1195
V O L U M E 25, NO. 8, A U G U S T 1 9 5 3 In order to be certain that no calcium pantothenate was removed by this treatment, recoveries were seen as follows: h 25-ml. aliquot of standard calcium pantothenate was diluted with 25 ml. of water. Four separate 25-ml. aliquots of the standard calcium pantothenate were diluted with 25-ml. portions of ascorbic acid solutions containing 25, 50, 125, and 250 mg. of ascorbic acid, respectively. Then the five solutions were treated with calcium hydroxide and cupric sulfate as described and the amount of calcium pantothenate in each was determined. Recoveries of 99.6 to 100.270 were obtained. Procedure for Vitamin Mixtures. Weigh an amount of the sample corresponding to about 100 mg. of calcium pantothenate into a 250-ml. glass-stoppered flask. Add exactly 100.0 ml. of distilled water and shake for at least 10 minutes, centrifuge or filter. Place the prepared Florisil column in a Fisher Filtrator containing a 125-ml. glass-stoppered Erlenmeyer flask. Accurately measure a 25.0-ml. aliquot of the clear sample solution onto the column. Collect the eluate in the flask and wash t,he column with successive portions of water, 25 ml. in all. Remove the flask containing the eluate and washings from the filtrator, add 1.0 gram of anhydrous cupric d f a t e , stopper the flask, and shake until all is dissolved. Then add 3.0 grams of calcium hydroxide, shake the mixture. and allow to stand for 30 minutes with occasional shaking. -It the end of the 30-minute period filter the mixture through a sintered-glass funnel fitted in a Fisher Filtrator containing a 250-ml. standard taper flask. Wash the original flask and filter with 25 ml. of water. To the combined filtrate and washings, add 5.0 ml. of concentrated sulfuric acid, connect the flask to o reflux condenser, and gently heat for 1 hour. (Add glass bcads to prevent bumping.) Cool to room temperature and proceed as described for calcium pantothenate. DISCUSSION
The procedures outlined above are based on the requirement of about 250 micrograms of calcium pant'othenate for the development of sufficient intensity of color to minimize instrumental error. By the use of cell paths of greater than 1 em. depth this sensitivity can be inrreased. With suitable modificat,ions based on the nature of the sample. the method should be applicable to many biological materials and food preparations since the separation on the chromatographic column and copper sulfate-calcium
hydroyide treatment have been found to be specific. I t would be necessary, of course, in the use of this procedure on complex biological materials to investigate samples of known concentrations. initially, to preclude the presence of interfering components, CONCLUSIONS
Riboflavin is quantitatively removed from vitamin mixtures by chromatographing on a Florisil column. The copper sulfatecalcium hydroxide treatment completely removes ascorbic acid, lactose, and glucose. The chemical method described was applied to a number of laboratory-prepared mixtures of vitamins and the recoveries obtained are listed. Table V shows the composition of these mixtures with the recoveries obtained. Several commercial vitamin mixtures and feed enrichment mixtures were also tested by the chemical method and compared with the microbiological method. These are shown in Tables VI and VII. Good agreement between the t\vo methods was found. This method is less sensitive than the microbiological method, and, further, the method does not differentiate pantothenic acid and its inactive lactone moiety. LITERATURE CITED
(1)
Crokaert, R., and Bigwood, E. T., Arch. intern. physiol., 56,
189
(1948).
Rubin, S. H., AXAL.CHEM, 21, 823 (1949). (3) Feigl, F., Anger, V., and Frehden, O., Miizrochemie, 15, 9 (1934). (4) Frost, D. V., IND. ENG.CHEX, AN.AL.ED.,15,306 (1943). (5) Saalkowski, C. R., Mader, W. J., and Frediani, H. A , , Cereal 12) DeRitter, E., and
Chcm., 28,218 (1951). (6) Tao, 11.E., Baumann, M.L., and Wask, Shirley, AN.AL.CHEM. 24.722 11952). ( 7 ) Walter, M., Jubilee Val. Dedicated to Emzl Christoph Barell, 1946 98; 2 Vztaminjorach., 18, 228 (1947) (8) Wollish, E. G., and Schmall, M.,A x i L . CHEM.22, 1033 (1950). RECEIVED for review January 3, 1953. Accepted M a y 19, 1933. Presented at the North Jersey Xeeting-in-1Iiniature of the S o r t h Jersey Section of the . h E a I c h r CHEMICAL SOCIETY. S e w a r k , X. 1..January 26, 1053.
Colorimetric Determination of Erythromycin JiRED H. FORD, GEORGE C. PRESCOTT, J. W.IIINJIAIV, AND E. LOCIS CAROU Research Laboratories, T h e Upjohn Co., IGzlamazoo, ItIich.
.4 rapid and precise method for determining erythromycin in all stages of proc-
essing from the beer to the crystalline antibiotic was desired in studies on the fermentation and extraction of this antibiotic. The results have been found to be reproducible to about *3% on beers and to about +lyo on aqueous solutions of the purified antibiotic. A chemical assay method which depends upon the development of intense absorption at 485 mp has been found satisfactory for the assay of fermentation liquors and other process samples. Its utility is limited by interference from certain degradation products of erythroniycin, but where the method can be used i t is more rapid and reproducible than the bioassay.
RYTHROLIYCIN is a basic antibiotic having an approximate molecular formula of C31-36H60-6jSOl1-14( 2 ) , which is now in wide use for the treatment of various infections due to Gram-positive bacteria. A rapid and precise method for its determination in all stages of processing from the beer to the crystalline antibiotic was desired to support studies on fermentation and extraction. During some preliminary experiments on degradation, it was obJerved that an intense yellow color was produced when erythromycin was heated with 6 -V hydrochloric acid. The maxiniuni absorption of the solutio~iwa-r found to occur a t about 485
mp. The intact antibiotic has been reported to be tratispnrent in the visible portion of the spectrum and to show only a weak absorption a t 280 mp ( 2 ) . In the authors' first assay experiments, the solutions of erythromycin in 6 N hydrochloric acid were heated for 30 minutes at 50" and the absorbance (optical density) a t 485 nip was measured. However, in subsequent work the hydrochloric acid was replaced by sulfuric acid to reduce the possihility of corroding the spectrophotometer. -2 similar method has been reported for the determination of aureomyin ( I ) , but in this case t'he antibiotic was heated with 0.2 .V hJdrochloric acid and the absorpt,ioii wa3 measured at 440 mp,
1196
ANALYTICAL CHEMISTRY
In assaying beers, the erythromycin was extracted into amyl acetate a t pH 9.5 and then removed from the amyl acetate by extraction with 0.1 N hydrochloric acid. The resulting extract was treated with sulfuric acid to develop the color. REAGENTS
27
Drain off the clear aqueous layer and pipet 5 ml. into a test tube. Add 5 ml. of 27 N sulfuric acid and proceed as with solid preparations.
If a spent beer is found to be below pH 9.0, it should be adjusted to pH 9.0 to 10.5 by the addition of a small volume of concentrated ammonium hydroxide.
N Sulfuric Acid. Add 750 ml. of reagent grade acid (spe-
cific gravity 1.84) to 350 ml. of distilled water slowly and cautiously with constant stirring and cooling. 0.1 M Carbonate Buffer, pH 9.5. Dissolve 7.47 grams of potassium bicarbonate and 3.50 grams of anhydrous potassium carbonate in distilled water and make up t o exactly 1.00 liter. Amyl Acetate. Shake reagent grade amyl acetate with 0.1 volume of 5% potassium bicarbonate solution.
OPTIMUM CONDITIONS FOR COLOR FORMATION
Effectof Sulfuric Acid Concentration. The results obtained by varying the concentration of sulfuric acid are listed in Table I. As the color intensity was found to be about the same for 12 N and 14 N acids, 27 N sulfuric acid was used as the reagent and added to an equal volume of water which contained the antibiotic. The resulting solution gave the same absorption niaximum a t about 485 mp (see Figure 1) and the intensity was found to be about 30% greater than that obtained with 6 N hydrochloric acid. Effect of Time and Temperature on Color Development. When 5-ml. portions of 27 N sulfuric acid were pipetted into 5-ml. samples of 1507 per ml. of erythromycin solution and the test tubes were placed in a 50" bath, the increase in absorbance was very slight after 30 minutes (see Table 11). Although a 30minute period of heating a t 50' gave satisfactory results, it was not necessary to use the 50" bath, as the heat of dilution of the 27 N sulfuric acid was sufficient to develop the color. In order t o determine whether or not the temperature of the solutions before mixing would have a marked effect on the color intensity, one experiment was carried out a t room temperature (26') and a sec-
1.40
-
i
g 1.00-
t
2 0.80. W
p 0.40 0 9 0.200.60
Figure 1.
Absorption Spectrum of Erythromycin Solution
5 ml. of erythromycin solution, lOOy per ml., mixed with 5 ml. of 27 N sulfuric acid
Q:
OV
M Phosphate Buffer, pH
Dissolve 2.72 grams of potassium dihydrogen phosphate and 6.84 grams of dipotassium hydrogen phosphate trihydrate in water and make up t o 500 ml. 0.1
7.0.
PROCEDURE
A. Solid Preparations. Prepare a 507 per ml. solution in the p H 7.0 phosphate buffer. Erythromycin base dissolves very slowly in water, The rate of solution is increased considerably by the use of the pH 7.0 buffer. Pipet 5 ml. into a test tube and add 5 ml. of 27 S sulfuric acid. After 30 minutes read the absorbance a t 485 mp in a 1-cm. cell, using distilled water as the blank, Determine the concentration of the solution by reference to a standard curve (see Figure 2). -4Beckman spectrophotometer, Model B, was used in the present investigation. A photoelectric colorimeter with a filter did not give satisfactory results, as the absorption band is a rather narrow one (see Figure 1). B. Standard Curve. Prepare a solution containing 2007 per ml. of pure erythromycin base in pH 7.0 phosphate buffer and dilute aliquots of this solution to give concentrations from 20 to 2007 per ml. Use the procedure described above. C. Beers. Dilute the whole beer with enough of the carbonate buffer to give a concentration of about 507 per ml. Filtration to remove the mycelium is unnecessary. Pipet 20 ml. of the diluted beer and 20 ml. of amyl acetate into a 50-ml. centrifuge tube, stopper the tube, shake vigorously for 30 seconds, and then spin the tube in a centrifuge. Pipet 10 ml. of 0.1 N hydrochloric acid and 10 ml. of the clear amyl acetate layer into a small separatory funnel and shake the mixture vigorously for 30 seconds.
20 40 60 60 160 i o I40 l i 0 tbo 2 K) MICROGRAMS PER MILLILITER ERYTHROMYCIN CONCENTRATION
-
Fighre 2. Standard Curve of Relation of Erythromycin Concentration to Absorbance at 485 Mp
Table I.
Sulfuric Acid Concentration us. Color Intensity
(Erythromycin concentration of 100 T/ml,; minutes) Sormality of HzSOa 6.0
8.0 10.0 12.0 14.0 16.0
Table 11.
480 mg 0,271 0.600
0,980 1.211 1.187 1.002
solutions a t 50' C. for 30 Absorbance 485 mp 0.278 0.620 1.015 1.276 1.260 1.073
490 mg 0.218
0.490 0.815 1.020 1.032 0,915
Change of Color Intensity with Time at 50" C Time of Heating, Min.
Adsorbance a t 485 l l g
1197
V O L U M E 25, NO. 8, A U G U S T 1 9 5 3 Table 111.
Effect of Temperature on Color Development
(5-ml. portions of 27 N sulfuric acid added t o 5-nil. portions of 52 /ml. erythroiriycin solution previously cooled t o temperatures indicated a n x test tube held in air a t specified temperature) Time, Air Temperature 26' Air Temperature 8' 31in. Temp. of Soln. Dm' Temp. of Soln. Dm 0.32 41 0.52 33 10 31 0.52 21 0.53 20 0.52 0.53 31 9 30 0.53 29 8 0.52 40 0.52 0.54 1.0 2.5 8 0.53 0.56 240 25 8 Control (in 50' \rater hatli) 30 50 0.55 ,j0 0.55
hoped that the amyl acetate extraction procedure as used in the assay of beers would eliminate the interference, but this did not prove to be the case. When the degradations were carried out in 0.1 N sodium hydroxide or 0.1 N hydrochloric acid, however, a larger proportion of the interfering substances was removed by the extraction procedure. The results of these experiments are given in Table IV.
Table V.
Composition of Sample Beer Beer Beer Beer Beer Beer Beer Filtered beer Spent beer Amyl acetate Pxtrart Buffer extract Spent buffer extrart Spent brer
orid experiment was conducted in a refrigerator a t 8". The results (see Table 111) indicated that the color intensity was reduced only slightly when the use of the 50" bath was omitted and that the increase in intensity was very small after 10 minutes. The fact that the color intensities were approximately the same
Table IV.
Comparison of Colorimetric and Biological Assays on Hydrolyzed Erythromycin
Hydrolysis Conditions 5.5 60' 44 hours 8 5' 28'" 30 days 9:O: 28': 30 days 9.5 28' 30 d a s N HCl, b3', 6 iV PiaOH, 23', 24 hours a B~ciZlussubtilis plate assay. b I3rt.r procedure used.
gaurs
3'% ilctivity Remaining Bioassap Colorimetric a r i a y b 4.8 95 74 34 32 68 26 9 6 0.4 1
