| Objective To establish the fingerprints of Shuanghuanglian(SHL) Powder-Injection and Oral-Liquid by high performance liquid chromatography (HPLC) and to evaluate of the comparability of 10 batches of SHL Powder-Injection and Oral-Liquid.To investigate the effect of different solvents on fingerprints of SHL Powder-Injection.To explore the in vitro metabolism of SHL Powder-Injection and Oral-Liquid in rat liver microsomes by establishing their metabolic fingerprints.Methods Agilent 1200 LC with quaternary pump and UV detection was used. A column of DIKMA C18 (150 mm×4. 6 mm, 5μm) was used for separation. By examining a variety of different mobile phases and gradient elution modes, finalize the mobile phase consisted of methanol (A) and 0.25% acetic acid (B). The optimal gradient elution mode was as follows: 0 15 min, A is 15% 35%; 15 20 min, A is 35%; 20 50 min, A is 35% 100%. The mobile phase was delivered at a flow-rate of 1.0 ml·min-1, and in a gradient elution mode. UV detection wave length was set at 350nm and column temperature was kept at 30℃. SHL Powder-Injection, SHL Oral-Liquid and the reference standards was dissolved by 50% methanol to a certain concentration. 20μl of every sample was injected into LC. The analysis time was 60 min. The fingerprints of SHL Powder-Injection dissolved in 0.9% NaCl injection, 10% glucose injection and 10 batches of SHL Powder-Injection and SHL Oral-Liquid were analyzed by using the method established above. The rat liver microsomes were prepared by ultracentrifuging method. Then the concentration of microsomes protein was quantified. 1 mg·mL-1 of microsomes protein were used in in vitro incubation. SHL Powder-Injection, SHL Oral-Liquid and the reference standards were taken in vitro metabolism. The samples 100μl were taken from the incubation system in 0 min,10 min,20 min,30 min and 60 min separately. twice of the amount of cold methanol was added to stop the reaction. The compound was centrifuged 10 min at 4000×g . The in vitro metabolic fingerprints were analyzed by using the method established above.Results Fingerprints of SHL Powder-Injection, SHL Oral-Liquid and the reference standards were established and the quality control indicating components were assigned. The peak of baicalin was taken as a reference peak. The precision and reproducibility of the method established were inspected. Then the stability of the samples in 16 hour was investigated. The variation of main peaks relative retention time and relative peak area RSD were less than 3%. Similarity factors of SHL Powder-Injection dissolved in different solvents were greater than 0.97. Similarity factors of 10 batches of SHL Powder-Injection and SHL Oral-Liquid were greater than 0.97. The in vitro metabolic fingerprints showed that the main active ingredient baicalin can be metabolised and the product is different from baicalein.Conclusion The HPLC method established for the fingerprints of SHL Powder-Injection and SHL Oral-Liquid has good reproducibility and specificity. The quality of SHL Powder-Injection and SHL Oral-Liquid can be represented objectively. The comparability of SHL Powder-Injection dissolved in different solvents was investigated. The effect was not embodied in the fingerprints. The method can be used for quality control of SHL Powder-Injection and SHL oral liquid. And can be used for safety studies of SHL. It also can be used to evaluate the comparability of 10 batches of SHL powder-injection or SHL Oral-Liquid. By using the in vitro metabolic fingerprints,the metabolism of SHL in rat liver microsomes was studied preliminary. The results might provide some basic and new ideas to explore the mechanism of the adverse drug reaction and to study the efficacy and safety of the medicine.
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