Quantitative Thin Layer Chromatography Technique and Application to Assay of a Steroid: 6-Chloro17a-hyd roxypregna-4,6-d iene-3,2O-d ione Acetate H. L. BIRD, JR., H. F. BRICKLEY, J. P. COMER, P. E. HARTSAW, and M. L. JOHNSON Control Division, Eli lilly and
Co., Indianapolis,
b Steroid samples and standards were chromatographed on thin layers of silica gel using a mixture of chloroform and ether. The steroid spots were located by ultraviolet light and a water spray. A technique is described for removing the adsorbent by vacuum, eluting the steroid, and filtering the eluate using a simple bent glass tube with a cotton plug. Absorbance a t 283 mp was used to quantitate the steroid. Studies of precision, accuracy, and linearity of the method, using the work of several analysts, showed quantitative thin layer chromatography to be sufficiently precise and accurate for practical quality control.
W
value of thin layer chromatography has become well recognized in recent years, its quantitative potential has not been widely investigated. The first quantitative use of the thin layer technique was reported by Kirchner, Miller, and Rice in 1954 (5), when they described the assay of biphenyl in citrus fruits and fruit products. Since then, only a few reports have been published on the quantitative use of this technique. Included among these reports were one by Ganshirt, KOSS,and Morianz (3) in 1960 on the use of thin layer chromatography for the quantitative assay of bile acids, and one by Zollner, Wolfram, and Amin (8) in 1962 on cholesterol esters. After presentation and submission of this manuscript, a technique using somewhat similar techniques on steroids was published (6). These are the only quantitative uses of the technique on steroidal compounds which have come to our attention. Because the application of quantitative paper chromatography has proved useful for steroids of various types, a procedure using thin layer chromatography quantitatively should be even more useful because of the advantages of speed and simplicity which the thin layer technique possesses over that of paper chromatography. The purpose of this paper is to introduce certain techniques, which we feel 346
HILE THE QUALITATIVE
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ANALYTICAL CHEMISTRY
Ind.
contribute to simplicity and accuracy, and to show the accuracy and precision obtainable from quantitative thin layer chromatography in assaying the purity of a steroid of the progestational type. EXPERIMENTAL
Materials and Techniques. Glass plates (200- X 200-mm.) were covered with a 250-micron layer of Silica Gel G (E. Merck, Darmstadt) by the usual procedure using a fixed thickness, commercial spreading device (Desaga, Heidelberg). The plates were air-dried overnight in a laboratory under constant temperature (25' C.) and humidity (40% rH) control. No further drying procedures were applied before use. Volumes of 50 pl. of sample and standard solutions were spotted in duplicate on each plate with a blank space (6 cm.) between each sample and standard. A single 50-p1. microsyringe with cemented needle was used for application of both standard and samples. All solutions were made in chloroform and a gentle current of air was applied to keep the spots of application less than 7 mm. in diameter. The chromatograms were developed in a mixture of nine volumes of chloroform and one volume of anhydrous ethyl ether in the usual ascending manner in a rectangular jar with a filter paper liner. Development was allowed to continue until the solvent front reached a point within 1 inch of the top of the plate, a period of 30 to 45 minutes a t 25" C. The chromatogram was then removed and dried with warm air from a blower. The dried chromatograms were examined rapidly under low wavelength ultraviolet light, where the steroid being assayed appeared as a dark spot on the slightly fluorescent background. The spots were outlined with a metal or wood stylus outside the periphery of the spot. To ensure removal of the same amount of adsorbent from the areas of standard, blank, and sample, these areas were marked, using as a guide a template having an opening large enough to include all of the largest steroid spot on the plate. The blanks were marked a t the same mobility as that of the steroid spots. The silica gel in each of the marked areas was transferred to a special
collection and elution tube using vacuum. These collection and elution tubes were modifications of a tube used by Cerny, Joska, and Labler (1) for preparative work on unbound thin layers. To make the modified tube, a 14-cm. length of 7-mm. 0.d. glass tubing was bent to an angle of 120' with one section, 9 cm., the other 5 cm. long. The shorter end was lightly firepolished and the longer end was slightly constricted. A small wad of absorbent cotton, previously washed with absolute ethanol, was sucked into the open end by applying vacuum to the constricted end. This cotton filtering plug was lightly packed into the constricted end with a flexible plastic rod. Vacuum was applied to the constricted end of the tube and, as the area of the plate was scraped clean by the edge of the open end of the tube, the silica gel was pulled into the tube and collected a t the cotton filtering plug and along the sides. When all the silica gel in the area marked was completely removed from the plate, the tube was turned upright and tapped lightly to dislodge the silica gel from the sides. Each collection tube was placed in the neck of a 10-ml. volumetric flask and the steroid was eluted by delivering 1- or 2-ml. portions of absolute ethanol into the open end of the tube with a hypodermic syringe until a total of 5 ml. was used for elution. The clear eluates were diluted to 10 ml. with absolute ethanol, and the absorbances were measured on a Beckman Model DU spectrophotometer a t 283 mp against absolute ethanol. Absorbance of the blank was subtracted from the corresponding standard and sample in each assay. These techniques were used in all of the experimental work except where otherwise noted. Procedures. The experimental procedures were designed to determine whether analysts, using the techniques described above, could obtain the necessary precision and accuracy for practical quality control. Precision, accuracy and linearity were studied. The compound chosen for these studies was the progestational steroid 6-chloro-17a-hydroxypregna-4,6diene-3,20-dione acetate ( 7 ) . PRECISION STUDY.A raw material sample of the steroid was assayed by four analysts performing two assays per plate on eight plates. The standard
Table I. Summary of Analysis of Variance on Assay or Raw Material Sample
Source of
Degrees of
Sum of
Variation freedom squares Ilnalyst s 3 19.48 Plates within analysts 2S 311.9 Replication 386.1 within plates 32 Relative standard dpriation of lion within a plate, f:3.1;‘2.
Table II.
Yean square 6.49 11.1 12.1
renlicn-
recovered from the spots of chromatographed standard compared to the absorbance of the same amount of unchromatographed standard. LINEARITY STUDY.Four solutions of the standard were made accurately a t the following approximate concentrations: 16, 14.5, 13, and 11.5 mg. in 10 cc. and were referred to as 100, 90, 80, and 7Oy0 solutions, respectively. The 90, 80, and 70% solutions were assayed using the 100% solution as the standard, by three analysts performing two assays per plate on one plate for each of the three concentrations. The 5O-pl. volume spotted in each case represented a range
Recovery of 6-Chloro-l7a-hydroxypregna-4,6-diene-3,20-dione tate after Chromatography of 85% Mixture
Analysts Plate 1 Plate 2 Analysts average Average = 99.194;.
A, 7%
B, %
98.4 95.1 95.7 101 97.6
101 95.3 105 95.1 99.4
was a highly purified, chromatographically homogeneous sample of the steroid. Solutions of sample and standard were made by placing an accurately weighed sample (approximately 16 mg.) into a 10-ml. volumetric flask and diluting to 10 ml. with chloroform. The results were submitted to a statistical analysis of variance. .~CCURACY STUDY.A synthetic m k ture of the standard steroid and an estrogenic steroid licr-ethynylestradiol3-methyl ether (9) was accurately prepared. The mixture contained approximately 85% of the standard steroid and 15% of the estrogenic artificial impurity. A solution was accurately prepared to contain approximately 16 mg. of total steroid in 10 ml. of solution. These two steroids previously had been shown to separate in the solvent system used in this method. Because the 17aethynylestradiol-3-methyl ether does not show under ultraviolet light, the chromatograms in this study were sprayed with distilled water to locate the steroids. Both appeared as dry white spots on the translucent wet silica background. The spots were outlined outside the periphery as with the ultraviolet method and the plate was dried with a blower before marking the areas of the major steroid and blanks with the template. This is the only variance from the previously described techniques. The 85% mixture was assayed by four analysts performing two assays per plate on two plates. Three of these analysts also measured the absorbance of 50-p1. aliquots of unchromatographed standard, a t the same time, to determine the actual recovery of the standard after chromatography and elution. The results on the mixture were calculated as per cent recovery based upon chromatographed standards while the results of the other part were based upon the amount of absorbance
c, % 98.6 99.8 96.8 95.i 97.7
Ace-
D, 70 105 101 105 98.8 102
Table 111. Recovery of Standard 6Chloro- 1 7a-Hydroxypregna-4,6diene-3,20-dione Acetate after Chromatography
Plate 1 Plate 2 Analysts average Average
=
97.4 97.6 98.5 94.7
95.6 99.4 96 6
96.9 98.7 98.4 99 1
97.0
97.4
96 3
98.0
97.6Yc.
Table IV. Linearity Study Results and Regression Analysis Conclusions
Actual yc
Observed yoa Analvst B“ C
A 70.1 72.1 69.5 78.8 79.9 80.3 86.4 89.3 89.8 Average of two replicates on one plate. Observed yo = 4 20 0.944 actual 7c. 70.1 50.4 89.8
+
F test data fitting straight line is acceptable ( 4 ) . 95Yc confidence limits for line at 80% actnnl = observed yo i~.763.
of 57 to 8 1 pg. of steroid. A regression analysis of the data was carried out to determine the linearity of the results. The compound studied was known to follow Beer’s law.
any possible losses from evaporation, filtration, or transfer were minimized. Linearity Study. Table I V presents a summary of the average results obtained on the assays of the RESULTS AND DISCUSSION 90, 80, and 70% solutions of standard Precision Study. Table I sumand also shows the conclusions of the marizes the results of analyzing variregression analysis of these data. ance of the data obtained in the The results fit the statistical requireprecision study. This analysis ments for a straight line (4) having an showed that the major source of intercept a t 4.20 and a slope of 0.944. variance was the replication within a The assay is therefore valid for amounts plate. When this variance of repliof the steroid from 57 to 81 pg. cation was considered, the variance The precision, accuracy, and linearity among analysts or among plates was studies showed that analysts trained not significant. The estimate of the in the described techniques could obtain relative standard deviation of ~t3.4797~ the necessary precision and accuracy for the replication within a plate may from quantitative thin layer chromatogbe used to calculate the number of raphy for practical quality control. replications necessary for the desired precision. The relative standard deviaLITERATURE CITED tion of the average from two replica(1) Cerny, V., Joska, J., Labler, L., tions per plate on two plates could be Collection Czech. Chem. Ccmm. 2 6 , 1658 expected to be *1.73%. The cause (1961). (2) Colton, F. B., Mysted, L. Tu’., Riegel, of variance of replication within a B., Raymond, A. L., J . Am. Chem. SOC. plate mould be difficult to determine. 79,1123 (1957). Accuracy Study. Table I1 sum( 3 ) Ganshirt, H., Koss, F. Xi., RIorianz, marizes the recoveries of the major K., Arzneimittel-Forsch. 10, 943 (1960). (4) Hald, A., “Statistical Theory with steroid from the 85y0 mixture. An Engineering Application,” p. 534, Wiley, average recovery of 99.19% was New York, 1952. shown. (5) Kirchner, J. G., Miller, J. M., Rice, Table I11 shows that the amount of R. G., J . Agr. Food Chem. 2 , 1031 (1954). steroid measured in the eluates was (6) Matthews, J. S., et al., J . Chromatog. 97.6y0 of the amount spotted. These 9, 331 (1962). satisfactory recoveries may be attrib(7) Ringold, H. J., Batres, E., Bowers, A,, uted to the method of elution. With Edwards. J.. Zderic. J.. J . Am. Chena. SOC.81,3486 (1959).’ ‘ this method of elution, the steroid on (8) Zollner, N., Wolfram, G., Amin, G., the silica gel was exposed to succesKlin. Wochschr. 40,273 (1962). sive applications of fresh solvent, thus RECEIVED for review October 22, 1962. favoring elution. With the volume of Accepted December 26, 1962. Division eluting solvent small and filtration of Analytical Chemistry, 142nd Meeting, taking place as the steroid was eluted, Atlantic City, N. J., September 1962. VOL. 35, NO. 3, MARCH 1963
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