Agilent 1100 series hplc equipment

Plasma samples were processed and analyzed with the same method, and the concentration of caffeine and paraxanthine was determined by HPLC based on the reported analytical method with some modifications [12 (link), 13 (link)]. Agilent 1100 series HPLC equipment (Agilent technologies Inc., Santa Clara, America) was used and a Hypersil BDS C₁₈ column (4.6 mm × 200 mm, 5 μm. Dalian, China) coupled with a phenomenex ODS-C18 guard column (4.0 mm × 3.0 mm, 5 μm. CA, America) was applied to separate the analytes. The detection wave length was set at 280 nm. The column temperature was 25°C. The mobile phase was modified by gradient control. The solvents used for elution were phase A (acetonitrile containing 0.2% formic acid (v/v)) and phase B (10 mM ammonium formate acetic acid containing 0.2% formic acid (v/v), pH 3.5). Typical conditions for elution were 98% B (0–4 min), 98–90% B (4–9 min), 90% B (9–15 min), 90–70% B (15–18 min), 70% B (18–21 min), 70–98% B (21–25 min), and 98% B (25–30 min). The analytical intraday coefficients of variation ranged 2.56%–9.48%. The interday coefficients of variation ranged 5.74%–10.91%. The limit of quantification was 10 nmol/L for each analyte. The standard deviation for quality control samples in all analysis batches was less than 15%, and the results of method validation were specific, precise, and repetitive.

The HPLC technique was employed for the determination of phenolic compounds by modification of the method by Rodriguez-Delgado et al. [27 (link)]. An amount of 5 g from each homogenized sample was diluted with distilled water (1:1) and centrifuged at 15,000 rpm for 15 min. The supernatants were filtered through a 0.45 µm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Burlington, MA, USA). HPLC analysis was realized by Agilent 1100 series HPLC equipment, with ODS column (250 mm × 4.6 mm, 4 µm (HiChrom, Chadds Ford, PA, USA)) and mobile phase composed of Solvent A (methanol/acetic acid/water (10:2:88, v/v/v)) and Solvent B (methanol/acetic acid/water (90:2:8, v/v/v) with a gradient program. For the identification of chromatographic data, a DAD detector (Agilent, Santa Clara, CA, USA) was used, with spectral determination at 254 and 280 nm. Samples were identified and quantified by standards. The results were expressed as mg/100 g fw.