V O L U M E 2 1 , N O . 11, N O V E M B E R 1 9 4 9 (7) Lecky and Ewell, IND. ENG.CHEM.,ANAL.ED., 12,544 (1940). (8) Peel, D.H., I.C.I. Report, “Billingham Method of Analysis of Petrols.” circulated orivatelv t o members of T.A.C. Soectrographic Committee -and to-officials of various British and American oil companies, May 31,1943. (9) Podbielniak. W. J., IND.ENG. CHEM.,ANAL.ED., 13, 639 (1941).
1383 (10) Tongberg, C. O.,Lawroski, S., and Fenske, M. R., Znd. Eng Chem.,29,957(1937). (11) Tongberg, C.O.,Quiggle, D., and Fenske, M. R., Zbid., 26, 213 (1934). Be?., 44,3121 (1911); 45,3678 (1912) (12) Zelinsky, N. D., (13) Zelinsky, N. Refiner, 20,73 271 (l941). RECEIVED March 20,1948.
Determination of Mannosidostreutomvcin and DihvdromannosidostreDtomvcin A Colorimetric Method JOHN A. KOWALD AND ROBERT B. RICCORJIACI< E . R . Squibb & Sons, New Brunswick, N . J .
Mannosidostreptomycin or dihydromannosidostreptomycin in samples of commercial streptomycin or dihydrostreptomycin solids may be determined colorimetrically by means of the anthrone reagent. This reagent (0.29” in 95% sulfuric acid) reacts with the mannose moiety, producing a characteristic color. The intensity of this color, compared with that produced by mannose standards, permits quantitative determination of mannosidostreptomycin and dihydromannosidostreptomycin. Streptomycin and dihydrostreptomycin at the low level of concentration used for this test (about 300 micrograms per ml.) do not interfere with the determinations.
T
HE procedure presented is based on the use of Dreywood’s anthrone reagent ( 1 ) in sulfuric acid for the determination of
carbohydrates. This method has been applied to the determination of the quantity of mannose derived from the hydrochloride or the sulfate salts of mannosidostreptomycin (streptomycin B)or dihydromannosidostreptomycin which is contained in the sample. [Subsequent to the authors’ routine application of this reagent, a similar use of anthrone was announced, but no details of the procedure were made available @).] This reagent is highly specific for carbohydrates, giving a characteristic blue-green color. S o ooncarbohydrate, except furfural, has been reported to give this color. It has been shown ( 4 )that the anthrone reagent in sulfuric acid gives the same depth of color with a compound of a sugar as if the compound were first hydrolyzed and then the determination made. Streptomycin and dihydrostreptomycin a t the low level of concentration used for these determinations do not interfere with the test while similar absorption spectra (450 to 700 millimicrons) are obtained for mannose, mannosidostreptomycin, and material containing both streptomycin and mannosidostreptomycin (see Figure 1). D(+)-Mannose is used as a standard for the determination and over the range indicated reacts with anthrone following Beers’ law (Figure 2). APPARATUS
A Coleman Junior spectrophotometer was used for the measurements of color intensity, but any type of instrument is suitable if absorption measurements a t a wave length of 620 millimicrons can be attained. The size of the cuvette used in the instrument will affect the range of the test. For the results here reported, 25 X 105 mm. cuvettes were used. When cells of smaller diameter are used, the concentration of sample solution will need to be increased to obtain suitable intensity of color for accurate measurement.
Pyrex test tubes (inside diameter 22 mm.) are suitable reaction tubes. The uniformity of these tubes is important for reproducibility of results, as variation in size may effect the development of the heat of reaction. Because of the sensitivity of the test, chemical cleanliness of glassware is important. Freedom from traces of lint or fibers from cotton or filter paper is especially necee sary in both glassware and water sources. Pipets or syringes are suitable for adding the reagent to the sample. The use of lubricants such as Cello-Seal with burets introduces errors. REAGEWTS AND SOLUTION5
Anthrone Reagent. The reagent for the test is 0.2% anthrone prepared according to the method in “Organic Syntheses” (3) in 95% sulfuric acid (reagent grade). Fresh solutions are to be prepared daily. Sulfuric Acid, 95%. The 95% sulfuric acid waa prepared ( 4 ) by cautiously adding 1 liter of concentrated acid to 50 ml. of water and cooling. D( +)-Mannose. C.P. reagent having a specific rotation of 14.25 O was obtained from the Pfanstiehl Chemical Company.
+
PROCEDURE
I t is necessary t o standardize the reagent with a known concentration of mannose each time a series of determinations is made. With the size of cuvette here used, concentrations of mannose in water ranging from 5 to 50 micrograms per ml. gave color density sufficient for accurate determinations. The concentration of streptomycin in solution used will be somewhat dependent upon the amount of mannosidostreptomycin or dihydromannosidostreptomgcin contained, and the limits of detection desired when pure streptomycin or dihydrostreptomycin is to be authenticated. Usually a total of 300 micrograms per ml. is satisfactory for initial determinations. Inasmuch as the yield of mannose from pure mannosidostreptomycin approximatea
1384
ANALYTICAL CHEMISTRY
20%, the concentration of the solution can be varied, depending upon the estimated content of the sample. For accurate determinations the sample should be of sufficient concentration so that the mannosidostreptomycin or dihydromannosidostreptomycin content will yield a t least 5 micrograms per ml. of mannose.
RESULTS
I n Table I are shown the results of several analyses. The anthrone procedure compares favorably with the countercurrent distribution procedure ( 5 ) which has been authenticated for these types of samples.
To 5 ml. of the streptomycin solution or mannose standard, 10 ml. of the anthrone reagent are added and immediately mixed. When the heat of reaction has subsided to room temperature (the period of standing should be kept uniform for each series of analyses, including the standard and blank), the solutions are transferred to matched cuvettes or colorimeter tubes for measurement of the density of absorption at 620 millimicrons. Included with each series of analyses is a blank for zero adjustment of the measuring instrument. This blank consists of 5 ml. distilled water plus 10 ml. of the reagent and is prepared concurrently with the other samples.
c t
1.000
0.900
I
,/
0.800
0.60 0.7$ CALCULATION OF RESULTS
-
For each series of determinations the conversion factor, K , is established using mannose standards (see Figure 2).
*
c
z
K = C (mannose concentration
n (density of absorption:
0.504
W
n
2 V
0.400
I
0.000
1
20
IO
I
30
I
40
I
I
60
50
CONCENTRATION OF MANNOSE (GAMMAS/kL.) Figure 2. Relation of Color Density Due to Anthrone Reaction Using Varying Quantities of D( +)-Mannose
Table I.
0.000 500
550
600
650
7C
Absorption Curves of Anthrone Reaction Products
I
25.0 46.0 52 0 57.0 58.0 97.0
Ka
67.0
o n
.T
WAVE LENGTH IN MILLIMICRONS Figare 1.
‘Z .\IannosidostreDtomvcin . ” Anthrone Countercurrent method distribution (colorimetric) method 0 0 0.5 2 0 2.3 6.5 6 0
Streptomycin Sample A B C D E F G H
0.1 00
450
.4nalgsis of Mannosidostreptomycin in Samples of Streptomycin
24 45 50 56 56 98
0
0 0 0 0 0
. .
Samples of dihydrostreptomycin preparations and corresponding per cent of dihydromannosidostreptomycin. a
D Mannose 0 Crystalline streptomycin hydrochloride (300 y/ml.) 0 Mannosidostreptomycin sulfate (300 -y/ml.)
A Mixture of streptomycin and mannosidostreptomycin
Psing this factor the mannose content of the streptomycin sample is determined. From the mannose content the mannosidostreptom! cin or dihydromannosidostreptomycin content may then be calculated. In the results here given the mannose content of mannosidostreptomycin hydrochloride is taken as 21.14%, while for the corresponding sulfate 20.28% is assumed. When the total weight of mannosidostreptomycin in the sample is known, the content in the original material can be computed.
LITERATURE CITED
(1) Dreywood, Roman, IND. ENG.CHEM,,. h . i L . ED..18, 499 (1946). (2) E m e r y , IT. B., and W a l k e r , 8 . D., S u t u r e , 162. 525 (1948). (3) M e y e r , K. H., “Organic S y n t h e s i s , ” C o l l e c t i v e Vol. I, 2nd ed., p. 60, X e w York, John W i l e y & S o n s , Inc., 1941. (4) Morris, D. L., Science, 107, 254 (1948). (5) P l a u t , G. W.E., and McCormack, R. B., J . Am. Chem. Sac., 71, 2264 (1949). RECEIVED M a r c h 23, 1949.
