Y of pregnenolone were detected. This color reaction can be used for the estimation of dehvdroetiandrosterone in human urine a n d plasma, if extracts of these fluids are purified by paper chromatography (6).
Morris and Renate Oertel for their technical assistance. LITERATURE CITED
~~
ACKNOWLEDGMENT
The authors wish to thank Mavis
(1) Allen, W. M., J . Clin. Endocrinol. 10, 71 (1950). (2) Dhscherl, Wilhelm, Zilliken, Friedrich, Naturwissenschuflen 31, 349 (1943). ( 3 ) Linford, J. H., Can. J . Biochem. and Physiol. 34, 1153 (1956).
(4) Munson, P. L., Jones, 31.E., McCall, P. J., Gallagher, T. F., J . Biol. Chem 176, 73 (1948). ( 5 ) Oertel, G. W., Eik-Kea, K. B., Ibid., in press, 1958. 16) . , Zaffaroni. Aleiandro. J . Am. Chem. SOC. 72, 3828 (1950).
RECEIVED for review February 20, 1958. Accepted August 4, 1968. Work supported by United States Public Health Grant CRTY-5000 Xational Institutes of Health, Bethesda 14, Md,
Determination of Novobiocin in the Presence of Isonovobiocin ARLINGTON A. FORIST, SUSAN THEAL, and WILLIAM A. STRUCK Department o f Physical and Analytical Chemistry,, The Upjohn Co., Kalamazoo,
b Novobiocin frequently contains varying amounts of isonovobiocin, which is devoid of antibiotic activity. The two isomers are indistinguishable by physical measurements. A chemical procedure for the determination of novobiocin in the presence of isonovobiocin is based on cleavage of the novobiocins with anhydrous trifluoroacetic acid to yield the respective 0carbamylnovioses, followed by determination of periodate consumption b y 3 -0-carba mylnoviose from novobiocin. 2-0-Carbamylnoviose from isonovobiocin does not interfere. Analysis of standard novobiocin samples indicates excellent accuracy and precision (mean recovery fstandard deviation 99.9 f 1.5%). Mean deviation for novobiocin in samples containing 0 to 20y0 isonovobiocin is f1.5%.
T
HE ANTIBIOTICnovobiocin
has been assigned structure I (3,6-8, 10-12). Hinman, Caron, and Hoeksema (4) have reported the isomerization of I to give isonovobiocin (11) by a carbamyl migration. Because I1 is inactive as an antibiotic, an analytical procedure is needed which will permit the determination of I in the presence of I1 without disturbing the ratio of the two forms,
100
ANALYTICAL CHEMISTRY
Mich.
REAGENTS
grams of potassium iodide is dissolved in 75 ml. of water and diluted to 1 liter. This solution is stored in a tightly stoppered bottle protected from light. Iodine, Working Solution, 0.01N. This is prepared by a tenfold dilution of the stock solution and is stored in a tightly stoppered bottle protected from light. This solution is standardized daily by the titration of 3.00-ml. aliquots of the 0.1000N sodium arsenite reagent, saturated with sodium bicarbonate, to a starch end point. Potassium Iodide, 5%. Five grams of potassium iodide are dissolved in 100 ml. of water and this solution is saturated with sodium bicarbonate. The reagent prepared in this manner is stable for several weeks.
Trifluoroacetic Acid, anhydrous. Sodium Arsenite, 0.1000N. A sample of 9.8910 grams of primary standard arsenious oxide is added to a 2-liter volumetric flask containing 20 grams of sodium hydroxide in 50 ml. of water. The solution is diluted with about 300 ml. of water and 42 ml. of concentrated hydrochloric acid are added, followed by 20 grams of sodium bicarbonate. The resulting solution is diluted to 2 liters. Periodic Acid, 0.2N. Twenty-three grams of o-periodic acid are dissolved and diluted t o 1 liter with water. This solution is stored in an amber bottle and standardized by the following modification of the method of Fleury and Lange ( 2 ) . Duplicate 4.00-ml. aliquots are saturated with sodium bicarbonate. A 10.00-ml. aliquot of the 0.1000N sodium arsenite solution is added to each, followed by 2 ml. of 5% potassium iodide. After a t least 15 minutes, the excess arsenite is titrated to a starch end point with the standard 0.0lhr iodine solution. Additional sodium bicarbonate may be required during the titrations to keep the solutions saturated (solid phase present). Weekly restandardization is recommended. Iodine, Stock Solution, 0.1N. A mixture of 12.7 grams of iodine and 60
d sample of 40 to 50 mg. of the material to be analyzed is accurately weighed into a 125-ml. glass-stoppered Erlenmeyer flask or alternatively, into a 50-ml. glass-stoppered centrifuge tube. Five milliliters of anhydrous trifluoroacetic acid are added to the sample, the vessel is tightly stoppered, and the resulting solution is allowed to stand a t room temperature in the dark. At the end of 2 hours, 20.00 ml. of water are slowly added with swirling. The precipitate which forms is separated a short centrifugation, followed by ration of the supernatant through a medium porosity glass-fritted funnel under vacuum. The vacuum is released as soon as the filtration is complete t o avoid loss of solvent by evaporation. The resulting filtrate contains the 0carbamylnovioses. Accurately weighed samples of approximately 20, 40, and 60 mg. of a standard novobiocin preparation are carried through the procedure above in parallel with the unknown. Periodic Acid Oxidation. A 20.00ml. aliquot of each aqueous trifluoroacetic acid filtrate is transferred to a
The two isomers are indistinguishable by ultraviolet or rotational measurements and shorn only slight differences in their infrared spectra. Published methods for the analysis of mixtures of I and dihydronovobiocin (9) and for the determination of 1 in plasma and serum (1) do not differentiate I and 11. The procedure reported here permits the direct determination of I in the presence of 11. It is based on the periodate oxidation of 3-0-carbamylnoviose produced from I by acid cleavage of the glycosidic bond.
PROCEDURE
Glycoside Cleavage.
2
separate 125-ml. Erlenmeyer flask, followed by 5.00 ml. of standard 0.2N periodic acid. A solvent blank (20 ml. of a mixture of 5 ml. of trifluoroacetic acid and 20 ml. of water) is treated similarly. Oxidation is allowed t o proceed at room temperature in the dark for 1 hour. Each solution is then partially neutralized with 2.5 ml. of 10N sodium hydroxide. Neutralization is completed by the slow addition of sodium bicarbonate until saturated solutions result. A 10.00-ml. aliquot of 0.1000N sodium arsenite is then added to each solution, followed by 2 ml. of 5% potassium iodide (volume of the latter need not be exactly controlled). After a t least 15 minutes, the excess arsenite is titrated to a starch end : point with the standard 0.01N iodine. The 15-minute reaction period is necessary to ensure complete reduction of periodate to iodate. Titrations may be carried out a t any convenient time after this, because the resulting solutions show no instability within 24 hours.
Calculations. Periodate consumption ( P , meq.) is calculated from the equation P = (5.00 X NIO,-)(1.0000) (VI, x N I , ) (2) where NIO,- = normality of the periodic acid = volume of iodine required VI, = normality of the iodine N,,
+
I n each case the apparent periodate consumption is corrected for the small amount of periodate consumed by the solvent blank. This blank is usually very small but varies for different batches of trifluoroacetic acid. Novobiocin carried through this procedure gives about 85% of the theoretical periodate consumption of 2 equivalents per mole. However, periodate consumption is a linear function of sample size and a working calibration curve is prepared daily and employed in the analysis of unknoF3rns. The per cent novobiocin (yo K) is calculated from the equation
where P = periodate consumed by the aliquot oxidized, corrected for the blank b = intercept of the calibration curve, meq. m = slope of the calibration curve, meq. per mg. in aliquot oxidized w = sample weight, mg.
A mixture of 20.00 ml. of water and 5.00 ml. of trifluoroacetic acid gives a final volume of 24.09 ml. The factor 0.83 corrects for the fraction of the total aqueous triffuoroacetic acid filtrate employed for periodate oxidation.
Figure 1. Production of 3-O-carbam y In ovi os e from novobiocin as function of reaction time with anhydrous trifluoroacetic acid at 25’ C.
kf 60 a 0
RESULTS AND DISCUSSION
This procedure is based on the cleavage of the glycosidic bonds of I and I1 with anhydrous trifluoroacetic acid to yield the respective O-carbamylnovioses (I11 and IV) and/or the corresponding trifluoroacetic acid esters and cyclonovobiocic acid (V), the precipitation and separation of V by the addition of water followed by filtration (any trifluoroacetic acid esters present will be hydrolyzed a t this point because such esters are highly unstable in aqueous solution), and the determination of periodate consumption by the mixture of I11 and IV in the aqueous trifluoroacetic acid filtrate. 3-0-Carbamylnoviose (111) from I contains a (1) t (11 ( a ) Anhydrous TFA ( b l H-0
1
50
IO0 150 200 REACTION TIME (MINUTES)
of reaction time is shown in Figure 1. Maximum reaction is reached within 2 hours a t room temperature and remains unchanged for an additional 2 hours. Reaction a t elevated temperatures, although more rapid, is less reproducible and the yield of I11 is not increased. Accordingly, a 2-hour reaction a t room temperature has been adopted. Periodate consumption by I11 in the aqueous trifluoroacetic acid filtrate as a function of oxidation time is shown in Table I. Oxidation is complete within 1 hour and no overoxidation occurs during an additional hour. Periodate consumption by I is a Effect of Oxidation Time on Table 1. Periodate Consumption b y 3-0-Carbamylnoviose
Time, Min. 30 60 90 120 Table II.
pair of vicinal hydroxyl groups which consume 2 equivalents of periodate per mole, Thereas 2-0-carbamylnoviose (IV) from I1 is not oxidizable by periodate. Success of a method based on this difference requires reaction conditions under which neither carbamyl migration nor decarbamylation occurs. Among the acidic systems tested, only trifluoroacetic acid possessed the desirable combination of excellent solvent properties and the ability to cleave I in good yield without the production of stable esters of 111. Production of I11 from I as a function
250
Maximal IO4Consumption,
%
97.4 99.0 100.4 99.5
Determination of Standard Novobiocin Samples
Taken, Mg. 20.33 31 88 41 29 51 60 71 03 81 66 20.76 30.74 42.67 48.03
61.09 67.61 84.11 22,05 33.18 41.21 53.92 63.66 75.41 Mean recovery
Found, Mg. 20 4.5 32 08 41 88 50 81 71 34 79 73 20.08 30.75 43.71 48.11 61.41 67,52 81.70 21.92 33.23 41.46 .54,93 63,46 75.51
Recovery, % 100.6 100 6 101 4 98 5 100 4 97 6 96.7 100.0
102.4 100.2 100.5 89.9 97.1 99.4 100.2 100.6 101.9 99.7 100.1 std. dev. 99.9 1 . 5
’401.31, NO. 1, JANUARY 1959
101
linear function of sample size. The slope of such a curve is about 85% of the theoretical value (observed, 0.00266 t o 0.00288; theory, 0.00327) and a small intercept is also obtained. Dayto-day variations in both the slope and intercept necessitate the inclusion of standard samples in parallel with unknowns for maximum accuracy. Periodate consumption by I1 is independent of sample size and equal to the intercept of the calibration curve for I, validating Equation 3. The intercept is apparently due to the presence of a small but constant amount (saturated solution) of V, or a degradation product in the aqueous trifluoroacetic acid filtrate. Results of the analysis of standard samples of I show a mean recovery of 99.9% with a standard deviation of & 1.5% over the range from 20 to 80 mg. (Table 11). Application of the procedure to the determination of I in mixtures of I and I1 is shown in Table 111. Over the typical range between 80 to 100% I, a mean deviation of & 1.5% is observed, confirming the validity and accuracy of the method. Analysis of an equilibrium mixture of I and I1 produced by alkaline isomerization has indicated 65% I. This agrees with the previously reported values of 65 to 70% obtained by bioassay, and 67% obtained by countercurrent distribution
(4).
cn300H y3
OR
OH OH
Taken
In)
100.0
The failure to achieve more than an 85% yield of 111apparently results from the simultaneous production of I11 and a substance not periodate oxidizable during cleavage with trifluoroacetic acid. A similar yield of I11 from ethyl 3-0carbamylnovioside indicates that the reaction does not involve V. Hinman, Caron, and Hoeksema (3) have reported that hydrolysis of methyl 3-0-carbamylnovioside with 0.5N sulfuric acid produces, in addition to 111, a substance thought to possess structure VI1 on the basis of its infrared spectrum. A similar reaction may occur during the glycosidic cleavage with trifluoroacetic acid.
ACKNOWLEDGMENT
The authors are indebted to Herman Hoeksema for pure isonovobiocin.
Any material capable of consuming periodate under the conditions employed constitutes a positive interference. Descarbamylnovobiocin (VI) consumes 2.3 times as much periodate as I on a molar basis when carried through the above procedure. Therefore, the error introduced by the presence of VI may be corrected by an independent determination of VI (IS).
111. Determination of Novobiocin in Novobiocin-lsonovobiocinMixtures Novobiocin, % Deviation,
Table
LITERATURE CITED
(1) Boxer, G. E., Shonk, C. E., Antibiotics & Chemotherapy 6, 589-97 (1956).
(2) Fleury, P. F., Lange, J., J. pharm. chim. 17, 107-13 (1933). (3) Hinman, J. W.,Caron, E. L., Hoeksema. Herman. J . Am. Chem. SOC.79. 37891-3800 (1967). (4) Hinman, J. W.,Caron, E. L., Hoeksema, Herman, Ibid., 79, 5321 (1957).
Found 99.0 93.7 95.3 92.1 94.7 92.3 87.7 91.0 89.2 88.0 83.3 83.9 80.8
95.2 95.1 94.6 93.0 90.8 89.6 88.9 86.9
86.4 85.4 83.2 81.4 79.9
I
70
-1.0 -1.5 +o 2
-2.5 +1.7 $1.5 -1.9 +2.1 +2.3 +1.6 -2.1 +0.7 -0.6 81.1 $1.2 Mean yo dev. & 1 . 5
(5) Hinman, J. W.,Hoeksema, Herman, Caron, E. L., Jackson, W. G., Zbid., 78, 1072-3 (1956). (6) Hoeksema, Herman, Caron, E. L., Hmman, J. W,,Ibid.,. 78,. 2019-20 (1956). ' (7) Hoeksema, Herman, Johnson, J. L., Hinman, J. IT., Ibid., 77, 6710-11 (1955). (8) Kaczka, E. A., Shunk, C. H., Richter, J. W., Wolf, F. J., Gasser, ?VI.M., Folkers. Karl. Ibid.. 78. 4125-7 (1956). (9) Sknsi, Piero ' Gdlo, G. G., Chiesa, Luigi, ANAL.&HEM. 29, 1611-3 (1957). (10) Shunk, C. H., Stammer, C. H., Kaczka. E. A.. Walton. Edward. Spencer, C. F., Ti'ilson, A. N., Richter; J . W..Hollv. F. W.. Folkers. Karl. J. Am: Chem: koc. 78, i770-1 (1956). ' (11) Stammer, C. H., Walton, Edward, Trenner, Wilson, A. N., Walker, R. W., N. R.. Hollv, F. W..Folkers, Karl, Ibid., 80, 137-'40 (1958). 112) Walton. Edward. Rodin. J. 0.. ' Stammer, 'c. H., Hofly, F. W:,Folkers; Karl, Zbid., 78, 5464-5 (1956). (13) Wolf, F. J., Nescot, R., Antibiotics Ann. 1956/57,'1035-9. RECEIVED for review April 25, 1958. Accepted August 20, 1968.
Differentiation of A4-3-Ketosteroidsand A"4-3-Ketosteroidswith isonicotinic Acid Hydrazide LELAND L. SMITH and THEODORE FOELL Chemical Process Improvement Department, lederle laboratories Division, American Cyanamid Co., Pearl River, N. Y. lsonicotinic acid hydrazide is useful in detecting A4-3-ketosteroids and A184-3-ketosteroids on paper chromatograms. The reagent may b e used for the differential detection of the two types of unsaturated steroids on chromatograms of mixtures resulting from microbiological fermentations and metabolic studies. The reagent does not interfere with the
702
ANALYTICAL CHEMISTRY
subsequent use of tetrazolium reagents for the detection of reducing a-ketolic steroids.
M
for the detection of Ad-3-ketosteroids on paper chromatograms with specific color tests are well established, mainly through studies of cortical steroid metabolism using chromatographic techniques. The imETHODS
portant Alp e3-ketosteroid hormones and their metabolites on paper chromatograms have generally been recognized by ultraviolet light absorption on paper, followed by direct exclusion of A4-3ketones through the failure of selective color tests. Thus, absorption of 2537 A. light, as determined with the Haines and Drake scanning device (9, 18) or photographically (8, S), together
