A method for obtaining a natural mixture of conjugated estrogens from pregnant mare urine containing major conjugated estrogen components and minor conjugated estrogen components comprising;
- (a) contacting a mare urine material having a pH of 8.5 to about 9.5 and freed of particulate matter with a quantity of a non-ionic resin to adsorb phenolic compounds contained in the mare urine material and separating the resin laden with the phenolic compounds form a first residual mare urine material;
- (b) contacting the first residual mare urine material with a quantity of weak cationic resin to adsorb the major conjugated estrogen components contained in the mare urine material and separating the resin laden with the major conjugated estrogen components form a second residual mare urine material;
- (c) contacting the second residual mare urine material with a quantity of a non-ionic resin to adsorb the minor conjugated estrogen components contained in the mare urine material and separating the resin laden with the minor conjugated estrogen components from a third residual urine material;
- (d) contacting the resin laden with the major conjugated estrogen components with a sufficient quantity of an elution liquid selected from the group consisting of isopropyl alcohol and methanol to desorb the adsorbed major conjugated estrogen components and recovering an eluant containing crude major conjugated estrogen components;
- (e) contacting the resin laden with the minor conjugated estrogen components with a sufficient quantity of an elution liquid selected from the group consisting of isopropyl alcohol and methanol to desorb the adsorbed minor conjugated estrogen components and recovering an eluant containing crude minor conjugated estrogen components;
- (f) concentrating the eluant containing the major conjugated estrogen components to a viscous liquid, mixing the viscous liquid with acetone and filtering the acetone mixture through diatomaceous earth to obtain a filtrate containing crude major conjugated estrogen components;
- (g) concentrating the eluant containing the minor conjugated estrogen components to a viscous liquid, mixing the viscous liquid with acetone and filtering the acetone mixture through diatomaceous earth to obtain a filtrate containing crude minor conjugated estrogen components;
- (h) evaporating the eluant containing the major conjugated estrogen components to produce a heavy black mass, dissolving the black mass in water, adding NaCl until saturation to produce a solution, mixing the solution with n-butanol, extracting the n-butanol layer, evaporating the n-butanol layer to dryness to produce a powder containing the major conjugated estrogen components, washing the powder with toluene to remove free estrogens and degradation products, dissolving the powder in acetonitrile and removing the acetonitrile to produce crystalline major conjugated estrogen components;
- (i) evaporating the eluant containing the minor conjugated estrogen components to produce a heavy black mass, dissolving the black mass in water, adding NaCl until saturation to produce a solution, mixing the solution with n-butanol, extracting the n-butanol layer, evaporating the n-butanol layer to dryness to produce a powder containing the minor conjugated estrogen components, washing the powder with toluene to remove free estrogens and degradation products, dissolving the powder in acetonitrile and removing the acetonitrile to produce crystalline minor conjugated estrogen components;
- (i) analyzing the crystalline major conjugated estrogen components to determine the quantity of major conjugated estrogen components present;
- (j) analyzing the crystalline minor conjugated estrogen components to determine the quantity of minor conjugated estrogen components present;
- and
- (k) blending the crystalline major conjugated estrogen components and the crystalline minor conjugated estrogen components to produce a natural mixture of conjugated estrogens.