Waters alliance 2695 hplc system

Equal amount of snap frozen cecal content was weighed, dissolved in HPLC grade water and supernatants were collected after centrifugation (12,000 g, 10 min), while processing on ice. Concentrations of SCFAs (acetate, propionate and butyrate) were determined using a HPLC system (Waters-2695 Alliance HPLC system, Waters Corporation, Milford, MA, USA) equipped with Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) and DAD detector (210 nm), and eluting with H₂SO₄ (0.005 N) mobile phase with a flow rate of 0.6 ml/min at 25 °C, as described elsewhere^{63 (link),65 (link),66 (link)}.

Preparation of Standard Solutions. A mixed standard stock solution containing uridine, inosine, guanosine, safflomin A, and brazilin was prepared in MeOH/H₂O (50 : 50, v/v). The standard solutions were filtered through a 0.22 μm membrane prior to injection. All solutions were stored in a refrigerator at 4°C before analysis.
HPLC Analysis of Sample. Analysis was performed on Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, and an autosampler. The raw data were detected by Waters 2998 PDA, acquired, and processed with Empower Software. A Waters X-select C18 column (5 μm, 4.6 mm × 250 mm) preceded by a RP C₁₈ guard column (5 μm, 3.9 mm × 20 mm) was applied for all chromatographic separation. Detection wavelength was set at 260 nm for all compounds. The mobile phase was composed of A (methanol) and B (0.1% aqueous ethylic acid) with a linear gradient elution: 0–20 min, 5% A; 20–75 min, 5–50% A, then keeping 50% A for 10 min to clean the column. MeOH/H₂O (10 : 90, v/v) were used as solutions for cleaning the injection needle. The flow rate was set at 0.80 mL/min and the injection volume was 10 μL. The column temperature was maintained at 30°C.

The levels of adenosine phosphates (ATP, ADP, and AMP) in the LT muscle were determined by high-performance liquid chromatography (HPLC) according to the method of Li et al. (19 (link)). Briefly, 0.5 g of frozen muscle sample was homogenized in 2.5 ml of 7% ice-cold perchloric acid at 13,500 rpm for 30 s in an ice bath and then centrifuged (15,000 × g, 4°C, 10 min). The supernatant (850 μl) was then neutralized with 0.85 M KOH (850 μl) and centrifuged again (15,000 × g, 4°C, 10 min) to remove KClO₄. The neutralized supernatant was filtered through a 0.45-μm filter before injection into a Waters-2695 Alliance HPLC system (Waters, Milford, MA, USA). The column was a Kromasil 5 μm C₁₈, 250 × 4.6 mm (Feinano, Tianjin, China). The chromatographic conditions were as follows: mobile phase A, HPLC grade methanol; mobile phase B, phosphate buffer (2.5 mM tetra-butylammonium hydrogen sulfate, 0.04 M potassium dihydrogen orthophosphate, 0.06 M dipotassium hydrogen orthophosphate, pH 7.0), filtered through a 0.45-μm membrane; mobile A/mobile B, 13.5%/86.5%. The column temperature was set at 30°C, the injection volume was 10 μl, and UV detection was at 254 nm. The characteristic running time was 15 min, the flow rate was maintained at 1.0 ml/min, and sample measurement was set to auto-sequence injection. Peaks were identified and quantified using standard curves.