piwes along with the samples. This procedure assured a platinum-to-sample ratio of 3 or more until the crucible was filled. after installing a new crucible, about 20 minutes of heating a t 1950’ C. was required for the procedure blanks to drop to the levels stated above for nitrogen and oxygen. A somewhat longer period was usually required for the hydrogen blank to level off, probably because of relatively slow desorption of H20contamination on the inner walls of the train. Successful application of the inert gas fusion method is, as in the case of the vacuum fusion me1 hod, dependent upon the free energies of the dissociation of nitrides and hydrides, the carbonreduction of oxides, 1,he solution of metals in platinum, and the formation of metal carbides, as mell as upon the solubilities of gaseous inpurities in the melt a t 1950’ C., and the diffusion rates of the g a through ~ the melt to the surface. The two methods differ in the environment encountered by the gas impurities as they reaci the surface of the mell, one being thal, of low pressure and the other, gas diluion. The coni-
parative results obtained on the NBS steel samples indicate that this difference is not significant so far as these materials are concerned. I n the inert gas method, the blank levels for the three gases are not dependent upon a high-quality vacuum system, and are not so sensitive to the thoroughness by which the line components have been outgassed. The components of the analytical train may be conveniently interconnected by greased joints. NIuch of the line is operated a t a positive pressure so that leaks into the system are not a potential problem. The metal sample is never subjected to low pressure prior to analysis, thereby minimizing the chance for loss of hydrogen from a sample in which it may reside as an unstable, transient impurity. ACKNOWLEDGMENT
The authors are grateful to John Marsh for his assistance in obtaining experimental data by the procedure of this method and to Carol Bloomquist for comparative data by the Kjeldahl method.
LITERATURE CITED
(I) Brown, E. H., IND.ENG. CHEM., ANAL.ED. 14, 551 (1942). (2) Coe, F. R., Jenkins, N., Iron Steel Inst. (London), Spec. Rept. 68, 229 11960). I - - -
I
(3) Elwell, W.T., Ibid., p. 19. (4) Fassel, V. A., Dallmann, W. E., Skorrerboe, R., Horrigan, V. M., ANAL. C H G ~34,’1364 ( i ~ s i j .’ (5) Holt. B. D., Ibid.. 27. 1500 (1955). (6) Zbid.; 28, 1153 (1956): ( 7 ) Leco Nitrox-6 Nitrogen Oxygen Analyzer, Form 1102, Laboratory Equipment Corp., St. Joseph, Mich. (1962). (8) Peterson, J. I., Melnick, F. A., Steers, J. E., ANAL. CHEM.30, 1086 (1958). (9) Shanahan, C. E. A., I r o n Steel Inst. ( L o n d o n ) , Spec. Repl. 68, 75 (1960). (10) Smiley, W. G., ASAL. CHEM.27, 1098 (1955). (11) Turovtseva, Z. M., Kunin, L. L., “Analysis of Gases in Illetala,” Publishing House of the Academy of Sciences of the USSR in Moscow and Leningrad, 1959. (Translated by Consultants Bureau, Kew York). RECEIVEDfor review March 22, 1963. Accepted June 26, 1963. Division of Analytical Chemistry, 144th hleeting, ACS, Los Angeles, Calif., April 1963. Based on work performed under the auspiFes of the U. S. Atomic Energy Commls81on.
New Fluorometric Micromethod for the Simultaneous Determination of Digitoxin and Digoxin IVAN M. JAKOVLJEVIC Analytical Development !aborafory, Eli l i l y and Co., Indianapolis, Ind.
i~ A new, fluorometric microprocedure for the simultaneous determination of digitoxin and digoxin in leaves and other pharmaceutical formulations has been developed. In most work previously reported these two glycosides were determined together and to determine them separately, chromatographic procedures had t o be employed. On the basis of different fluorescence spectra, the method described here offers a means of determining them simultaneously in the same sample. Under the conditions proposed, gitoxin does not react. Not more than 90 minutes of actual work i s necessary for the assay of digitalis tincture. URISG
the past 70 years, analytical
.tudies of cardiac glycosides have
qhon 11 that sspccific reactions for each g1~. c o d e cannot be ex1 ected from the
augar or butenolide moicity. Specificity can be expected from a reaction which tnkes place somenhere in the steroid inwty, Iiic-h I I I : ~ ~contain sever:tl hydroxyl groups arranged differently
for each glycoside (Figure 1). Position 14 is common to all the gly-cosides. The sugar at position 3 may be any one of several; Digitoxose, cymarose, sarmantose, glucose, rhamnose, galactose, xylose, etc., have been identified. The methods for the determination of cardiac glycosides can be divided into three general groups: based on (A) the sugar moiety, (B) the butenolide moiety, and (C) the steroid moiety. Group A. As far back as 1885. Lafon (17‘) published a colorimetric method using equal amounts of sulfuric acid and ethanol with the addition of ferric chloride. Kiliani (14) used n solution of ferric sulfate in concentrated sulfuric acid: and Keller (12) applied ferric chloride to a solution of the glycoside in acetic acid, and then underlaid the Kiliani reagent. Pesez (19) used xanthydrol as a reagent for digitoxose and cymarose. Group B. The colorimetric method of Baljet ( l ) ,based on the reaction in the butenolide side chain, is the most frequently used. The reagent contains picric acid in alkaline ethanol. There
are many modifications and applications of the Baljet reaction (2, 3, 5, 13, 16, 21). Kedde (11) studied the use of 3,5dinitrobenzoic acid and Legal’s reagent (sodium nitroprusside) in a buffered (pH 11) solution. Morel (18) was first to apply m-dinitrobenzene to digitalis and other glycosides. Kimura (15) applied a methanolic solution of 1,3,5-trinitrobenzene in alkaline medium as a reagent for quantitative determination of some cardioactive glycosides. Tattje (65) used 2,4dinitrodiphenylsulfone in ethanol and alkali. Warren (26) used sodium 2naphthoquinone-4-sulfonate for the colorimetric assay of digitoxin. Group C. Methods based upon the reaction in the steroid moiety are for the most part fluorometric, such as that of Petit et a2. ($0) which uses sirupy phosphoric acid. Jenseri ( 6 ) used equal parts of hydrochloric acid and glycerol as a dehydration agent. Fruytier and van Pinxteren ( 6 ) improved .Tensen’s reagent by the addition of ethanol. Jensen (0) developed VOL. 35, NO. 10, SEPTEMBER 1963
0
1513
SUGAR
STEROID 7 -
BUTENOLIDE I
SUk.4R
I
Figure 1. Molecular structure cardiac glycosides Name Digitoxin Digoxin Gitoxin Strophanthin Ouabain
of some
OH position 14 14,12 14,16 14, 5
14, 1 , 5, 1 1 , 1 9
another fluorometric method for digitoxigenin using hydrogen peroxide, hydrochloric acid, and methanol. Wells, Katzung, and Meyers (27) modified Jensen's method and were able to determine digitoxin with a fluorescence maximum a t 570 mp, but digoxin and gitoxin exhibited very close maxima a t 470 and 490 mp. Sasakawa (23) combined paper partition chromatography and Jensen's reagent. Kaiser (10) and Rigby (2%')also used chromatographic procedures. Tattje (2$) made a reagent by adding sulfuric acid and ferric chloride to phosphoric acid. Houk ( 7 ) published a fluorometric method for gitatoxin (16-formylgitoxin), using equal amount of propylene glycol and hydrochloric acid. Windaus and Schwarte (28),Cloetta (4),and others explained these reactions as dehydration processes in which unsaturated mono- or dianhydro compounds are formed. The reagent proposed in this paper is a mixture of acetic anhydride, acetyl chloride, and trifluoroacetic acid. Studies of ultraviolet, nuclear magnetic resonance, infrared, and fluorescence activation spectral data support the theory that when the reagent reacts with digitoxin, a low yield of a highly conjugated fluorophor of substituted 3,4-benzpyrene type is obtained, while the activation spectrum of the fluorophor from digoxin suggests a mixture of chrysene and the 3,4-benzpyrene type. Further studies of this fluorophor will be reported in a forthcoming publication.
(maximum transmittance a t 436 mp) was employed as a primary filter and filter 2A-12, which transmits light a t 510 mp and above, was employed as and lansecondarv filter for digitoxin atoside A. For digoxin and lanatoside C assavs. filter 7-600 (transmitting light betwekn 350 and 400 mp) was used as the primary filter, and filter 2A (transmitting at 415 mp and above), in combination with No. 2 neutral density filter to reduce to 1% the emission energy, as the secondary filter. All filters are available through G. K. Turner Associates. Reagents. U. S. Pharmacopeia reference standards of digitoxin and digoxin. Acetic anhydride, analytical reagent, Mallinckrodt Chemical Works. - Acetyl chloride, analytical reagent, Code 1006, Baker and Adamson. Trifluoroacetic acid, Code 6287, Eastman Organic Chemicals. The same results were achieved with trifluoroacetic acid, Code 7454, Matheson Coleman and Bell, supplied in ampoules, but not with trifluoroacetic acid, Code K 7454, from the same manufacturer in 1-pound bottles. Dichloromethane, Code 342, Eastman Organic Chemicals. Solution A. Equal parts, usually 10.0 ml. of each, of acetic anhydride and acetyl chloride are mixed in a glassstoppered bottle and kept in a refrigerator. Such a mixture was active for 3 weeks. Solution B (Reagent). Prior to use, 4 ml. of solution A are pipetted into a IO-ml. volumetric flask, and the volume is made up to 10 ml. with trifluoroacetic acid. Two milliliters of Solution B were used in all experiments described below. Procedure. STANDARD CURVE. Using a microbalance, 5 mg. of USP digitoxin standard were weighed, transferred t o a 100-ml. volumetric flask, dissolved, and diluted to vol40 -
2 -
30-
z
3
>
3
20-
l\ I'I 1I
/ \
I
k
m
LT
Q '
10 I
\
\ \.
EXPERIMENTAL
Apparatus. An Aminco-Bowman spectrofluorometer (American Instrument Co., Inc., Silver Spring, Md.)) equipped with 1-cm. square silica cells, was used to determine all excitation and emission spectra. A Turner Model 110 fluorometer (G. K. Turner bssociates), equipped with a set of matched borosilicate glass test tubes, was employed for all fluorometric measurements. Filter 47B 15 14
ANALYTICAL CHEMISTRY
400 500 600 WAVELENGTH I N mp
Figure 2. Excitation and fluorescence spectra of digitoxin fluorophor A.
AI.
Excitation spectrum of fluorophor, peak at 470 mp Fluorescence spectrum of fluorophor, peak a t 500 mp
WAVELENGTH I N m y
Figure 3. Excitation and fluorescence spectra of digoxin fluorophor 6, C. Excitation spectra of fluorophor, peaks 61, C1.
at 3 4 5 and 470 mp, based on fluorescence measured at 500 m p Fluorescence spectra of fluorophor, peaks a t 4 3 5 and 500 mp, based on excitations a t 3 4 5 and 470 mp, respectively
ume with chloroform. Aliquots of 1.0, 2.0, 4.0, and 6.0 ml., corresponding to 50, 100, 200, and .300 t g . of digitoxin, were pipetted into 90-ml. volumetric flasks and evaporated under a stream of air. The air mas circulated by means of glass tubes inserted deep into the necks of the flash to facilitate the evaporation. To the dry residue in each flask, 2 ml. of the reagent were pipetted, and the flasks mere placed in a water bath a t 45" & 1' C. After 1 minute in the bath, the flasks were firmly stoppered. They were heated for 30 =t 1 minutes in the bath and cooled to room temperature, and the solution was diluted to 50 ml. with dichloromethane. ,4 reagent blank was carried through the procedure simultaneously. After heating and cooling, the samples were allowed to stand 25 minutes a t room temperature before reading against the reagent blank. As seen from Figures 2 and 3, the fluorescence peaks for both digitoxin and digoxin occur a t 500 mp, with the digoxin fluorescence being approximately 30% of the digitoxin fluorescence. The same relationship holds for the Turner instrument. Thus, when the fluorescence is read using the 4 i B 2.4-12 filter combination, the reading gives the sum of the fluorescence of digitoxin and digoxin. To correct this reading for digoxin, the fluorescence 2X - 2XD is read nith the 7-60 filter combination and this reading, which is due t o digoxin only, because of its excitation a t 345 mp, must be subtracted from the combined reading. This is possible, since the readings of digoxin are practically the same under both filter combinations. The standard curves of both glyco-
+
+
sides are linear in concentrations from 0.5 to 6 pg. per ml. and pass through the origin.
DETERMINATION OF DIGITOXINS c B s T A m E . Five milligrams of the digitoxin sample were weighed, transferred into a 50-ml. volumetric flask, dissolved, and diluted t o volume with chloroform. A 1-ml. aliquot, corresponding to 100 pg. of digitoxin, was run simultaneously with 2 ml. of digitoxin standard as directed for the standard curve, beginning with “and ev;iporated under the stream of air.” Applications.
DETERhIIiiATIOPi C F DIGITOXIN-DIGOXIN RATIOIN LEAYES. One hundred
milligrams of well pulverized digitalis leares, containing 11. USP unit, as measured by bioassay were weighed into a IO-nil. glass-stoppered Erlenmeyer flask. Five rnilliliters of 80% ethanol mere added, and the sample was placed in a water bath a t 70” C. for 10 minutes with constant swirling. After that time, the flask mas tightly stoppered and shaken for 1 hour in a n automatic shaker a t room temperature. It was established that a n overnight extraction, recommended by many pharinacopeias, gives t’lies5,me results. Using 20 ml. of water, the !:ample was transferred into a 60-nil. separatory funnel and extracted five %nee with P m l . aliquots of chloroforin. Each extract, was passed through ,x small funnel, 3 cm. in diameter, fitted with Whatinan Xo. 1 filter paper ccntaining about 3 grams of anhydrous sodium sulfate. The filtrate wa? colltxted in a 25-mI. volumetric flask. ‘Ihe volume was niade up to the mark with chloroform, which was allowed to pass through the same filter. X 10-ml. aliquot was transferred into a E’O-ml. volumetric flask, and evaporated to dryness under a stream of air. To :;he dry residue, 2 nil. of the reagent m.e:.e added, and the procedure was conti.iued as for the standard curve, begiming with “and the flasks were placed. . .” The final solution, diluted with dichloromethane to 50 ml., is read against both filter combinations, and the digitoxin-digoxin ratio calculated as for the digitalis tincture. Simultaneously a standard was run through the same procedure. Six niilliliters of the digitoxin :.tandard solution, corresponding to 300 fig., were transferred into a 10-ml glass-stoppered Erlenmeyer flask and evaporated to dryness. -1fter addition of 5 ml. of 80% ethanol, the prclcedure was continued as for the sample. DETERMIXATION OB’ DIGITOXIN-DIGOXIN RATIOi s DIGITALIS TINCTURE. One millilter of the tincture, corresponding t o I USP unit by bioassay, was pipetted into a 60-ml. :jeparatory funnel containing a mixture of 20 ml. of water and 4 ml. of 80% ethanol. The mixture (vas extracted five times with 4 ml. of chloroform each time. The assay was continued as for the determination of digitoxin-digoxin ratio in leaves, beginning with: “Each extract mas passed through. . .,” and using the same amount of standard solution.
CALCULATIONS.For the digitalis tincture, labeled as 1 USP unit per ml., a reading of 35.1 was obtained using the first filter combination (47B 2A-12), which measures both digitoxin and digoxin. Using the other filter combination (7-60 2il - 2ND), which nieasures only digoxin-like material, a reading of 4.7 was found for the sample. If the reading of 4.7, corresponding to digoxin-like substances, is subtracted from the 35.1 value (readings for dignuin are practically the same Kith both filter combinations), a calculation which gives only the content of digitoxin like material is possible:
+
+
35 1 - 4 7 2.4 23 __ 27 X -X - X 50 =
x-
1000 10 0 328 mg. of digitoxin-like material per ml. of tincture
Digoxin content can be calculated from the reading obtained with the 7-60 2-1 - 2 5 D filter combination, and the digitouin reference solution, by virtue of the fact that the digoxin readings are practically the same with both filter combinations and are 30% of the digitoxin fluorescence. The following equation !vas used for thi. calculation.
+
2 4 25 -- X 50 = 1000 10 0.169 mg. of digoxin-like mat,erial per ml. of tincture 4 7
2i 8,j X __ X
The value 2.4 in both equations represents micrograms of digitoxin in the final standard solution. The digoxin content can be estimated more precisely and accurately by remoring the interference filter and replacing it by a more transparent filter or by changing to a larger aperture on the fluorometer and comparing this reading to a digoxin standard processed simultaneously with the sample. DETERMINATION O F DIGITOXIX IN TABLETS.One tablet contaiiiing approximately 0.1 mg. of digit,oxin was placed in a IO-ml. Erlenmeyer flask and pulverized with a glass rod, 5 ml. of 80% ethanol were added, and the solution was heated a t 70” C. for 10 minutes with const’ant swirling. The mixture was transferred by means of 20 ml. of mater into a 60-ml. separatorp funnel, and extracted with five 4-ml. portions of chloroform. The procedure was continued as for determination of djgit?xin-digoxin ratio in leaves, beginning with: “Each extract waq passed through. . . I J Instead of being collected in a 25-ml. volumetric flask as outlined in the procedure for leaves, the filt’rate was collected in a 50-ml. volumetric flask. Then the entire amount of filtrate was evaporated in the same flask and the reagent was added to the dry residue. After this, the remainder of the leave,? procedure was followed.
DETERMINATION OF DIGITOXIN IN AMPOULES. One milliliter of the ampoule content, corresponding to 0.1 mg. of digitoxin, was pipetted into a 60-ml. separatory funnel containing 20 ml. of water. The usual procedure for chloroform extraction was followed. DISCUSSION
A solution of digitoxin treated \vith the reagent proposed here exhibits a yellow fluorescence under a laboratory ultraviolet lamp. Cnder the same conditions digoxin gives a light blue fluorescence. Gitoxin does not fluoresce. Because of the sensitivity of the eye to the blue fluorescence, even the smallest amounts of digoxin can be detected if present in digitoxin. In daylight, and in very low concentrations, the digitoxin fluorophor exhibits a reddish color which remains even after dilution with dichloromethane. At higher concentrations, the color of the fluorophor is more violet. SPECTR-4 OF FLUOROPHORS. Gitoxin, under the conditions givrn above, does not fluoresce. Figure 2 illustrates the excitation and fluorescence spectra of the digitoxin fluorophor. -1s seen from Figure 3, digoxin has two excitation peaks: one at the same 17-avelength as digitoxin-i.e., 470 mpplus a second at 345 mu. The fluore+ cence, corresponding to t,he excitation peak a t 470 mp, is very cl,:se to the digitoxin fluorescence at 500 inp, but represents only 30% of digitoxin fluorescence. At the excitation peak a t 345 mp, there is a new fluorescence spectrum with a peak at 435 mp where digitoxin does not exhibit any fluorescence. REBGENT. The misture of equal parts of acetic anhydride and acetyl chloride, as well as trifluoroacetic acid, n-as kept in the refrigerator, but it was unneceisary to bring them to room temperature before combining 4 ml. of the mixture with R ml. of trifluoroacetic acid. The reaction is highly inhibited and behares differently if solvents like chloroform, ethanol, acetone, and even dichloromethane are present during the fluorophor formation. Therefore t,he reagent must be added to the dry residue only. ‘The reagent blank bcing constant, and very low, can be replaced in most routine analyses by dichloroniethane as a blank. The ratio of 4 nil. of the mixture and 6 inl. of trifluoroacetic acid is critical for the reaction, and to avoid tiiscrepancies in the results, a standard digitoxin determination should be run together with the sample. Trifluoroacetic anhydride used instead of trifluoroacetic acid did not produce enough fluorescence. The quantity of the reagent itself V‘OL.35, NO. 10, SEPTEMBER 1963
1515
is not very critical, but 2 ml. were selected as optimum. PRECISION AND ACCURACY. At a level of 2 pug. per ml. based on nine independent determinations, the accuracy was 99.2% of theory with a standard deviation of 0.19 HEATING Trim. Thirty [minutes was determined to be the optimum heating period. Time differences of i l minute in the total heating period of 30 minutes caused no changes in readings. SIGNIFICANCE O F h O U N T OF FvATER.
The fluorescence depends very much upon maintaining anhydrous conditions, as shown in Table I. STABILITYOF FLUOROPHOR. Immediately after the dilution with dichloromethane, the fluorophor has maximum intensity, which slowly fades for the next 15 minutes, remains constant for about 10 minutes, and then again slowly decreases, as shown by Table 11. The recommended time for reading is 25 minutes after dilution with dichloro-
Table I. Influence of Water Content in Total Volume of 50 MI.
Anhydrous
condi- 0.025 0.05 0 . 1 tion ml. rnl. ml.
Galvanometer readings 31.5
Table 11.
30.8 17.2 1.6
Stability of Fluorophor
Time, min.
Galv. readings
25 30
34.3 3.4.1 32.8 32.5 32.5 32.0
60
29.3
a 10 1.i 20
Table Ill.
Formulation Digit. tincture, unit/ml.
methane. In chloroform the fluorophor is much less stable. Table 111 compares results obtained by assaying several different formulations. To determine whether the method measures the decomposition products of digitoxin, the stability of digitoxin in chloroform was studied. One sample was kept in the refrigerator, one in the dark a t room temperature, and the third on a window ledge in occasional sunlight During a month the withdrawn samples showed little difference from the initial solution. After 2 months, the room temperature and sunlight samples indicated a drop of only 2.3%. More drastic sample treatment, such as exposure to ultraviolet light for 1 and 3 hours, caused a decomposition of 20 and 30%, respectively. An overnight-autoclaved sample showed a decomposition of about 30%. These results are in good agreement with the USP method. To prove that the reaction takes place within the steroid moiety, pure digitoxigenin was treated in the same concentration as digitoxin. The same fluorophor was formed, except that the reading was about twice as high as that of digitoxin, which corresponds to the molecular weight ratio. Under ultraT-iolet light the digitoxigenin fluorophor exhibited the yellow greenibh fluorescence of digitoxin. Lanatoside A differs from digitoxin in having one additional molecule of glucosc and one more acetyl group. It yields the same fluorophor as digitoxin, but readings are about 207, lower, corresponding to the 20% increase in molecular weight. Lanatoside C readings are also in agreement with the molecular weight difference between digoxin and lanatoside C. Both exhibit a sky-blue fluorescence under ultraviolet light. Lanatoside B, like gitoxin, does not react. The reagent was applied to several steroids; estrone showed a very intense
Comparison of Data
Theory Bioassay
USP X KI
1
1.03
...
Digit. leaves, unit/100 mg.
1
1.07
...
Digitoxin tablets,
0.05 0.1
mg.
Digitoxin ariipoules, mg.
1516
0.2
ANALYTICAL CHEMISTRY
...
ACKNOWLEDGMENT
The author thanks J. Klemm and H. Boaa of the spectroscopy group for all spectral data and their interpretation. He acknowledges the assistance of S.R. Kuzel and J. BI. Fose in preparing this paper. Thanks are eytended to J. I?. Condon, Sandoz Pharmaceuticals, Division of Sandoe, Inc , Ilanover, K. J., for the generous gift of most glycosides used in this work. 1lTERATURE CITED
Baljet, H., Schweiz. Apoth. Ztg. 56, 71, 84 (1918). (2) Bell, F. K., Krantz, A. C., Jr., J . (1)
Am. Pharm. Assoc., Sei. Ed. 37. 297 (1948).
(3) Bell, F. K., Iirantz, A. C., Jr., J . Pharmacol. Exptl. 7‘hernp. 88, 14 (1946). (4) Cloetta, AI, Arch. Exptl. I’athol. Pharmahol. 112, 261 (1926). (5) Dyer, F. J., Quart. 1.Pharm. 5, 172 (1932).
(6) Fruytier, J. F. A., Pinxteren, J. A. C. van, Pharm. &*eekb/ad. 89, 99 (1954). (7) Houk, A. E. H., J . Assoc. Oj% Agr. Chemzsts 43, 793 (1960). (8) Jensen, K. R , Acta Phnrmncol. Toxtcol. 8. 101 f1932). (9) Ibad., 9,‘66 (195:3).’ (10) Kaiser, F., Chon. D e r . 88, 556 (1955). (11) Kedde, 1). L., Phurm. 1T’eekblad. 82, 741 (1947). (12) Keller, C., Ijer. Pharm. Ges. 5 , 275 (1895). \ - - - - I
(13) Kennedy, E. E., J . A m . Pharm. Assoc., Sci. Ed. 39, 25 (1950). (14) Kiliani, H., Arch. Pharoi. 234, 273 (1896).
(15) Kimura, XI., J . Piiarm. SOC.J a p a n 71, 991 (1961).
(16) Knudson, .\., Dresbach, AI., J . Pharmacol.
Exptl.
Therap. 2 0 , 205
(1923). (17) Lafon, P., Compt. Rend. 100, 1463 (1885).
(18) lforel, A.. Bull. SOC.China. France . ( 5 ) 2.949 11935). (li)’Pksez, YI., .4nn Pharin ~ r a n c .IO, 104 (1952). (20) Petit, A , PPaez, 31, Bellct, P , hmiard, G , Bull. SOC.Chain. Fiance 17, 288 1\ *1. 4.,-.,. ifll (21) Pinx beren. J. A. C. van. Pharin. Weekblad. 69, 4 (1932). (22) Rinhv. G . Bellis, I). AI., .\‘ntui.e (LOndon) 178; 415 (1956). (23L fSasakawa, Y., Yakugaku Zassiii 79, - - - . , I
Fluorometric determination 0.323 mg. digitoxin-like niaterial/ml. 0.169 mg. digoxin-like material/ml. 0,400 nig. digitoxin-like niateria1/100 mg. 0.185 mg. digoxin-like materia1/100 mg.
0.049 0.101
0,048
0.208
0.216
0.200
0.2
fluorescence, about ten times stronger than digitoxin, with three characteristic activation peaks.
0.104
0.206
51
1, (1959).
rattje, 13. 11. E., J . Pharm. Pharmacol 8 , 4 i 6 (1954). (2.5) Tattie. I 1. 13. E., Pharm. Weekblad. ’ 93, 248‘( 19%). (26) Warren, A. T., Ilowland, F. 0 . ) Green, L. K . , tJ. Am. Pharnc. Assoc., Sci. Ed. 37, 1% (19-4S). ( 2 7 ) Wells, TI., Iiatzung, B., JIeyers, F. H., J . I’itnrin. Phnririncol. 13, 380 (1901). (28) JT’induus, A , , Schwarte, G., Ber. Deut. Chem. Ges. 58, 1515 (1925). (24)
RECEIVED for review S ~ J V C30, II~ l M~2J . CI Accepted May 7, 1963.
