exponent in the second term on the RHS of the equation is based on fitting the equation to experimental data, assuming no dependence of the resistance to mass transfer in the mobile phase on k'. This equation, it can be seen, can in some circumstances be reduced to either the Snyder or the Knox equation. The Huber equation allows one to minimize the analysis time for a given resolution in a manner similar to Knox's. Since the equation relates the plate height to the experimental conditions (such as Dm, dp etc.) the researcher can choose the system that best suits his needs. Other Efficiency Related Expressions In addition to plate height, several other param-

eters are used by chromatographers to evaluate their systems. Among them are: number of effective plates, height equivalent to an effective theoretical plate, number of plates generated in a given time, column performance factor (discussed briefly previously), resolution per unit time, etc. Although these parameters are becoming more widely used, they are not employed as regularly as the plate height. This is unfortunate since valuable information can be gained from such quantities. In a subsequent paper, these efficiency parameters will be discussed and analyzed as to their advantages and disadvantages in general and vis-a-vis one another. • Manuscript received July 18,1974

Quantitative Determination of Conjugated or Esterified Estrogens In Tablets By Thin Layer Chromatography by Irma Schroeder, Gulliver Lopez-Sanchez, Jorge C. Medina-Acevedo, Ma. del Carmen Espinosa,

Syntex, S. A. Pharmaceutical Division, Analytical Development Department, Apartado Postal 10-821 Mexico 10, D. F. Mexico Abstract

Experimental

A method has been developed using thin layer chromatography to separate and determine quantitatively estrone, equilin, equilenin and estradiol after hydrolysis of their sodium sulfate salts in tablets.

Shandon equipment for thin layer chromatography and a Unicam Spectrophotometer Model SP-800 were used. Separation of the estrogens was achieved on 20 x 20 cm thin layer chromatography plates coated to a thickness of 0.25 mm with silica gel F234 (E. Merck, Darmstadt) and the solvents in the system (chloroform rcyclohexane: acetone: ammonium hydroxide (30: 54:8:8)) were reagent grade. The estrogen sodium sulfates were synthetic, supplied by Syntex, S.A. Chemical Division, Cuernavaca, Estado de Morelos, Mexico. Aqueous hydrolyses with sulfuric (4) and hydrochloric acids under identical conditions (5% v/v, 60 min at 60° C) were carried out. As the results obtained with hydrochloric acid were superior, this was the one adopted as hydrolyzing agent.

Introduction

Conjugated or esterified estrogens are the sodium sulfates of estrogens of the type excreted in urine by pregnant mares (1,2). The main components according to the amount in which they are found, are: estrone, equilin, equilenin and estradiol. Existing analytical methods to quantitate these estrogens separately using colorimetry (2,3) or gas liquid chromatography (4) are much too complicated and time-consuming. The infrared method (5) is subject to many errors due to interfering absorptions by substances present with the estrogens which do not separate completely and lead to high results. Because of their similar polarities, the chromatographic separation has always presented many difficulties. The isolation of estrone and equilin has been achieved by paper chromatography for 32 to 37 hours (6). Very good results have been obtained with thin layer chromatography using continuous and dual elution systems (7). The method described achieves the quantitative separation and determination of the four estrogens by multiple elution (8) in a strong alkaline solvent system. JOURNAL OF CHROMATOGRAPHIC SCIENCE



VOL. 13

1. Savard, K. Endocrinology, 68, 411 (1961). 2. The United States Pharmacopoeia, Eighteenth Revision, p. 242 (1970). 3. The National Formulary, Twelfth Edition, p. 158 (1965). 4. Schroeder, I., Medina, J. C. and Lopez, G., J. Chromatog. Sci., 10, 183 (1972). 5. Higuchi, T. and Brochmann-Hanssen, E., "Pharmaceutical Analysis," Interscience Publishers, New York 1961. 6. Cox, J. E., J. Chromatog., 61, 193 (1971). 7. Knol, G. J., Bayden, G. R, Moody, R. H., Commeau, J. C. and Kho, B. T., J. Chromatog., 61, 187 (1971). 8. Bennett, R. D. and Heftmann, E., J. Chromatog., 21, 488 (1966). JANUARY 1975

37

To verify that no degradation occurred during the hydrolysis, free estrogens were treated under the same conditions, obtaining satisfactory recoveries. Hydrolysis with simultaneous benzene or chloroform extraction (9) and hydrolysis followed by chloroform extraction were performed. Since in all cases the recoveries were analogous, the last method was adopted for its ease of application. Before extraction with chloroform, the acidic hydrolysis medium was neutralized with IN sodium bicarbonate solution against methyl orange test solution (10). By developing the plate in a strongly alkaline system saturated with water, the quantitative separation of these four estrogens was obtained (Figure 1). The plate was eluted in the chromatographic chamber at room temperature equilibrated 30 min before use with the solvent system (chloroform :cyclohexane: acetone:ammonium hydroxide (30:54:8:8)). After the solvent front reached the top of the plate, it was allowed to dry in air ten minutes and eluted two times more with the same solvent system (3 to 3V£ hours). The water which separates from the solvent system was originally part of the ammonium hydroxide (Mallinckrodt A.R.) 58% aqueous solution. This second phase was absorbed both on a thin layer of cotton placed on the bottom of the chamber and on filter paper placed around the chamber walls. This water acting as a stationary phase delayed the movement of steroids up the plate so that extended multiple elution times can be employed. The estrogens were quantitatively determined by

absorption under ultraviolet light after development of the plate and elution from the silica gel. Estrone, equilin and estradiol were eluted into a 1% aqueous sodium hydroxide solution. The maximum absorption of estrogens in this alkaline solution occurred at 296 nm, considerably higher than the value at 280 nm when methanol was the solvent. Absorption of equilenin at 231 nm in a methanolic solution was high enough to permit its elution and determination in this solvent. In order to check the efficiency of recovery from the plate, an artificial mixture of the four estrogens was applied to the plate together with controls and the above process repeated. The results are given in Table I.

Table I. Recoveries of Estrogens Separated by Thin Layer Chromatography. Solvent System— Chloroform:Cyclohexane:Acetone:Ammonium Hydroxide (30:54:8:8)

Estrogen Estrone Equilin Equilenin Estradiol

meg. Applied to Plate meg Found 145.5 73.0 18.0 34.0

144.6 75.6 18.8 33.1

or /o

Recoveries 99.4 103.7 104.4 97.6

Analytical Method

»V

IT

ST

1. Addition of 10 ml of water to an amount of sample corresponding to 5 mg of estrogen sulfates. 2. Hydrolysis by addition of 0.5 ml of concentrated hydrochloric acid and heating under nitrogen atmosphere 60 min at 60° C. 3. Neutralization after cooling at room temperature with IN sodium bicarbonate solution using methyl orange test solution (10) as indicator, followed by chloroform extraction of the free estrogens from the aqueous suspension. 4. The chloroform extract was evaporated under nitrogen atmosphere and quantitatively made up to 2 ml. 5. 200 microliters were spotted on a thin layer chromatography plate. 6. The plate was placed in the solvent chamber and removed when the solvent reached 16 cm from the point of application. After drying at room temperature, the plate was developed twice more in the

Figure I. Separation of estrone, equilin, equilenin and estradiol after hydrolysis of the synthetic sodium sulfates in tablets. Estrone Rf = 0.55, equilin Rf = 0.45, equilenin Rf = 0.34, estradiol Rf = 0.22. Solvent system: chloroform: cyclohexane:acetone:ammonium hydroxide (30:54:8:8). ST = Standard Mixture, A,B = Uncoated Tablets, C,D = Coated Tablets. Spray reagent = CoCI2 4% in H2SO4 10% in water. 38

JANUARY 1975

9. Vestergaard, P. and Claussen, B., Acta Endocrin. Supp. 64, 35 (1962). 10. The United States Pharmacopoeia, Eighteenth Revision, p. 1028 (1970). JOURNAL OF CHROMATOGRAPHIC SCIENCE

• VOL. 13

Table II. Recoveries of Estrogens from Coated Placebo Tablets Spiked with Estrogens' Sodium Sulfate Salts* Estrone Sodium Sulfate 0.75 mg % Recovery 99.4 100.8 98.6 101.4 100.0 101.0 97.8 96.8 99.7 100.2 98.7 98.7 97.7 99.5 X = 99.29 (T= 1.35 CI.,,,% = 99.29 ± 0.98

Equilin Sodium Sulfate 0.375 mg % Recovery 100.8 99.5 97.0 98.5 96.8 96.8 100.8 97.3 102.3 97.0 102.0 105.0 105.0 102.0 99.98 2.92 99.98 ±1.69

Equilenin Sodium Sulfate 0.06 mg % Recovery 97.0 96.3 96.0 95.6 101.4 99.5 101.0 102.3 102.9 104.4 96.4 94.5 105.3 101.9 99.6 3.58 99.6 ± 2.06

Estradiol Sodium Sulfate 0.125 mg % Recovery 102.0 97.9 94.0 98.0 99.0 91.0 99.0 92.5 102.0 98.3 100.0 97.5 100.0 98.5 97.87 3.25 97.87 ± 1.87

*Each value corresponds to a duplicate analysis. Sets of two values were determined on different days.

Table III. Recoveries of Estrogens from Uncoated Placebo Tablets Spiked with Estrogens' Sodium Sulfate Salts* Estrone Sodium Sulfate 0.75 mg % Recovery 101.0 99.3 98.5 101.5 99.3 97.5 100.0 100.0 101.0 99.0 99.0 97.0 X = 99.42

Quantitative determination of conjugated or esterified estrogens in tablets by thin layer chromatography.

exponent in the second term on the RHS of the equation is based on fitting the equation to experimental data, assuming no dependence of the resistance...
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