Pharmacokinetic studies in rats

Pharmacokinetic studies of the test (optimized AP-PLGA-NPs) and control (AP suspension containing 0.1% CMS-Na) formulations were conducted in male Wistar rats (200–250 g). After fasting for 12 hours with free access to water, the rats were randomly divided into two groups (n=6 in each group) by weight and were orally administered AP formulations at a dose of 30 mg/kg. At 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 hours after administration, 0.3 mL of blood sample was collected from the jugular vein into a heparinized tube. Plasma samples were obtained after centrifugation at 3,000× g for 10 minutes and then stored at −20°C prior to analysis.

AP was metabolized to PUE after oral administration in our previous study,15 so our present pharmacokinetic study was based on the quantification of PUE in the rat plasma. The plasma concentration of PUE was determined using a validated liquid chromatography–tandem mass spectrometric method, as described in our previous report.22 In short, the plasma samples were pretreated by precipitating the protein using a mixture of methanol and acetonitrile (50:50, v/v). The chromatographic separation was carried out on a CAPCELL PAK C₁₈ MGIII column (150×2.0 mm, 5 µm; Shiseido, Kanagawa, Japan) in which the mobile phase consisted of a mixture of 0.1% formic acid in water (containing 5 mM ammonium formate) and methanol (35:65, v/v). The analyte was detected using an API 4000 triple-quadrupole mass spectrometric instrument (Applied Biosystems, Framingham, MA, USA) in multiple reaction monitoring mode. The precursors to the product ion transitions of m/z 417.5→297.6 and 271.3→153.0 were used to quantify PUE and the internal standard (genistein), respectively. The weighted (W=1/x²) calibration curve was linear over a plasma concentration range of 2.5–5,000 ng/mL, with a regression equation of

The results of precision, accuracy, extraction recovery, matrix effects, and sample stability were found to be within the accepted variable limits.

The rats used in the experiments received care according to University Unit for Laboratory Animal Medicine guidelines. All animal experiments complied with the requirements of the National Act on the Use of Experimental Animals (People’s Republic of China) and were conducted using protocols approved by the Shandong University Institutional Animal Care and Use Committee.