Determination of plasma calcium dobesilate concentrations
POSITIVE
The concentration of calcium dobesilate in plasma was determined by a validated LC-MS/MS method [The bioanalytical method and validation results are briefly described as follows: an aliquot (10 μL) of IS working solution (10 μg/mL CaD-D3-10% methanol-water solution) was added to 100 μL of plasma and mixed well, then 400 μL of acetonitrile was added into each sample and mixed well (vortex 30 s at 1400 rpm). The mixture was transferred to a dephosphorized sheet and made pressure filtration by Positive Pressure-96 Processor. The filtrate was diluted 10 times with water and mixed for 30 s at 2000 rpm. Then the filtrate was injected into the chromatographic system for analysis.Chromatographic conditions were as follows: The column was LunaCalcium dobesilate and the IS were quantitated by multiple reaction monitoring (MRM) mode with negative electrospray ionization. The detection ions of calcium dobesilate and the internal standard were 188.9→79.9 amu and 191.9→79.9 amu, respectively.There was no impurity peak in blank plasma affecting the determination accuracy. Under the selected LC-MS/MS conditions, the retention time of calcium dobesilate and the internal standard was stable, and the response could be reproduced. The linear range of calcium dobesilate is 0.2–20 μg/mL, and the lower limit of quantitation is 0.2 μg/mL. The ratio of concentration and peak area has a good linear relationship.The developed method was validated for selectivity, linearity, accuracy, precision, sensitivity, recovery, reproducibility, and stability according to the guidance for bioanalytical method validation of FDA/CFDA/EMA.