Progress in biochemical investigations. I. Steroids: 1961

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NATELSON, Roosevelt Hospital, Department of Biochemistry, New York, New York

This review should be compared to a similar review published in 1959l where the analytical chemistry of the steroids was included in a broader study including other substances. The most, significant advance in the field of the analytical chemistry of the steroids is the development of practical techniques for gas In the discussion below the extensive use of chromatography.2-4 this technique will become apparent. Thin-layer chromatography has been long used abroad but is first beginning to be applied generally in this country. In this technique a supporting chromatographic medium such as silica gel or alumina is spread on glass in a thin layer, usually with a binder, to keep t,he exchange material coherent. The technique used is then similar t,o paper ehromatography.5 A pattern develops rapidly, usually in l/t hr., and a wide variety of reagents are practical for developing the spots. In effect, this technique is similar to column chromatography long used in this field. A gradient elution technique has been described for continuously separating the corticoids and recording the color produced with tetrazolium blue on a strip chart.6 For the steroids having some solubility in water, such as the glucuronides of the steroids7 the saponins,8 and other glyeosides of the steroids,g paper chromatography is practical for separation. For those which are relatively insoluble in water but soluble in organic solvents, reversed-phase paper chromatography is commonly used.lo,ll A new stable color reaction has been reported for the conjugated steroids, namely p-aminodimethyl aniline as the H&!nCl~ salt.12

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Cholesterol For convenience in the reading of this review the structure of cholesterol as a typical steroid is drawn in Figure 1. The four rings of cholesterol are pictured schematically lying at an angle to the paper so as to illustrate the location of the different groups with respect to the plane of the rings. Actually ring B is distorted to the “half chair” configuration because of the double bond at posit’ions 5, 6. Solid 18 22-27

~~6

H13

Fig. 1. Cholesterol.

lines are drawn to those groups above the plane, such as the methyl groups at positions 18 and 19, the OH group at position 3, the side chain at position 17, and the hydrogen at position 8. These groups are said to be in the beta (0) position. The hydrogens at positions 3, 9, 14, and 17 are below the plane or in the alpha (CX)position. According to convent’ion, these are connected to the ring by a dotted line. The structure is rigid and several isomers exist. The basic struct,ure of cholesterol, with the double bond reduced and the OH replaced by hydrogen is called cholestane; thus cholesterol could be called 3-p-01 cholestene A.5,6 Where there are two hydrogens on the ring such as in position 1, one is above and one is below t’he ring. When cholesterol is reduced it will be noted that only one hydrogen will exist at position 5. In cholestane this is below the ring or alpha (dotted line). Cholesterol is most commonly est,imated by the LiebennanBurchard reaction. This comprises dissolving the cholesterol in chloroform and adding a mixture of glacial acetic acid, acetic anhydride, and concentrated sulfuric acid to produce a blue-green color.13-17 In one variation, toluenesulfonic acid is used with or in place of the sulfuric acid to produce a color more stable with time.18 Elemental iodine has been added to a mix of glacial acetic acid and sulfuric acid to produce a sensitive reagent, for cholesterol.lg The MICROCHEMICAL

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combination, sulfosalicylic acid, acetic anhydride, and sulfuric acid, has been suggested for the same purpose.20 Usually, proteins are removed before analysis, but reagents have been suggested for estimating cholesterol in human serum without deproteinization. In this case the acetic anhydride concentration is increased to react with the water present.zls22 Ferric ion in glacial acetic acid in the presence of sulfuric acid produces a color suitable for quantitation for cholestero1.23-*8 This is known as the Kiliani reagent. This method has been developed for the estimation of 10 ~1. of serum, phosphoric acid being used in the mix.2g It has been shown that nitrate ion interferes in this reaction.30 The Tschgaeff reagent (acetyl chloride in the presence of zinc chloride) continues to be used by some for cholesterol estimation.31 Digitonin forms an insoluble complex with free cholesterol and is used for separation of cholesterol from its esters.32T33 It is claimed that tomatine is more specific for this purpose.34 Cholesterol may be assayed by the turbidity produced with digit’onin.35 A variation of the procedure is t,o precipitate the cholesterol with digitonin and determine the carbohydrate moiety in the digit,onin by a carbohydrate reagent such as anthrone.z6 It has been pointed out that a chromatographic separation of steroids is more effective than with the use of digitonin.57 Cholesterol readily adds bromine to the double bond t#o form a Heating with zinc in glacial acetic readily crystallizable dibromide. acid regenerates the cholesterol. This has been suggested as a method of purification of small amounts of cholesteroL3* Cholesterol may be readily separated from its esters on an alumina column using Ccl, to remove the esters and CHCl, to elute the free cholesterol.3g Ether-chloroform mixtures are also used for sequential elution of esters and free cholesterol from alumina columns.“0 By paper chromatography it has been demonstrabed that cholesterol esters in serum are predominantly oleic, palmitic, and st,earic acid esters.41 SbC& is a suitable reagent for staining cholesterol derivaIX is added t’o tives on paper chromatograms.42 If protoporphyrin serum, cholesterol can be located as the electropherogram develops.43 Gas chromatography has been used for the separation of cholesterol and its derivatives from scalp sebum, liver oil, and various other oils on an Apiezon L column at 330°C.44 The absorption spectrum of various aj-3-hydroxy steroids has been

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determined after addition of strong sulfuric acid. The different steroids exhibit distinctive spectra in t,he visible region.45

Pregnanes Pregnane is the hydrocarbon that would be obtained if the side chain in Figure 1, from CZZto C27, were oxidized away, the double bond reduced, and the OH group at position 3 replaced by hydrogen. In progesterone C-20 is attached to an oxygen to form a ketone, and the OH at position 3 has also been oxidized to a ketone. The double bond at position 5,6 shifts to position 4,5 to be conjugated with the ketone at position 3. Thus progesterone may be called 3,20-diketopregnene-A.4J Reduction of the ketone groups at positions 3 and 20 to produce the alcohols and reduction of the double bond produces pregnandiol. With an OH group on position 21 replacing a hydrogen in pregnandiol we have one pregnantriol isomer. This particular isomer would arise from reduction of hydrocortisone. Aldosterone, cortisone, and hydrocortisone are also pregnane derivatives, as will be seen below. Progesterone may be fractionated and isolated on an alumina column.46 It can then be assayed by its characteristic absorption at 240 mH. Isonicotinic acid hydrazide has been used as a reagent for progesterone, to produce a yellow color with an absorption maximum at 380 rnp. This reaction is not specific, as reaction with this reagent occurs with numerous other ketosteroids, such as testosterone and cortisone.47 Pregnandiol is most commonly assayed in the urine as an indirect measure of progesterone production by the pregnant woman. The usual method is to extract the urine with organic solvents and treat with concentrated sulfuric acid containing the sulfite ion to produce a color. Glycol sulfite can substitute advantageously for the sulfite, and methyl alcohol added to the mix improves the stability of the color.48 Separation of pregnandiol from pregnantriol is readily achieved by glass fiber paper chromatography. The glass fiber is impregnated with potassium silicate, and a cyclohexane-acetone mixture is used for the separation. The color is developed on the paper with the sodium bisulfite-sulfuric acid reagent.kg Pregnandiol exists as the glucuronide in the urine. If the glucuronic acid is not split off but the whole molecule is extracted with a butanol-isoamyl alcohol mix, the naphthoresorcinol test may be applied to estimate MICROCHEMICAL

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the glucuronide and thus the pregnandiol.60 Thin-layer chromatography with alumina on the glass plate may also be used for fractionating the pregnandiols and pregnantriols, the fluorescence produced with 70y0 phosphoric acid being used.51 The glucuronides move in the electrical field, and electrophoresis has been applied to separate pregnandiol glucuronides.52 The methods for assay of pregnandiol have been reviewed and evaluated.sa The corticoids are related to progesterone in that they have the same basic structure but an hydroxyl group replaces one of the hydrogens on carbon 21. Carbon 20 still retains the ketonic structure. In eorticosterone hydroxyl groups replace the hydrogens of progesterone at carbons 11 and 21. Hydrocortisone has an additional hydroxyl group replacing the hydrogen at position 17. Thus hydrocortisone is a 17-hydroxyeorticosterone. In cortisone the hydroxyl group of hydrocortisone at position 11 has been oxidized to a ketone. The most commonly used method for the estimation of the 17hydroxycorticosteroids is the reaction with 62ye sulfuric acid in the presence of phenylhydrazine to produce a yellow color with a maximum absorption at 410 rnp (Porter-Silber reaction).64-57 This reaction is often applied after fractionation of the steroids on a Florisil column.5* During the last few years the fl~Iorescence of the 17-hydroxycorticoids when treated with concentrated sulfuric acid, usually in the presence of ethanol, has been extensively used because of its greater sensitivity.5g-63 This reaction has been applied to the 17hydroxycorticoids after fractionation on silica ge1.64 In the corticosteroids one is dealing with a structure similar to the first two carbons of a ketose. Thus, in sequence,one has an hydroxy and keto group on carbons 21 and 20, respectively. These substances are readily oxidized. The reducing action of these materials, such as with phosphomolybdic acid, has been used for assay.66 Oxidation of the hydroxyl group on carbon 21 to the aldehyde and assay with aldehyde reagents has been suggested for the 17-deoxycorticosteroids.@J If the corticosteroids are oxidized with sodium bismuthate, the carbons 20 and 21 are removed, and a keto group appears at position 17. The 17-ketosteroids so formed are readily assayed with dinitrobenzene reagent. These are called the 17-ketogenic steroids. The

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procedure usually proceeds by reduction of the 17-ketosteroids initially present with sodium borohydride, and then oxidation with sodium bismuthate.67~6s An alternative is to apply the dinitrobenzene calorimetric reaction before and after oxidation with sodium bismuthate obtaining the 17-ketogenic steroids by subtraction. Sodium periodate may replace sodium bismuthate as the oxidizing agent .6g The corticosteroids may be fractionated on columns70J1 or by paper chromatography.72,73 The RI values for several solvent mixtures and different corticosteroids are listed in one publication.74 The Porter-Silber reaction may be used in paper chromatography to locate the spots.75 Tetrazolium blue will locate the glucocorticoids on paper after chromatography.76s77 Separation by thin-layer chromatography separates the corticosteroids rapidly. Silica gel has been used in this technique with 5% ethanol in chloroform as the irrigating solution.78 Gas chromatography is being vigorously investigated as a most practical method for assay of minute amounts of corticosteroids. At 222°C. and with a silicone polymer supported on Chromasorb W, excellent separations are claimed.79 A combination of paper chromatography followed by acetylation of the corticosteroids and then gas chromatography after labeling with tritium has served to assay the corticosteroids in blood.*0 Several reviews have appeared on the various methods for the separation and assay of the corticosteroids81-a5 Aldosterone has the same general structure as hydrocortisone except that the angular methyl group at position 18 has been oxidized to the aldehyde. The aldehyde is connected to the hydroxyl group In urine the aldoson the 11 position in the form of an hemiacetal. terone is conjugated as the glucuronide or sulfate. Acid hydrolysis at pH l-l.5 in the presence of an extractant, such as chloroform or methylene chloride, serves to free the aldosterone.s6t87 The aldosterone is separated from other steroids by reversed-phase paper chromatography.88 When acetylated, aldosterone can be separated from cortisone and other steroids by gas chromatography.89 The C-19 Steroids If the side chain is completely removed, the residue with the double bond reduced and the hydroxyl group replaced by hydrogen MICROCHEMICAL

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is called androstane. This is the parent hydrocarbon of the male sex hormones. The 17-ketosteroids in the urine are derived partly from the testes in the male and partly from the adrenal cortex. In the female they are derived solely from the adrenal cortex. In testosterone, the male hormone of the testes, there is a ketone at position 3 in conjugation with a double bond at position 4,5. An hydroxy group exists at position 17 (beta). In the urine, however, this hydroxy group is usually found oxidized to the ketone group. Similarly, some of the corticosteroids are oxidized to produce a ketone group at position 17. The 17-ketosteroids are a complicated mixture of numerous products and their stereoisomers. The 17-ketosteroids are commonly assayed by their production of a purple color with dinitrobenzene in alkaline solution (Zimmerman reaction).sO,gl Pyridine has been used as the alkali to produce a more stable color.g2 Column chromatography serves to fractionate the 17-ketosteroids into some of their compo~ents.g3 Florisil,s4 alumina,s5-97 and silicie acids8 have been employed by sequential or gradient elution. In the urine, the 17-ketosteroids occur as sulfate esters and as glucuronides. This requires acid hydrolysis before estimation. If left in the form of their glucuronide or sulfate ester, they are somewhat water-soluble, and ordinary paper chromatography can be used. In this manner androsterone and dehydroisoandrosterone sulfates have been fractionated.ss Gas chromatography serves to separate the complicated mixtures of 17-keto-steroids derived from urine.lOO~lO1 The estrogens are also C-19 steroids lacking the side chain at position 17. Ring A is benzenoid, and t)herefore the hydroxyl at position 3 is phenolic in nature. The estrogens (female sex hormones) are thus separated from the androgens by extraction with alkali from an organic solvent. Since ring A is benzenoid there is no methyl group at position 19. There is no double bond in ring B. If there is a ketone group at position 17 we have estrone. If this ketone group is reduced to an alcohol we have estradiol-3,17. If there is an additional hydroxyl group at position 16 we have estriol$16,17. The parent hydrocarbon of the estrogens is estrane, and hence the nomenclat~lre. In human urine the estrogens occur conjugated with sulfate and glucuronic acid, and acid hydrolysis serves to split them free. An enzyme from the snail containing a sulfatase and a glucuronidase

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serves the same purpose with less side reaction.ln2 The color reaction most commonly used is that obtained by strong sulfuric acid or strong sulfuric acid in the presence of phenol (Kober reaction). Generally, the estrogens are first purified on a column before the color reactions are applied.103-10g Treatment with sulfuric acid produces a Auorescent material, and this has also been used as a method of assay.110-112 The conjugated estrogens may be chromatographed directly without hydrolysis on Sepahadex.‘13 Thin-layer chromatography wit’h silica on glass permits the use of concentrated acidic and corrosive materials for development of a color. Antimony trichloride is useful for locating spots due to the estrogens in thin-layer chromatography.l14 Reversed-phase paper chromatography permits a wide separation of the estrogens. For example, with ligroin-methyl alcohol as the irrigating solution, the R, values of estrone, estradiol, and estriol are 0.44, 0.29, and 0.07, respectively.ln Acetylated paper has also been used in paper chromatography.116 If the estrogens are methylated or acetylated, they are stable at high temperatures and are readily separated by gas chromatography.117~11* Stilbestrol has been separated in a similar manner. llI!

Vitamin D In the antirachitic vit’amins (vitamin D), ring B is split open at the 9,lO position and a double bond at the 7,8 position conjugates with the double bond at the 5,6 position. The side chain is intact and can vary in detail without affecting the nature of the antirachitic action except in the relative activity of the molecule. Antimony trichloride produces a characteristic color with vitamin D.120 However, vitamin A gives a similar color. Column chromatography is therefore necessary before the color reaction is applied.‘2l The steroids may be removed with digitonin before application of the antimony trichloride reaction.lz2 During gas chromatography the vitamins D are cyclized, yielding several isomers.‘z” A review of paper chromatographic methods for the fat-soluble vitamins has appeared.124

Bile Acids The bile acids all have the basic cyclic structure of cholesterol, hut substituents on the rings may vary. The side chain is oxidized at some point to produce a carboxylic acid. This acid may be conjuMICROCHEMICAL

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gated in a peptidelike bond with glycine or taurine. The bile acids produce a color when concentrated sulfuric acid is added to them in t’he presence of a sugar concentrated HC1.1z5 A pink color is obtained with 70y0 sulfuric acid in the presence of furfural.lz6 These two color reactions are probably the same. The esters of the bile acids also lend themselves t’o separat’ion by gas chromatography.

References 1. Natelson, S., Micro&em. J., 3, 351 (1959). “Progress in biochemical investigations: 1958.” 2. Van den Heuvel, W. J. A., E. 0. A. Haahti, and E. C. Horning, J. Am. Chem. Sot., 83, 1513 (1961). “A new liquid phase for gas chromatographic separations of steroids.” 3. Chen, C., and C. D. Lantz, Biochem. Biophys. Res. Communs., 3, 451 (1960). “Gas chromatography of some steroid hormones and metabolites.” 4. Van den Heuvel, W. J. A., J. Sjovall, and E. C. Horning, Biochim. Biophgs. “Gas-chromatographic behavior of trifluoroacetoxy ilctu, 48, 596 (1961). steroids.” 5. Hermanek, S., V. Schwarz, and Z. Cekan, Collection Czech. Chem. Communzs., 26, 1669 (1961). “Steroid derivatives. XII. Chromatograhy of neutral steroids on a t,hin aluminum oxide layer.” 6. Anderson, F. O., L. R. Crisp, G. C. Riggle, G. G. Vurek, E. Heftmann, I>. F. Johnson, I). Francois, and T. D. Perrine, Anal. Chem., 33, 1606 (1961). “Steroid analyzer.” 7. Crepy, O., B. LaChese, and 0. Judas, Rev. Franc. Etudes Clin. Biol., 6, “A new method for the detection of steroid glucuronides by paper, 601 (1961). chromatography.” “Paper chromatography of natural 8. Pasich, B., Nature, 190, 830 (1961). saponins.” 9. Herman, R. H., Nature, 190, 268 (1961). “Reaction of digitalis compounds with antimony pentachloride.” 10. Carr, H. E., Jr., and W. J. Reddy, Anal. Biochem., 2, 152 (1961). “Reversed phase partition chromatography of steroids.” 11. Starka, L., and Z. Matis, Suvremenna Med., 12, 123 (1961). “Paperchromatographic fractionation of steroids.” 12. Huettenrauch, R., Z. Ph,ysioZ. Chem., 326, 166 (1961). “Colored steroids. III. Color reaction of conjugated unsaturated steroids.” 13. de Traverse, P. M., G. H. Lavergne, and R. Depraitere, Repert. Med. “Determination of total serum cholesterol.” Pmt., 1, 1 l-12 (1960). 14. Assous, E., and M. Girard, Compt. Rend., 252, 2462 (1961). “Simple and rapid calorimetric determination of free cholesterol in the presence of fatty acid esters.” 15. Schwarz, H., Aerztl. Lab., 6, No. 5, 133 (1960). “Total cholesterol determination in serum.”

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16. Macapinlac, M. P., J. Philippine Med. Assoc., 37, 782 (1961). “A micromethod for serum total cholesterol.” 17. Nath, R. L., and N. K. Ghosh, Clin. Chem., 7, 678 (1961). “Effect of high room temperature on estimation of blood cholesterol by Bloor’s method.” 18. Boy, J., N. Bonnafe, and J. B. Mazet, Ann. Biol. Clin. (Paris), 18, 66!+ of total cholesterol by a modification of the Pearson (1960). “Determination method.” 19. Tamura, T., and T. Toya, l’okyo Toritsu Eisei Kenkyusho Nempo, 9, 148 (1957). “Calorimetric quantitative analysis of cholesterol.” 20. Ciampi, C., Progr. Med., 17, 133 (1961). “A new calorimetric method direct determination of serum cholesterol.” 21. Huang, T. C., C. P. Chen, V. Wefler, and A. Raftery, Anal. Chem., 33, Appli1405 (1961). “A stable reagent for the Liebermann-Burchard reaction. cation to rapid serum cholesterol determination.” 22. Hopper, Q. R., U.S. Pat. 3,001,950, August 18, 1958. “Reagent for determining cholesterol in blood serum.” “A method for measurement 23. Mann, G. V., Clin. Chem., 7, 275 (1961). of cholesterol in blood serum.” 24. Koval, G. J., J. Lipid Res., 2,419 (1961). “Cholesterol measurements on normal and lipemic serums. Elimination of an extraneous chromogen.” “A new cholesterol 25. Zak, B., and E. Epstein, Cl&. Chem., 7, 368 (1961). reagent.” 26. Weller, H., Aerztl. Lab., 7, 215 (1961). “The errors and variations in various methods for cholesterol determination, with emphasis on the ferrous sulfate method.” 27. Ciampi, G., and 0. Urcialo, Progr. Med., 16, 148 (1960). “Cholesterol determination in blood.” “A note 28. Bowman, R. E., and R. C. Wolf, Clin. Chim. Acta, 6,146 (1961). on the Searcy-Bergquist cholesterol reaction.” “Rapid spectro29. Shin, P. S., and J. C. Lee, Anal. Chem., 33,122O (1961). photometric determination of total cholesterol in small amounts of blood and cerebrospinal fluid.” “Prob30. Kitamura, M., and Y. Arimatsu, Nisshin Zgaku, 48, 456 (1961). lems on the determination of total cholesterol in serum with special reference to the interference of nitrate in commercial sulfuric acid.” 31. Sheff, N. F., M. D. Grete, and J. B. McMarlin, Clin. Chem., 7, 504 (1961). “Estimation of cholesterol in small quantities of cerebrospinal fluid.” 32. Anici, G., and D. Franzini, Progr. Med., 15, 170 (1959). “Cholesterol determination in blood.” 33. Brown, W. II)., Australian J. EzptE. Biol. Med. Sci., 39, 223 (1961). “Filtration procedure for determining serum cholesterol with digitonin.” 34. Kabara, J. J., J. T. McLaughlin, and C. A. Riegel, Anal. Chem., 33, 305 (1961). “Quantitative microdetermination of cholesterol using tomatine as precipitating agent.” “A turbidimetric method 35. Krinitsku, A. F., Lab. Delo, 7, No. 4,27 (1961). for determination of blood cholesterol.” 36. Vohouny, G. V., C. R. Borja, R. M. Mayer, and C. R. Treadwell, Anal. MICROCHEMICAL

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56. Jenkins, J. S., J. Clin. Puthot., 14, 185 (1961). “The effect of corticntropin zinc on pIasma 17-hydroxy corticoids as a test of adrenal cortical function.” 57. Serrano, M., A. Pedro, Prensa Med. &fez., 26, No. 5, 185 (1961). “Determination of corticoids in blood.” 58. Lavanchy, E., and S. Neukomm, Bull. Sot. Vandoise Sci. Nat., 67, No. 304,467 (1961). “A critical study of a method of determining urinary 17-hydroxy ~o~icos~roi~.” 59. Stewart, C. P., F. -~lbe~-Re~ht, and L. M. &man, C&i%.Ch&z. Aeta, 6, 696 (1961). “The simultaneous fluorimetric microdetermination of eortisol and corticosterone in plasma.” 60. Braunsberg, H., and V. IT. T. James, Anal. Bioehem., 1, 452 (1960). “The fluorometric determination of adrenocortical steroids.” 61. Hedner, P., Acta Pham~~ol. Tozicol., 18, 65 (1961). “Experiences with a fluorometric method for determining ca~e~~roids in man and rat.” 62. Giuliani, G., and A. Maffei-Faccioli, Atti. Accud. Med. ~~b~r~, 15, No. 1, 45 (1960). “Fluorometrie assay for plasma corticosterone, even in the presence of other steroids.” 63. Rudd, B. T., J. M. Cowper, and N. Crawford, Cl&. Chim. dcta, 6, 686 (1961). “The determination of plasma free hydrocortisone and corticosterone by a combined fluorimetric and modified Porter-Silber procedure.” 64. Lado, P., and F. Negri, F&a ~~~~~~Z., 13, 83 (1960) “chloroform extraction in the determination of urinary reducing corticosteroids.” 65. Braunsberg, H., and V. H. T. James, J. EncZocrinoZ.,21, 327 (19601. “The determination of adrenocortical steroids in blood. Results in normal individuals and adrenal hyperfunction.” 66. Exley, D., S. C. Ingall, J. H. Norymberski, and G. F. Woods, Biochem. J., 81, 428 (1961). “Indirect analysis of corticosteroids. V. The determinat,ion of 17-deoxy eorticosteroi~.” 67. Jorgensen, M., Uges~~~~t Lueger, 119, 1631 (1957). “Determination of urinary 17-ketogenic steroids.” 68. Brabencova, H., 2. Matys, R. Reisenauer, and L. Starka, &sop& L&aru reslcych, 99, 729 (1960). “Estimation of 17-ketosteroids, a simple method for determining corticosteroids.” 69. Few, J. D., J. Endocrinot., 22,31 (1961). “A method for the analysis of urinary 17-hydroxy corticos~roids.” 70. Cavins, G., and G. Nicholosi, Boll. See. ftal. BioZ. Sper., 35, 2119 (1959). “Chromatographic study of the steroids present in chloroform extracts of urine, fractionated between benzene and water (11-hydroxycorticoids and ll-deoxycorticoids).” Technol. Eng., 3, 21 71. Mizsei, A., and A. Szab6, J. Biochem. Microbial. (1961). “Rapid in determination of 17-a-hydroxy-l-deoxycorticosterone biological oxidation.” 72. Ganis, F. M., M. Hendrickson, P. D. Giunta, and J. W. Howland, U.S. At. Energy Comm., UR-601, 17 pp. (1961). “Evaluation of factors in the dution of hydrocortisone from paper chromatograms.” 73. Weisz, I?., and E. Glas, Med. Exptl., 3, 264 (1960). “A simple paper MICROCHEMICAL

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