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The Polarographic Determination of Riboflavin
The Polarographic Determination of Riboflavin*,' By W. J. SEAGERS Polarographic assay procedures have been developed for the determination of riboflavin (Vitamin Bz) i n riboflavin U. S. P., partially purified riboflavin, solubilized riboflavin, and vitamin mixtures containing riboflavin. Comparisons are made between the results using this assay method and those with the usual fluorometric assay method o n production samples.
C
OLORIMETRIC,
(4) was used, care being taken that the height of
the mercury head remained constant. In all cases, tank nitrogen, purged of oxygen by passage through an alkaline solution of pyyogallol, was bubbled through the cell solution for five minutes in order to remove oxygen. A Leeds and Northrup Electrochemograph Type E was used t o record the polarograms. Procedure.-The materials assayed can be divided into three groups containing those preparations which could be assayed by a common procedure. Group A included riboflavin U. S. P. and partially purified riboflavin since both compounds may be assayed directly. A sample of 20-30 mg. of the material was accurately weighed and transferred into a 50-ml. volumetric flask, and 5 mi. of 0.4 M sodium hydroxide was added. The flask was swirled until solution was complete, and 20 ml. of aqueous buffer, 0.2 M with respect t o acetic, boric, and phosphoric acids and 1.0 M with respect .to potassium chloride, was then added. The solution was made up t o volume with water and thoroughly mixed. A sample of 20-30 mg. of dried riboflavin reference standard U. S. P. was treated in the same manner. The solutions (PH about 2.8) were placed in the polarographic cell in turn, purged with nitrogen for five minutes, and polarographed over the potential range of 0 t o - 1 v. The diffusion currents were calculated in microamperes and the percentage of riboflavin was calculated from the following formula :
fluorometric, a n d microbio-
logical methods have been used for the
determination of riboflavin (vitamin B2). A polarographic method h a s been suggested (1) for the assay of riboflavin in yeast b u t details of t h e proposed method or results of assays were not given. The present paper reports the procedures used for the assay of various products containing riboflavin, t h e assay results obtained, a n d a comparison of these data with those obtained using the fluorometric technique (2). Over t h e pH range 1 t o 12, riboflavin exhibits a well-defined wave (1, 3) which is suitable for assay purposes. In t h e following procedures the pH of the solution polarographed depends on the extraction procedure used a n d the subsequent treatment. T h e extractive procedure used will depend on the material being assayed but, in general, three methods have been used which are described in detail. Because the solution obtained on extraction of a vitamin mixture is of unknown composition, a standard addition method of analysis was used. For the sake of consistency, this same method was used for the other groups of materials.
Per cent Riboflavin = id sample (pa.) ad std. (pa.)
EXPERIMENTAL Materials.-The preparations assayed were regular production lots of riboflavin U. S. P., partially purified riboflavin, vitamin mixtures, and solubilized riboflavin. All chemicals were reagent grade and were used without further purification. Riboflavin reference standard U. S. P., dried at 100" for three hours, was used as a standard for all procedures. Apparatus.-The polarographic cell was a 35 x 50-mm. weighing bottle provided with a four-hole rubber stopper in which were inserted a nitrogen inlet tube, the dropping-mercury electrode, a lead for contact with the. mercury anode, and a thermometer. A standard dropping-mercury electrode
* Received
Sept. 27, 1952, from the Analytical Research Department, Chemical Control Division, Merck Lk Co., Inc., Rahwav. N. 1. I Af
mg. of std. mg. of sample
x
100
One standard was polarographed for each four det'erminations in order t o correct for any changes in room temperature since all formulas used are based on the assumption that the temperature and the mercury head were constant. The results obtained with this procedure on representative samples are shown in Table I.
TABLE I.-A COMPARISON OF THE POLAROCRAPHIC AND FLUOROMETRIC ASSAYMETHODSFOR RIBOFLAVINI N GROUPA MATERIAL3 Sample
U. S. P. A U. S. P. B Partially purified A Partiallv Durified B
Polarogra hic Assay,
98.0; 99.2; 91.6; 89.2:
%,
Fluorometric Assay, %
97.2 98.0 93.2 92.2
99.5 99.3 92.1 91.7
Group B included only solubilized riboflavin since the criterion of effective riboflavin content in this preparation is its solubility in water. A sample of 5004300 mg. of material was accurately weighed into a 100-ml. volumetric flask and diluted to volume with water; 10.0-ml. aliquots were pipetted
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JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
into two 50-ml. G. S. Erlenmeyer flasks. T o one flask was added 10.0 ml. of a solution of one part 0.4 M sodium hydroxide to four parts of aqueous buffer (rf. Group A). To the other flask was added 10.0 ml. of standard, prepared by accurately weighing about 20 mg. of dried riboflavin reference standard U. S. P . , adding 10 ml. of 0.4 M sodium hydroxide, swirling to dissolve, and diluting t o 50.0 ml. with aqueous buffer. Each solution (PH about 2.8) was placed in the polarographic cell, purged with nitrogen for five minutes, and polarographed over the potential range 0 t o - 1 v. The diffusion currents were calculated in microamperes and the appropriate values were substituted in the following formula: Per cent riboflavin = i, (pa)-mg. of std. weighed is - ia ( p a ) 5
100 mg. of sample
where i, is the diffusion current for the sample and i b is the diffusion current for the sample plus standard. The results obtained using this procedure on representative samples compared with results of the fluorometric assay are shown in Table 11.
Mg. of riboflavin per oz. = . i a ( W ) Ib
Fluorometric Assay,
Lot No.
%
%
1 2
8.27 9.50; 9.43
8.09; 8.29 9.17; 9.35
- ia (Ma)
mg. riboflavin (U.S.P.)/50 ml. X 28.35 wt. of sample in Gm.
where ia is the diffusion current found for the sample and i b is the diffusion current for the sample plus standard. The results obtained by this procedure are compared in Table 111 with those obtained by the fluorometric assay for a representative group of vitamin mixtures. TABLE 111.-A COMPARISON OF THE POLAROGRAPHIC A N D FLUOROMETRIC ASSAY METHODSFOR RIBOFLAVIN IN GROUPC MATERIALS Designation
Diluents
A B C
Cornstarch” CornstarchG Cornstarch
D E F TABLE 1L-A COMPARISON OF THE POLAROCRAPHIC AND FLUOROMETRIC METHODSFOR RIBOFLAVIN IN GROUPB MATERIAL G Polarographic Assay.
Vol. XLII, No. 5
CDDG” CDDGb Corn meal CDDGbvc
Polarographic Assay. Mg./Oz.
Fluorometric Assay, Mg./Oz.
1,035; 1,073 260; 265 1,088 1,078; 1,095 1,115; 1,150 279; 284 281; 283 444; 462 477; 483 457; 458 135; 141
1,160 262 1,080 1,100 1,100 273 299 4 52 469 430 134
a Also contains calcirrm pantothenate.
b CDDG is corn distiller’s dried grains. c Also contains calcium pantothenate. niacin, and choline chloride.
DISCUSSION Group C included vitamin mixtures containing riboflavin, one or more diluents, and, in some cases, one or more other vitamins. The sample size used in this group will depend on the amount of riboflavin present per ounce, i. e., 1,000 mg. per oz., 1-Gm. sample; 500 mg. per oz., 2-Gm. sample; 250 mg. per oz., 4-Gm. sample. An accurately weighed sample of suitable size mas placed in a Waring Blendor, 100 ml. of a solution of equal parts of acetone and 0.1 M aqueous hydrochloric acid was added, and the mixture was blended for five minutes. The contents of the Blendor were poured into a 150-ml. beaker, covered with a watch glass, and allowed to cool to room temperature (about twenty minutes). Ten milliliters of the supernatant liquid was pipetted into each of two G. S. Erlenmeyer flasks. The solution pipetted does not have to be clear. To one flask was added 5.0 ml. of a solution of four parts of the acetone-hydrochloric acid used previously and one part of 0.4 M sodium hydroxide. To the other flask was added 5.0 ml. of a standard solution prepared as for Group B. Each solution (pH about 2.9) was placed in the polarographic cell, purged with nitrogen for five minutes, and polarographed over the potential range 0 to -1 v. The diffusion currents were calculated in microamperes and the appropriate values were substituted in the following formula:
T h e fluorometric assay method is usually considered to have a precision of about *2 relative per cent whereas the polarographic assay has a precision of about =!=3 relative per cent. T h u s the agreement between the results obtained by the fluorometric assay method a n d by the polarographic method is within t h e precision of the respective methods. T h e polarographic method is most useful for the assay of riboflavin in mixtures, since in this case t h e pretreatment before the instrumental determination is much less t h a n for t h e fluorometric method. T h e polarographic method is a reliable alternate assay method for riboflavin and its use b y laboratories equipped for such determinations is urged.
REFERENCES (1) Lingane. J. J.. and Davis, 0. L., J . B i d . Chcm., 137, 567(1941). (2) “United States Pharmacopeia,” Fourteenth Revision, Mack Publishing Company, Easton, Pa., 1960, p. 7.50. (3) Brdicka, R., and Knobloch, E., 2. Elektrochem., 47; 721 (1941). (4) Kolthoff. I. M.. and Lingane, J. J., “Polarography,” 2nd ed.. Vol. 1 . Interscience Publishers, Inc., N ew York, 1952, p. 353.
C
OLORIMETRIC,
(4) was used, care being taken that the height of
the mercury head remained constant. In all cases, tank nitrogen, purged of oxygen by passage through an alkaline solution of pyyogallol, was bubbled through the cell solution for five minutes in order to remove oxygen. A Leeds and Northrup Electrochemograph Type E was used t o record the polarograms. Procedure.-The materials assayed can be divided into three groups containing those preparations which could be assayed by a common procedure. Group A included riboflavin U. S. P. and partially purified riboflavin since both compounds may be assayed directly. A sample of 20-30 mg. of the material was accurately weighed and transferred into a 50-ml. volumetric flask, and 5 mi. of 0.4 M sodium hydroxide was added. The flask was swirled until solution was complete, and 20 ml. of aqueous buffer, 0.2 M with respect t o acetic, boric, and phosphoric acids and 1.0 M with respect .to potassium chloride, was then added. The solution was made up t o volume with water and thoroughly mixed. A sample of 20-30 mg. of dried riboflavin reference standard U. S. P. was treated in the same manner. The solutions (PH about 2.8) were placed in the polarographic cell in turn, purged with nitrogen for five minutes, and polarographed over the potential range of 0 t o - 1 v. The diffusion currents were calculated in microamperes and the percentage of riboflavin was calculated from the following formula :
fluorometric, a n d microbio-
logical methods have been used for the
determination of riboflavin (vitamin B2). A polarographic method h a s been suggested (1) for the assay of riboflavin in yeast b u t details of t h e proposed method or results of assays were not given. The present paper reports the procedures used for the assay of various products containing riboflavin, t h e assay results obtained, a n d a comparison of these data with those obtained using the fluorometric technique (2). Over t h e pH range 1 t o 12, riboflavin exhibits a well-defined wave (1, 3) which is suitable for assay purposes. In t h e following procedures the pH of the solution polarographed depends on the extraction procedure used a n d the subsequent treatment. T h e extractive procedure used will depend on the material being assayed but, in general, three methods have been used which are described in detail. Because the solution obtained on extraction of a vitamin mixture is of unknown composition, a standard addition method of analysis was used. For the sake of consistency, this same method was used for the other groups of materials.
Per cent Riboflavin = id sample (pa.) ad std. (pa.)
EXPERIMENTAL Materials.-The preparations assayed were regular production lots of riboflavin U. S. P., partially purified riboflavin, vitamin mixtures, and solubilized riboflavin. All chemicals were reagent grade and were used without further purification. Riboflavin reference standard U. S. P., dried at 100" for three hours, was used as a standard for all procedures. Apparatus.-The polarographic cell was a 35 x 50-mm. weighing bottle provided with a four-hole rubber stopper in which were inserted a nitrogen inlet tube, the dropping-mercury electrode, a lead for contact with the. mercury anode, and a thermometer. A standard dropping-mercury electrode
* Received
Sept. 27, 1952, from the Analytical Research Department, Chemical Control Division, Merck Lk Co., Inc., Rahwav. N. 1. I Af
mg. of std. mg. of sample
x
100
One standard was polarographed for each four det'erminations in order t o correct for any changes in room temperature since all formulas used are based on the assumption that the temperature and the mercury head were constant. The results obtained with this procedure on representative samples are shown in Table I.
TABLE I.-A COMPARISON OF THE POLAROCRAPHIC AND FLUOROMETRIC ASSAYMETHODSFOR RIBOFLAVINI N GROUPA MATERIAL3 Sample
U. S. P. A U. S. P. B Partially purified A Partiallv Durified B
Polarogra hic Assay,
98.0; 99.2; 91.6; 89.2:
%,
Fluorometric Assay, %
97.2 98.0 93.2 92.2
99.5 99.3 92.1 91.7
Group B included only solubilized riboflavin since the criterion of effective riboflavin content in this preparation is its solubility in water. A sample of 5004300 mg. of material was accurately weighed into a 100-ml. volumetric flask and diluted to volume with water; 10.0-ml. aliquots were pipetted
317
318
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
into two 50-ml. G. S. Erlenmeyer flasks. T o one flask was added 10.0 ml. of a solution of one part 0.4 M sodium hydroxide to four parts of aqueous buffer (rf. Group A). To the other flask was added 10.0 ml. of standard, prepared by accurately weighing about 20 mg. of dried riboflavin reference standard U. S. P . , adding 10 ml. of 0.4 M sodium hydroxide, swirling to dissolve, and diluting t o 50.0 ml. with aqueous buffer. Each solution (PH about 2.8) was placed in the polarographic cell, purged with nitrogen for five minutes, and polarographed over the potential range 0 t o - 1 v. The diffusion currents were calculated in microamperes and the appropriate values were substituted in the following formula: Per cent riboflavin = i, (pa)-mg. of std. weighed is - ia ( p a ) 5
100 mg. of sample
where i, is the diffusion current for the sample and i b is the diffusion current for the sample plus standard. The results obtained using this procedure on representative samples compared with results of the fluorometric assay are shown in Table 11.
Mg. of riboflavin per oz. = . i a ( W ) Ib
Fluorometric Assay,
Lot No.
%
%
1 2
8.27 9.50; 9.43
8.09; 8.29 9.17; 9.35
- ia (Ma)
mg. riboflavin (U.S.P.)/50 ml. X 28.35 wt. of sample in Gm.
where ia is the diffusion current found for the sample and i b is the diffusion current for the sample plus standard. The results obtained by this procedure are compared in Table 111 with those obtained by the fluorometric assay for a representative group of vitamin mixtures. TABLE 111.-A COMPARISON OF THE POLAROGRAPHIC A N D FLUOROMETRIC ASSAY METHODSFOR RIBOFLAVIN IN GROUPC MATERIALS Designation
Diluents
A B C
Cornstarch” CornstarchG Cornstarch
D E F TABLE 1L-A COMPARISON OF THE POLAROCRAPHIC AND FLUOROMETRIC METHODSFOR RIBOFLAVIN IN GROUPB MATERIAL G Polarographic Assay.
Vol. XLII, No. 5
CDDG” CDDGb Corn meal CDDGbvc
Polarographic Assay. Mg./Oz.
Fluorometric Assay, Mg./Oz.
1,035; 1,073 260; 265 1,088 1,078; 1,095 1,115; 1,150 279; 284 281; 283 444; 462 477; 483 457; 458 135; 141
1,160 262 1,080 1,100 1,100 273 299 4 52 469 430 134
a Also contains calcirrm pantothenate.
b CDDG is corn distiller’s dried grains. c Also contains calcium pantothenate. niacin, and choline chloride.
DISCUSSION Group C included vitamin mixtures containing riboflavin, one or more diluents, and, in some cases, one or more other vitamins. The sample size used in this group will depend on the amount of riboflavin present per ounce, i. e., 1,000 mg. per oz., 1-Gm. sample; 500 mg. per oz., 2-Gm. sample; 250 mg. per oz., 4-Gm. sample. An accurately weighed sample of suitable size mas placed in a Waring Blendor, 100 ml. of a solution of equal parts of acetone and 0.1 M aqueous hydrochloric acid was added, and the mixture was blended for five minutes. The contents of the Blendor were poured into a 150-ml. beaker, covered with a watch glass, and allowed to cool to room temperature (about twenty minutes). Ten milliliters of the supernatant liquid was pipetted into each of two G. S. Erlenmeyer flasks. The solution pipetted does not have to be clear. To one flask was added 5.0 ml. of a solution of four parts of the acetone-hydrochloric acid used previously and one part of 0.4 M sodium hydroxide. To the other flask was added 5.0 ml. of a standard solution prepared as for Group B. Each solution (pH about 2.9) was placed in the polarographic cell, purged with nitrogen for five minutes, and polarographed over the potential range 0 to -1 v. The diffusion currents were calculated in microamperes and the appropriate values were substituted in the following formula:
T h e fluorometric assay method is usually considered to have a precision of about *2 relative per cent whereas the polarographic assay has a precision of about =!=3 relative per cent. T h u s the agreement between the results obtained by the fluorometric assay method a n d by the polarographic method is within t h e precision of the respective methods. T h e polarographic method is most useful for the assay of riboflavin in mixtures, since in this case t h e pretreatment before the instrumental determination is much less t h a n for t h e fluorometric method. T h e polarographic method is a reliable alternate assay method for riboflavin and its use b y laboratories equipped for such determinations is urged.
REFERENCES (1) Lingane. J. J.. and Davis, 0. L., J . B i d . Chcm., 137, 567(1941). (2) “United States Pharmacopeia,” Fourteenth Revision, Mack Publishing Company, Easton, Pa., 1960, p. 7.50. (3) Brdicka, R., and Knobloch, E., 2. Elektrochem., 47; 721 (1941). (4) Kolthoff. I. M.. and Lingane, J. J., “Polarography,” 2nd ed.. Vol. 1 . Interscience Publishers, Inc., N ew York, 1952, p. 353.