The LC-MS method for determining glycyrrhetinic acid in dog plasma and magnesium isoglycyrrhizinate involves taking 0.5ml of plasma, respectively, and adding 1 and 2 volumes of reference solution to create solutions containing concentrations of 10, 30, 50, 100, 300, and 500 ng/ml. A linear solution was prepared with concentrations of 30, 100, 250, 500, 1000, and 3000 ng/ml for regression analysis. The regression equations obtained were as follows: R = 5.093 × 10^-3 + 1.221 × 10^-1, r = 0.9997; R = 2.281 × 10^-a c + 9.324 × 10^-b, r = 0.9999. The results showed that the concentration-linear ranges for plasma components 1 and 2 were 10~500 ng/ml and 30~3000 ng/ml respectively.
In precision testing, blank plasma samples were paired with high-concentration samples (20, 200, 400 ng/ml for component 1; 60, 1200, 2400 ng/ml for component 2). According to the % calculation, the intra-day RSD values were 9.0%, 6.6%, and 7.2% for component 1, with accuracies of 107.4%, 92.79%, and 98.2%. For low, medium, and high concentrations tested daily over five days, the inter-day RSD values for component 1 were 11.3%, 5.7%, and 4.7%, with accuracies of 101.4%, 103.9%, and 109.6%; the inter-day RSD values were 8.9%, 9.2%, and 6.4%, with accuracies of 105.8%, 96.9%, and 101.8%.
In recovery testing, blank plasma samples were prepared at high concentrations for components 1 and 2 and subjected to direct injection and recovery tests under identical conditions. Results are shown in Table 2.
For stability testing, three replicates of blank plasma samples at low and high concentrations for components 1 and 2 were analyzed under various conditions including room temperature for 4 hours, -40°C for six weeks, freeze-thaw cycles at -40°C, and storage for 24 hours. Stability in the autosampler was also investigated. Under these conditions, the RSD values for components 1 and 2 were less than 15%, indicating that the target compounds were stable in the samples.
In plasma determination studies on Beagle dogs given a single oral dose (15 mg/kg) of HE2 ~ U, blood plasma samples were collected before administration and at 1, 2, 3, 4, 6, 8, 10, 14, 24, 36, 48, and 72 hours post-administration. The resulting drug-time curve is shown in Figure 2.
Two water-soluble components could not be effectively extracted from plasma using liquid-liquid extraction methods. Literature reports mostly use methanol-induced protein precipitation followed by concentration for analysis, which can extract impurity peaks. In this study, an Oasis HLB solid-phase extraction column with appropriate hydrophilic-lipophilic balance was used to eliminate interference from proteins, lipids, salts, and other impurities in the plasma by leaching. Methanol was used as the eluent, and in this study, 0.5% ammonia-methanol was used. The absolute recoveries were greater than 70%.