| Objective: 1. A selective and sensitive high performance liquid chromatography(HPLC) method was developed and validated for the simultaneous determination of loganin, feruilic acid, stilbene glucoside in Bushen Tongluo Formula. 2. To establish an HPLC method for extracting and determining the loganin, ferulic acid and stilbene glucoside in rat plasma after ig administered with Bushen Tongluo Formula. 3. To establish a AMS method for the semi-quantitative of Salviandic acid B in Bushen Tongluo Formula, and an HPLC method to quantitative.Methods: 1. Chromatographic separations were successfully performed on a C18 stationary phase with a mobile phase composed of methanol–water(35:65, v/v) with 0.1% glacial acetic acid at a flow of 1 mL/min. The UV detection wavelength was using a switching technology for the maximum absorption wavelength of each marked compounds. The temperature of column was 30 oC. 2. The plasma was extracted and treated with a mixture of methanol and ethyl acetate(3:1=v:v), then plasma extract evaporated to dryness under a stream of nitrogen flow, and the residue was reconstituted in HPLC mobile phase to inject. The conditions of chromatography: the chromatographic separations was performed on Johnson spherigel C18 column(250 mm×4.6 mm, 5 μm particle size) with a mobile phase composed of methanol–water(from 30:70 to 50:50, v/v) containing 0.01% glacial acetic acid at a flow of 1 mL/min. loganin was determined at 240 nm, ferulic acid and stilbene glucoside were determined at 320 nm. The temperature of column was 30 oC. The inject volume was 10 μL. 3. Using ambient ionization mass spectrometry(AMS) to semi-quantitative and HPLC to quantitative. The ion source was high voltage source, helium flow rate of 0.6 L/min, corona voltage of 3.5 kV, heating temperate of corn source was 100 oC, full scan in negative ion model, the inject volume was 10 μL; The chromatographic separations was performed on Johnson spherigel C18 column(250 mm×4.6 mm, 5 μm particle size) with a mobile phase composed of acetonitrile-water containing 0.05% phosphoric acid at a flow of 1 m L/min with gradient elution, the UV detection wavelength was 285 nm, the temperature of column was 25 oC. The inject volume was 10 μL.Results: 1. The method showed good linearity and no endogenous material interfered with the marked compounds peaks. The calibration curves of loganin, ferulic acid and stilbene glucoside were in good linearity over the ranges of 5~250 μg/m L(r=0.9997) for loganin, 0.5~25 μg/m L(r=0.9998) for ferulic acid, and 6~300 μg/m L(r=0.9999) for stilbene glucoside, respectively. The average recoveries(n=6) of loganin, ferulic acid and stilbene glucoside were 93.1%, 96.3%, and 101%, respectively. 2. The method showed good linearity and no endogenous material interfered with the marked compounds. The linear range of loganin, ferulic acid and stilbene glucoside was 0.03~3.00, 0.03~3.00 and 0.02~2.00 μg/m L respectively(r≥0.999), the limit of quantification was 0.020 μg/m L, 0.015 μg/m L, and 0.010 μg/m L, the extraction recovery was over 79%, over 74% and over 58%, within-day and between-day precisions were less than 10%, 7.3% and 9.7%. 3. A AMS method of rapid detect Salviandic acid B in Bushen Tongluo Formula was established with optimization experiments, the linear range of Salviandic acid B was 2~200 μg/m L(r=0.9961), the RSD(%) of precision of 5 repeated injects was less than 23%, the detection results of AMS method and HPLV method were consistent, which proved that the AMS method can be used to rapid qualitative and semi-quantitative of Salviandic acid B in Bushen Tongluo Formula.Conclusion: 1. The proposed method was precise and convenient, and can be used in determination of loganin, ferulic acid and stilbene glucoside in Bushen Tongluo Formula. 2. The established method is accurate, sensitive and convenient for detecting blood concentration of loganin, ferulic acid and stilbene glucoside, and studying in pharmacokinetics. 3. The established AMS method is quickly and efficiently for detecting Salviandic acid B in Bushen Tongluo Formula, it can be used as a semi-quantitative method for rapid detection of target components. |
PDF Full Text Request