Validated UHPLC-MS/MS Method for Rat Plasma Analysis

The UHPLC-MS/MS method developed for determining the 10 components in rat plasma (Figure 1)—namely genipin-1-β-D-gentiobiodide, scandoside methyl ester, geniposide, geniposidic acid, chlorogenic acid, capillarisin, rhein, emodin, caffeic acid, and 4-hydroxyacetophenone—was validated in terms of specificity, accuracy, precision, linearity, lower limit of quantitation (LLOQ), stability, recovery, and matrix effect, in accordance with United States Food and Drug Administration guidelines. Calibration curves were constructed using the peak area ratios of the analytes to peoniflorin and by applying a weighted (1/x²) least squares linear regression analysis. The LLOQ was determined at the lowest concentrations at which the signal-to-noise (S/N) ratio was 10. Specificity was determined by comparing chromatograms of blank rat plasma obtained from five individual subjects with chromatograms of plasma samples obtained after YCHD administration at a dose of 12 g/kg. Precision [expressed as the relative standard deviation (RSD)] and accuracy [expressed as the relative error (RE)] were calculated for three QC points (low, medium, and high). Five replicates of each QC point were analyzed to determine the interday accuracy and precision. This process was repeated three times over three consecutive days to determine the intraday accuracy and precision. Recovery was evaluated in five replicates at three different QC concentrations (low, medium, and high). The percentage recovery was determined by comparing the concentrations of the pre-extraction spiked QC samples prepared in blank matrix (by adding analytes and peoniflorin to blank matrix prior to extraction) with the peak area of the post-extraction spiked QC samples prepared in an extracted blank matrix (prepared by adding analytes and peoniflorin to blank matrix extract). Matrix effects were investigated on five independent sources of blank rat plasma by calculating the ratio of the peak area in the presence of matrix to the peak area in absence of matrix at three different QC concentrations (low, medium, and high).
The stability of standard analytes in rat plasma was evaluated under various conditions (time and temperature) by analyzing five replicates of the QC samples at three concentrations (low, medium, and high). Stability was investigated in terms of short-term and long-term stability, freeze and thaw stability, and post-preparative stability by using the developed method. Short-term stability was evaluated by storing QC samples at room temperature (25°C) for 24 h. Long-term stability was assessed after 60 days of storage at -20°C. Freeze and thaw stability was determined after three freeze–thaw cycles at -20°C. In addition, post-preparative stability during storage in an auto sampler at 4°C for 24 h was investigated.