Studies On Purification Process And Fingerprint Analysis Of Conjugated Estrogens

Objectives: To retain the all common peaks of FDA regulation while enhance the general conjugated estrogens (CE) purity, isolate and purify the CE extract with MCI GEL CHP20P -one kind of meticulous separation filler.Methods: (1) Inspect the static adsorption amounts of 3 kinds of MCI GEL meticulous separation fillers.(2)Inspect the MCI GEL CHP20P's dynamic saturated adsorption amount for the CE extract. (3)Inspect the concentration of elution solvent. (4)According to the results of dynamic saturated adsorption amount and the concentration of elution solvent, ascertain the purification process parameters by single factor experiment and orthogonal experiment. (5)Under the best conditions of purification process, do the validation experiment and the resin stability experiment.(6)Establish the HPLC fingerprint about 8 batches of CE refined substances derived from the validation test and evaluate its similarity by《2004 edition similarity evaluation software of traditional Chinese medicine fingerprint chromatogram》, meanwhile, analyze the retaining situation of 21 common peaks of FDA regulation. (7)Establish the GC fingerprint about 8 batches of CE refined substances derived from the validation test and evaluate its similarity by《2004 edition similarity evaluation software of traditional Chinese medicine fingerprint chromatogram》,meanwhile,analyze the retaining situation of 20 common peaks of FDA regulation. Results: (1)MCI GEL CHP20P showed best static adsorption capability among the 3 kinds of MCI GEL.(2)MCI GEL CHP20P's dynamic saturated adsorption amount for the CE extract is 90mg·mL^-1 (wet resin volume). (3)Water is the best elution solvent of another common peaks of FDA regulation excluding the 10 CE components; 10% methanol aqueous solution is the best elution solvent of 10 CE components. (4)The best adsorption and elution condition is: amount of CE extract/ wet resin volume=45mg·mL^-1;primary solution concentration= 0.16g·mL^-1; Amount of elution solvent/ amount of CE extract: water 45 mL·g^-1, 10% methanol aqueous solution 450mL·g^-1. (5)Under the best purification process, the resin adsorption and desorption are stable and it can be used 8-9 times repeatedly before regeneration; the purity of 10 CE compounds was enhanced obviously and the recovery could reach more than 90%; In addition, the color of CE refined substances become lighter and it has no peculiar smell compared with CE extraction. (6)HPLC fingerprint chromatogram about 8 batches of CE refined substances showed good similarity and the 21 common peaks parameters are same or closer compared with which FDA supply. (7)GC fingerprint chromatogram about 8 batches of CE refined substances showed good similarity and the 20 common peaks parameters are same or closer compared with which FDA supply.Conclusion: MCI GEL CHP20P showed good capability of static and dynamic saturated adsorption ;it begins to leak when the effluent volume reached 12 times of resin's;it becomes balanceable when the effluent volume reached 15 times of resin's. MCI GEL CHP20P showed well adsorption and desorption ability when isolating and purifying the CE extract. The purification process is simple, stable and credible. The CE refined substances showed high purity, light color and little peculiar smell; In addition, through the HPLC and GC fingerprint analyzing, the CE refined substances retained the characteristic common peaks, which include active substances of FDA regulation.This purification process has provided the solid basis for the improvement of the CE industrial production in the future.