2.5. Method validation
We evaluated the selectivity by comparing the chromatograms of six
batches of blank rat plasma with spiked rat plasma. The linearity of six
calibration curves was determined through a weighted
(1/x2) analysis. By analyzing three samples (n = 6) of
low-, medium-, and high-concentration QCs on different days, we
determined intraday and interday precisions (relative standard
deviation, RSD) and accuracy(relative error, re). The matrix effect was
studied by comparing the peak areas of the analytes in low-concentration
and high-concentration postextraction spiked blank plasma samples with
those of the corresponding standard solutions. In order to determine the
recovery rate, six extracted samples were compared to six extracted
samples with low, medium, and high QC concentrations. Stability was
evaluated by comparing the repeatability of low and high QC samples (n =
6) during storage and processing. A freeze-thaw stability test was
performed after three freeze-thaw cycles. By analyzing the QC samples at
4 °C in one day, the stability of the samples during postpreparation was
assessed. For long-term and short-term stability evaluations, six
aliquots of QC samples were stored at 20 °C for two weeks and at ambient
temperature for four hours.
2.6. Pharmacokinetic (PK) study in rats
Male Sprague–Dawley rats weighing 250-300 g were selected for the PK
study. All animal experiments were conducted according to the guidelines
of the institutions and with the approval of the Use and Care of Animals
of Guangxi Medical University. Twelve rats were individually treated
with RA and A1 aqueous solutions at 10 mg/kg. We collected blood samples
(0.5 ml) at 0, 5, 15, 30, 45, 60, 90, 120, 240, 360, 480, and 720 min
following oral administration of the drug. These samples were stored in
heparinized tubes. After centrifugation at 8049.6×g and 4 °C for 10 min,
plasma was collected and frozen at −20 °C until analysis. Using TopFit
2.0 software, pharmacokinetic parameters were assessed in a
noncompartmental model. The elimination half-life (t1/2)
is 0.693 ke, where ke is the elimination
rate constant, which is calculated by fitting the mean data of the
plasma concentration curve at 4 end points with a logarithmic linear
regression equation using the least squares method. Based on the
observed data, we calculated the maximum drug plasma concentration
(Cmax) and time to reach Cmax (Tmax). The area under the plasma
concentration-time curve from zero to the final measurable sample time
(AUC0–t) was calculated using the linear trapezoidal
rule.