Vp ods c18 column

ATP-related compounds were determined by a RP-HPLC procedure with the method of Yu et al. (28 (link)). 2.0 g of minced fish flesh and 7.5 mL precooled perchloric acid solution (6%, v/v) were homogenized and then centrifuged at 11,960 × g at 4°C for 5 min. The precipitate was extracted again with the same condition. The supernatants were collected and neutralized with KOH solutions to the final pH ranges of 6.5–6.8. After that, the neutralized solution was centrifuged at 3,040 × g at 4°C for 5 min and the supernatant was made up to 25 mL with deionized water. Subsequently, the prepared solution was filtered through a 0.22-μm filter membrane and analysized using HPLC (Waters 2695, Milford, USA) furnished with a Shim-pack VP-ODS C18 column (150 × 46 mm). Each sample was measured in triplicates. K-value was calculated according to Equation (1).
where HxR, Hx, ATP, ADP, AMP, IMP, are hypoxanthine riboside, and hypoxanthine, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, respectively.

E2-a was analyzed by HPLC using a Waters HPLC system (Milford, MA, USA) with Waters 1525 binary HPLC pump, Waters 2998 photodiadearry (PDA) detector and VP-ODS-C18 column (5 μm, 250 mm × 4.6 mm) [18 ]. Isocratic elution was used to analyze all samples in this experiment (flow rate of 1.0 mL/min; UV detection; column temperature 35 °C). The mobile phase was comprised of 25% A (acetonitrile) and 75% B (0.02 mol/L ammonium acetate containing 0.05% triethylamine). As a comparison, a mixture of standards, matrine and sophocarpine were also analyzed with the same HPLC conditions. In the analysis of E2-a, identification of analytes was based on matching the retention times against those of standard compounds and was confirmed by spiking standards to the samples. Matrine and sophocarpine standards were prepared at concentrations of 500 μg/mL in HPLC grade methanol as stock solutions. Work solution contained 50 μg/mL of standard was prepared from the stock standard solution and filtrated by a filter (pore diameter was 0.22 μm). The quantitation analysis was based on the peak area using the internal calibration method. According to the principle, the concentration of a compound is proportional to the peak area provided by HPLC, and therefore the concentration of the compound was determined. The analysis was performed in triplicate.