 99A/1B in 10 min to 95A/5B and changed in another 10 min to 92.5A/7.5B. Each run was followed by a 5-min wash with 70B/30 parts 0.1% phosphoric acid. Detection wavelength, flow rate, and column temperature were set to 254 nm, 1 mL/min, and 20 °C, respectively [26 (link)].

Optical rotations were measured with a JASCO P-2000 automatic polarimeter in MeOH at 20 °C. The CD spectra were recorded on a JASCO P-1500 spectropolarimeter. NMR spectra were recorded in CDCl₃ using residual solvents as internal standards with a Bruker AVANCE NEO 600 spectrometer with a 5 mm inverse detection triple resonance (H-C/N/D) cryoprobe having z-gradients. The chemical shift values are given in parts per million (ppm) relative to TMS at 0.0 ppm, and the coupling constants are in Hertz. High-resolution electrospray ionization mass spectra (HRESIMS) were measured on an Agilent (Santa Clara, CA, USA) 6545 Q-TOF instrument. Reversed-phase HPLC purification was carried out on a Waters HPLC equipped with a 1525 binary pump, and a Thermo Scientific (Waltham, MA, USA) ODS-2 Hypersil column (5 µm, 250 × 10 mm). Normal phase column chromatography and thin-layer chromatography were performed using silica gel (200–300 mesh) and GF254 (10–20 mm) (Qingdao Marine Chemical Company, Qingdao, China). YMC*GEL ODS-A (AA12S50; YMC Co., Ltd., Japan) was used for reverse phase column chromatography. Sephadex LH-20 was a product from GE Biotechnology, USA.

Analyses of arginine were performed using the Agilent HPLC system 1260 (Palo Alto, CA) equipped with an ODS-2 Hypersil column (4.6 × 150 mm × 5 μm) (Thermo Scientific, CA) as previously described32 (link).