C18 guard pre column

For individual anthocyanins, the protocol of Martínez-Esplá et al. [20 (link)] was adapted to our conditions. Freeze-dried (0.5 g) tissue was pulverized and homogenized with 10 mL solution containing methanol/formic acid/water (79:1:20, v/v/v). After centrifugation (10 min at 10,500× g), the upper phase was subjected to filtration (0.45 µm filter) and an aliquot of 20 µL sample injected into a HPLC. The individual anthocyanins were separated in a 25 × 0.46 cm Luna C18 column equipped with a C18 guard pre-column (Phenomenex, Macclesfield, UK). The concentrations of both anthocyanins (cyanidin 3-glucoside and cyanidin 3-(6″-malonylglucoside)) were calculated (mg L⁻¹) after detection at 520 nm and compared with known standards. HPLC chromatograms of individual anthocyanins are presented in Figure S1. The precision and recovery of HPLC data for individual anthocyanins are presented in Table S1.

HPLC analyses were performed using a Shimadzu LC-20AT pump equipped with a SPD-20AV variable-wavelength UV detector, an SIL-20A autosampler, a CTO-20AC thermostat and an LC solution system (Shimazu Co., Kyoto, Japan) (15 ). A Kromosil C18 column (250 ×4.6 mm, 5 μm; Shimadzu Co.) was used as the chromatographic column with a C18 Guard Precolumn (Phenomenex, Torrance, CA, USA), and the temperature was maintained at 50°C. The mobile phase consisted of 50 mmol/l acetonitrile, 1 mol/l citric acid and ammonium acetate (19:80:1, v/v). The detection wavelength was 295 nm, with 0.01 AUFS for all samples. The flow rate of the mobile phase was 1.0 ml/min, and the injection volume was 20 μl. The total running time was 6 min (16 ).
The linearity of the levofloxacin (standard purchased from the National Institutes for Food and Drug Control, Beijing, China) was investigated between 1 and 100 μg/ml in serum, gastric juice and gastric mucosa. Only 100 μl sample was required for detection. Subsequently, 100 μl 100 g/l trichloroacetic acid or methanol was individually added into the serum and the other samples to precipitate the proteins. The mixture was vortexed for 2 min and centrifuged for 10 min at 9,180 × g. The filtrates were obtained and 20 μl was detected for the drug concentration. Selectivity, linearity, accuracy, precision, stability and sensitivity were evaluated for method validation.