ANALYTICAL CHEMISTRY
776 Table I. Relative Fluorescence of Steroids Steroid
Fluorescence per Microgram, Galvanometer Reading 100.0b 100.0
Steroid I 17-Hydroxycorticosterone ( F X ) ~ Ia 4-Pregnene-lla 17a 21-triol-3 20-dione (Epi F K ) I1 Pregnane-llg i7u,' 21-triol-3, 20-dione diacetate (dihydro F& 5.0 I11 Allopregnane-3~,llg, 17a, 2l-tetrol-3-one, 3, 21 diacetate (tetrahydro FK) 14.3 I V 17-Hydroxy-11-desoxycorticosterone 12.5 V 17-Hydroxy-1I-dehydrocorticosterone (cortisone) 7.0 VI Corticosterone (BK) 87.0 VIa 4-Pregnene-lla 21-diol-3 20-dione (Epi BK) 88.0 V I 1 Allopregnane-3& llg, 2i-triol-20-one, 21 acetate (tetrahydro BK) 14.6 VI11 11-Dehydrocorticosterone 0.8 I X 11-Desoxycorticosterone 4.5 X Allopregnane-21-01-3 20-dione acetate 0.5 X I .4llopregnane-3~ 21-hiol-20-ode diacetate 7.5 X I 1 4-Pregnene-118, '17a, 208, 2l-ietrol-3-one (tetrol E R )0 8O.Od XI11 4-Pregnene-l7a, 208, 2l-triol-3-one, 20, 21 diacetate (pregnenetriol) 3.2 4.1 XIV 1I-Hydroxyprogesterone XV 4-Pregnene-3 20-dione (progesterone) 0.8 XVI 4-Pregnene-lja-01, 3, 20-dione (17-hydroxyprogesterone) 0.6 XVII Allopregnane-3a, 17a, 21-triol-20-one (pregnanediol) 4.5 15.0 X V I I I 4-Pregnene-12 6,21-diol-3,20-dione, 21 acetate 32.0 X I X Pregnane-12 6,21-diol-3,20-dione, 21 acetate acetate 2.0 X X 4-Pregnene-21-01-3,2O-trione, X X I Testosterone 0.5 1.6 X X I I 4-Androstenedione-3, 17 X X I I I Estrone 31.0 106.0 XXIV Estradiol Subscript K refers to Kendall's designation. b Galvanometer set a t 100 with 1.0 y of 17-hydroxycorticosterone. Subscript R refers to Reichstein's designation. d Estimated value due to tendency of substance to form hydrated crystals. SO.
extract, purify, and resolve the cortical steroids. The cortical steroids may be readily separated from fats, cholesterol, and estrogens by partitioning techniques. They may be separated from each other by paper chromatography (2,IO) or by a silicic acid microcolumn (7'). The steroids listed in Table I include only a small fraction of known cortical steroids. Cortical steroids, other than those listed in Table I, may be found to fluoresce with sulfuric acid, The fluorescent technique in conjunction with a silica gel microcolumn ( 7 ) has been applied to the quantitative analysis of 17-hydroxycorticosterone and corticosterone in biological fluids and tissues. h e a t ( 6 ) has employed the technique t o determine the rate of enzymatic synthesis of 17-hydroxycorticosterone from 17-hydroxy-1 I-desoxycorticosterone in adrenal mitochondrial preparations. The technique has been utilized by
Sweat and Farrell (8) to determine the concentration of 17hydroxycorticosterone and corticosterone in the adrenal venous blood of the dog and rat. The fluorescent method is accurate and reproducible. I t is more sensitive than the phenylhydrazine technique of Porter and Silber ( 4 ) . The fluorescent procedure is relatively simple and appears to be less susceptible to background interference than is the phenylhydrazine reaction. Quantities of steroid between 0.05 and 5.0 y produce a rectilinear relation between amount and galvanometer reading. This is a suitable range for analytical work. A range between 0.25 and 1.25 y has been found to be most advantageous for analysis of biological materials. For quantities of steroid greater than 5.0 y i t must be remembered that self-absorption is great enough to produce significant deviation from the rectilinear relation between quantity of steroid and galvanometer reading. .4 typical standard curve for the quantitative analysis of 17hydroxycorticosterone is shown in Figure 5. ACKNOWLEDGMENT
The author wishes to acknowledge the valuable technical assistance of Ella Sandberg and the helpful advice and friendly criticisms of George Sayers and Gordon Farrell throughout the course of this investigation. The crystalline steroids used in these studies were kindly supplied by Leonard R. Axelrod, Karl Folkers, Thomas F. Gallagher, William J. Haines, Dwight Ingle, Tadeus Reichstein, and George Rosenkranz. This investigation was supported by research grant A-331 from National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U.S. Public Health Service. LITERATURE CITED
(1) Euw, J. von, and Reichstein, Tadeus, Helv. Chin. Acta, 25, 988 (1942). (2) Farrell, G. L., Federation Proc., 12,41 (1953). (3) National Technical Laboratories, South Pasadena, Calif.,
Bull. 89-B. (4) Porter, C. C., and Silber, R. H., J . Biol. Chem., 185, 201 (1950). (5) Reichstein, Tadeus, and Shoppee, C . W., Vitamins and Hotmones, 1,345 (1943). (6) Sweat, 11.L., J . Am. Chem. SOC.,7 3 , 4056 (1951).
(7) Sweat, X I . L., unpublished data.
M. L., and Farrell, G. L., J . Clin. Endocrinol. and Metabolism, 12,968 (1952). (9) Wintersteiner, O., and Pfiffner, J. J., J . Bid. Chem., 116, 291 (8) Sweat,
(1936). (10) Zaffaroni, A., Burton, R. B., and Kentmann, 111,6 (1950).
E. H., Science,
RECEIVED for review September 21, 1953. Accepted January 4, 1954.
Reactions of Certain Unsaturated Steroids with Acid Iron Reagent BENNIE ZAK, Depdrtment o f Pathology, Detroit Receiving Hospital, Detroit, Mich. NORMAN MOSS', A. J. BOYLE, and ALBERT ZLATKISZ, Department o f Chemistry, W a y n e
T
HE present-day prominence of sterols containing different kinds of double bond configurations has increased the importance of both the qualitative and quantitative analysis of these compounds. The chemistry of the As unsaturated sterols have been thoroughly investigated ( 2 , 3 ) and up to this time there have been a few main reactions commonly used to develop colors from which the quantity of the sterols may be determined (1, 5, 6, IO). Many other reactions have appeared which give qualitative evidence, usually involving the formation of a colored ring a t the interface of two liquids of different densities (4,7-9,11). The purpose of the present investigation is to show the color 1 Present address, Medical School, University of Michigan, Ann Srbor, RIich. Present address, Research Department, Shell Oil Co., Houston, Tex.
University, Detroit,
Mich.
reaction of iron with steroids in a glacial acetic acid-sulfuric acid medium (12). The main emphasis is placed on those sterols which have A5 unsaturation with the idea of determining specificity for these particular compounds. A large number of different steroids were used in the study. REAGENTS
Ferric Chloride Solution. Weigh out 1 gram of ferric chloride hexahydrate and dissolve it in 10 ml. of glacial acetic acid. Color Reagent. Pipet 1.0 ml. of the ferric chloride solution into a 100-ml. volumetric flask and dilute to the mark with concentrated sulfuric acid with mixing. Glacial Acetic Acid. Steroid Solutions. Weigh out 25 mg. of the steroid to be tested. Transfer to a 100-ml. volumetric flask and dissolve and dilute to the mark with glacial acetic acid.
777
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 Table I. Colors of Steroids Tested with Iron Reagent Compound Tested Cholesterol Cholestanol Dehydroisoandrosterone Isoandrosterone 11ethyl-As-36-hydroxyetiocholenate As-Pregnenolone A b 18-Preenenolone acetate Testosterone A6-Androstene-l7,8-acetory-3-ethylenecyclic hemithioketal Cholesterol acetate Cholesterol oleate Cortisone Kryptogenin acetate 7-Dehydrodiosgenin acetate Stigmasterol Sitosterol A4~~-22-Isospirostadiene-3-one Cholesteryl is0 methyl ether Estradiol Ergosterol acetate Pregnane-3B-01-20-one 17-Methyl-androstan-3B-17,9-diol 17-Methyl-A~-androsten-3~-17B-diol
Color Formed Purple Yellow Red Yellow Green Reddish-purple Brownish-oranee Pale yellow
-
Blue-violet Purple Purple Pale yellow Pink Purple Purple Blue-violet Yellow Red-violet Pale yellow Brownish Yellow Yellow Purple
PROCEDURE
Pipet 1.0ml. of steroid solution into a test tube. Add 2.0 ml. of glacial acetic acid. Pipet in 2.0 ml. of the color reagent by carefully allowing it to flow down the side of the test tube, thus producing two layers. Record the colors of the several rings ormed for aid in qualitative analysis and then strike the bottom of the tube sharply while holding it a t the top between the thumb and forefinger to effect mixing. The color of the solution formed may be used as a qualitative test or the measurement of the absorbance a t the proper wave length ma be used as a quantitative test for the A6 sterols such as c%olesterol, pregnenolone, stigmasterol, sitosterol, and others. The colors are stable and show no change over a period of several hours. DISCUSSION AND RESULTS
The compounds tested were several saturated and unsaturated steroids where the latter included A’, A‘J7, A6, A6,’, and unsaturated steroids. The colors all appear almost immediately after mixing, and Table I shows the colors obtained with the
available compounds which xere reacted with the iron reagent. The A d and the A4r7 steroids give a brownish-yellow or purple ring, while only a thin yellow ring appears a t the interface of the two liquids with the saturated compounds. The AsJ the A%’, and the steroids give a multicolored ring (usually 3 to 4 colors) which may include any combination of purple, green, yellow, red, pink, orange, violet, and brown. Most A6 compounds such as cholesterol (12) and A6 pregnenolone produced linear curves up to 100 p.p.m. obeying Beer’s law over the entire range. The authors used a Coleman Jr. spectrophotonieter for measurements. This preliminary work was carried out with a large variety of pure compounds but the work in progress ie directed toward more specific determinations. ACKNOWLEDGMENT
The authors wish to express their appreciation to the Shering Corp., Bloomfield, S. J., and to Carl Djerassi, Department of Chemistry, Wayne University, Detroit, Mich., for their liberal donations of the steroids used in this investigation. LITERATURE CITED
(1) Burchard, H., Chenz. Zentr., 61 (l),25 (1890). (2) Fieser, L. F.,“Chemistry of Natural Products Related to Phenanthrene,” pp. 11 1-86, New York, Reinhold Publishing Corp., 1936. (3) Gilman, Henry, “Organic Chemistry, An Advanced Treatise,” 2nd ed., pp. 1341-532,New York, John Wiley & Sons, 1948. (4) Lange, W., Folzenlogen, R. G., and Kolp, D. G . , J . Am, Chem. SOC.,71, 1733 (1949). (5) Liebermann, C., Ber., 18, 1803 (1885). (6) Salkowski, Hoppe-Seyler’s 2.physiol. Chem., 57, 523 (1908). (7) Schaltegger, H., H e h . Chim.Acta, 29,285(1946). ( 8 ) Scherer, Ibid,, 22, 1329 (1939). (9) Teuber, H., ANAL.CHEM.,24, 1494 (1952). (10) Titchugaev, L., and Gastev, A., Ber., 42,4631 (1910). (11) Woker, G., and Antener, I., Helv. Chim. Acta, 22, 1309 (1939); 22, 511 (1939). (12) Zlatkis, A,, Zak, B., and Boyle, A. J., J. Lab. Clin. Med., 41,486 (1953). RECEIVED for review September 8, 1953. -4ccepted January 6 , 1954. Presented before the Division of Biological Chemistry at the 123rd Meeting of the AMERICAX C H E \ I I C ~SOCIETY, L Los Angeles, Calif., March 1953
Kjeldahl Method as Applied to Determination of Nitrogen in Nitrates WILLIAM E. DICKINSON F. S. /?oyster Guano Co., Atlanta, Ga.
T
HE fact that nitrates in excess of the salicylic acid capacity in the modified Kjeldahl method give nitrogen results that are only slightly low has resulted in acceptance of unscientific practices. I n this study are recorded some developments indicating the need for corrections in current Kjeldahl methods prior to 1950, and in the Gunning method current today, as applied to materials of high-nitrate content. Incidental to this work are some previously unreported findings enabling us to deal more informatively with Kjeldahl procedure. Some applications of these developments are suggested. The modified Kjeldahl method is designed to deliver the correct percentage of nitrogen i n nitrates. Most other nitrogenbearing compounds succumb to the reduction and/or digestion features of the method. The seventh (1950) edition of the Association of Official .4gricultural Chemists’ methods for the determination of nitrogen in fertilizers by the modified Kjeldahl procedure ( 2 ) increased the salicylic acid from 1 gram per 30 ml. of sulfurir acid to 2 grams.
The Gunning variation of the method was retained without change a t 1 gram per 30 ml. of sulfuric acid. Prior to this edition, both variations of the method (1) directed, for converting the nitrates to nitrosalicylic acid: “Place 0.7 t o 3.5 grams, according to the nitrogen content of material to be analyzed, in a Kjeldahl digestion flask. Add 30 ml. of sulfuric acid containing 1 gram of salicylic acid, shake thoroughly until mixed, allow to stand a t least 30 minutes with frequent shaking. . .” If these variations of the method are applied literally to the analysis of sodium nitrate, both will give nitrogen results about 0.20% low.
.
DEFICIENCIES O F KJELDAHL 4fETHOD
Since 1 gram of salicylic acid is converted to nitrosalicylic acid by 0.6160 gram of sodium nitrate, a 0,0840-gram residue of unconverted sodium nitrate w-ill be left from a 0.7-gram sample to f o l l o ~through in the method, unless some dinitrosalicylic acid is formed. If the residue does not go into dinitrosalicylic acid, the
