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Spectrophotometric determination of cyanocobalamin (as cobalt)
M,CROCHF~,ICAL J”“RXAI.
22, 479 -483 (1977)
Spectrophotometric Cyanocobalamin G. S. Laboratory
TH.
VASILIKIOTIS,
A.
Determination (as Cobalt) A.
KOUIMTZIS,
of
AND
VOULGAROPOULOS
of Analytical
Chemistry, University Thessaioniki, Greece
Received
of Thessaloniki,
May 5. 1977
INTRODUCTION The determination of cyanocobalamin in pharmaceutical preparations is usually done either directly by measuring the absorbance of its water solutions at 361 nm (1,2) or indirectly by a method which involves the determination of cobalt (on a weight percentage basis, cobalt being 4.35% of the total Bi2 molecule). The direct method is applied to pharmaceutical preparations that give clear water solutions. In the presence of other compounds that interfere with its determination, there have been suggested various methods for the separation of cyanocobalamin prior to its determination (3,4). For indirect determination various methods have been proposed. They are based on the decomposition of the samples and the subsequent determination of cobalt by spectrophotometric (5-7) and other methods such as isotopic dilution (8) and A.A.S. (9,10). The last method can also be used without decomposition of the samples by direct aspiration of their water solution into the appropriate flame. In this case, the absorbance by cobalt into the flame could be affected by the presence of various organic compounds which enhance the absorption (9). The spectrophotometric determination of cobalt after the decomposition of the sample is usually done by use of a reagent such as nitroso-R salts (5)) I-benzoyl-4-phenylthio-semicarbazidlosung (61, or fast navy 2R (7). With these reagents and the presence of other metal cations, interference in some extension was experienced in the determination of cobalt (7,111.
In a recent paper (12) a new specific and very sensitive spectrophotometric method for cobalt determination was proposed. The method is based on the formation of a complex between cobalt and 2,2’-dipyridyl-2pyridylhydrazone (DPPH). This reagent also forms colored complexes with a number of metal cations in solutions having pH>3. Addition of a strong acid (H$O,, HCIO,) to solutions of these complexes results in the decomposition of all complexes except that of cobalt, which only changes its color from orange-yellow to pink. At 25% perchloric acid the molar 479 Copyright @ 1977 by Academic Press, Inc
ISSN 0026-265X
480
VASILIKIOTIS,
KOUIMTZIS,
AND
VOULGAROPOULOS
absorptivity of the cobalt complex is 42,000 liter mol-’ at 500 nm. At this acidity, the presence of other metal cations does not interfere with the cobalt determination. The reagent itself does not absorb at 500 nm and this offers a considerable advantage over the group of nitrosonaphtholes and nitroso-R-salts. In the work presented here, the application of the DPPH method for the indirect determination of cyanocobalamin in pharmaceutical preparation was studied. EXPERIMENTAL Reagents
2,2’-Dipyridyl-2-pyridylhydrazone (DPPH) was prepared as described previously (12). Solutions of DPPH were prepared by dissolving the required weight in ethanol. The ethanolic solution of DPPH is stable and could be kept for several weeks in an amber glass bottle. Standard solutions of cobalt(II) were prepared by dissolving the appropriate amount of cobaltous chloride hexahydrate in distilled water. The solutions obtained were standardized by EDTA titration. Cyanocobalamin (Merck) was dried for 4 hr over silica gel at 50°C before its use. Various amounts of cyanocobalamin were accurately weighed and transferred to a 50-ml beaker for the wet ashing. A standard solution of cyanocobalamin containing 4.35 ppm Co was prepared by dissolving 100 mg of cyanocobalamin in 1000 ml of distilled water. These solutions were tested either by the recommended procedure or by atomic absorption spectroscopy. All other reagents used were of analytical grade. Apparatus
Absorbance measurements were made in lo-mm quartz cells with a Zeiss M4QII spectrophotometer. A Perkin-Elmer Model 403 atomic absorption spectrophotometer equipped with an air-acetylene burner head was also used in the experimental work. Recommended
Procedure
The appropriate amount of the pharmaceutical preparation (so as to contain 7 to 100 pg of cobalt) is weighed and transferred to a 50-ml beaker. Then 5 to 20 ml of concentrated sulfuric acid and about 0.2 g of potassium perchlorate are added. The solution is heated slowly with constant swirling of the beaker and is then boiled. If the hot digest is not colorless, a few crystals of potassium perchlorate are added, and the solution is boiled again. Finally the bulk of sulfuric acid is volatilized. At this stage all organic matter should have been completely decomposed. After cooling the digest, about 10 ml of distilled water are added followed by a concentrated solution of ammonium hydroxide until the pH exceeds 5. The solution is transferred to a 50-ml volumetric flask and 2 ml of 10m2M
DETERMINATION
481
OF RI2
TABLE I CoB.4L.r DE~ERMISATION IS CYANOCORLAMIS B,, taken
SL~SDARDS Cobalt found
Cobalt calculated
(mg)
(I*g)
(/a)
0.200 0.400 0.800 1.384 1.809 2.191
8.7 14.4 34.8 60.2 78.7 9.5.3
7.9 IS.8 32.4 60.8 76.5 94.4
ethanolic solution of DPPH are added. Then 10 ml of concentrated sulfuric acid are added and the flask is filled with distilled water. The absorbance of the resulting solution is measured at 500 nm and compared with the calibration curve which is obtained using standard solutions of cobalt. In the case of injectable pharmaceutical preparations, an appropriate volume is transferred into the beaker, boiled nearly to dryness, and then treated as described above. RESULTS AND DISCUSSION
The method developed has been applied to the determination of cobalt in cyanocobalamin standard samples. The results obtained are summarized in Table 1. For the first three samples listed in the table the appropriate volumes of the standard cyanocobalamin solution were taken and transferred to the beaker for the wet ashing. The other three samples were taken by weighing cyanocobalamin. The results show that the recovery of cobalt from the wet ashing is complete. The method has also been applied to the determination of cyanocobalamin (as cobalt) in two pharmaceutical preparations coded as A tablets and B injections. The analysis of these two preparations has also been performed by A.A.S. The results obtained are summarized in Table 2. TABLE 2 CYANOCOBALAMIN DETERMINATIOS (AS COBALT) IN PHARMACEVTICAL PRwAu.rtoNs Cobalt found (ppm)” Sample
Cobalt calculated
(ppm)
Present method
A.A.S
A 1251pg B,Jtablet
17.6
16.2 t 1.2
16.5 t 1.4b
B I mg B,,/ml
43.5
42.4 k 2.3
45.7 T 1.8r
ClFive determinations * After wet ashing. c Direct aspiration.
482
VASILIKIOTIS,
KOUIMTZIS,
AND
VOULGAROPOULOS
These results confirm the recovery of cobalt and the precision of the method developed. The procedure described is simple to use for routine testing in pharmaceutical preparations, mainly when these products are not dissolved in water or the solutions obtained produce cloudiness. It offers advantages over the other reagents used for this purpose because of the lack of interferences. Since the present method determines the total cobalt present, the presence of cobalt compounds other than cyanocobalamin will give higher values. This is applied to all methods that determine cyanocobalamin as cobalt. In this case, ionic cobalt, if present, could be extracted into a CHCl, solution of 8-hydroxyquinoline prior to the wet ashing (9). The method developed could also be applied to the determination of cobalt in various biological materials or food after the appropriate decomposition of the samples. SUMMARY A spectrophotometric method for the determination of cyanocobalamin (as cobalt) in pharmaceutical preparations has been developed. The sample is first decomposed with sulfuric acid and potassium perchlorate. The liberated cobalt is then determined using 2,2’dipyridyl-2-pyridylhydrazone as reagent in solutions containing 20% sulfuric acid. The presence of other metal cations does not interfere with the determination of cobalt. The method has been applied to the determination of cobalt in pure cyanocobalamin and in two pharmaceutical preparations.
REFERENCES 1. Bayer, J., Spectrophotometric examination of cyano- and hydroxocobalamines. Pharmazie 19, 602-605 (1964). 2. Bruening, C., Hall, W., and Kline, O., Rapid determination of the relative purity of vitamin B,, (cyanocobalamin) in pharmaceutical products. J. Amer. Pharma. Assoc. Sci. Ed. 47, 15-20 (1958); Anal. Abstr. 5, 3124 (1958). S. Tortolani, G., Bianchini, P., and Mantovani, V., Separation and determination of cobalamins on an SP-Sephadex column. J. Chromatogr. 53, 577-579 (1970). 4. Marini-Scotti, M., Determination of vitamin B,, and uridine-5’-triphosphate associated in lyophilised preparations. Farmaco Ed. Praf. 18, 332-334 (1963); Anal. Absrr. 11, 4509 (1964). 5. Monnier, D., Chaliounghi, V., and Saba, R., Determination of traces of vitamin B,,. Anal. Chim. Acta 28, 30-40 (1963). 6. Craciuneanu, R., and Florean. E., Photometric procedure for determining cyanocobalamin [as cobalt]. Pharmazie 24, 462-464 (1969). 7. El Raheem, Abd, and Dokhama, M., Calorimetric assay of cobalt and vitamin B,, with fast navy 2R. Z. Anal. Chew. 189, 389-396 (1962). 8. Rosenblum, C., Analytical application of radioactive vitamin B,,. Talanra 11, 255-270 (1964). 9. Mandrou, B., and Bres, J., Determination of vitamin B,, [cyanocobalamin] in some pharmaceutical preparations by atomic-absorption spectroscopy. J. Pharm. Eefg. 25, 3-25 (1970); Anal. Abstr. 20, 1272 (1971).
DETEKXIISAI-ION
OF R,.’
483
10. Diaz, F. J., Determination of cyanocobalamin by atomic-absorption spectrophotometry with a pre-mix air-acetylene flame. Am/. Chin?. AC/U 58, 455-458 (1972). 11. Charlot, G., “Calorimetric Determination of Elements.” p. 235. Elsevier, New York, 1964. 12. Vasilikiotis, G. S., Kouimtzis, Th. A., Apostolopoulou, C. and Voulgaropoulos, A.. Spectrophotometric determination of cobalt(H) with 2,2’-dipyridyl-2-pyridylhydrazone. Ancrl. Chim. Acta 70, 319-326 (1974).
22, 479 -483 (1977)
Spectrophotometric Cyanocobalamin G. S. Laboratory
TH.
VASILIKIOTIS,
A.
Determination (as Cobalt) A.
KOUIMTZIS,
of
AND
VOULGAROPOULOS
of Analytical
Chemistry, University Thessaioniki, Greece
Received
of Thessaloniki,
May 5. 1977
INTRODUCTION The determination of cyanocobalamin in pharmaceutical preparations is usually done either directly by measuring the absorbance of its water solutions at 361 nm (1,2) or indirectly by a method which involves the determination of cobalt (on a weight percentage basis, cobalt being 4.35% of the total Bi2 molecule). The direct method is applied to pharmaceutical preparations that give clear water solutions. In the presence of other compounds that interfere with its determination, there have been suggested various methods for the separation of cyanocobalamin prior to its determination (3,4). For indirect determination various methods have been proposed. They are based on the decomposition of the samples and the subsequent determination of cobalt by spectrophotometric (5-7) and other methods such as isotopic dilution (8) and A.A.S. (9,10). The last method can also be used without decomposition of the samples by direct aspiration of their water solution into the appropriate flame. In this case, the absorbance by cobalt into the flame could be affected by the presence of various organic compounds which enhance the absorption (9). The spectrophotometric determination of cobalt after the decomposition of the sample is usually done by use of a reagent such as nitroso-R salts (5)) I-benzoyl-4-phenylthio-semicarbazidlosung (61, or fast navy 2R (7). With these reagents and the presence of other metal cations, interference in some extension was experienced in the determination of cobalt (7,111.
In a recent paper (12) a new specific and very sensitive spectrophotometric method for cobalt determination was proposed. The method is based on the formation of a complex between cobalt and 2,2’-dipyridyl-2pyridylhydrazone (DPPH). This reagent also forms colored complexes with a number of metal cations in solutions having pH>3. Addition of a strong acid (H$O,, HCIO,) to solutions of these complexes results in the decomposition of all complexes except that of cobalt, which only changes its color from orange-yellow to pink. At 25% perchloric acid the molar 479 Copyright @ 1977 by Academic Press, Inc
ISSN 0026-265X
480
VASILIKIOTIS,
KOUIMTZIS,
AND
VOULGAROPOULOS
absorptivity of the cobalt complex is 42,000 liter mol-’ at 500 nm. At this acidity, the presence of other metal cations does not interfere with the cobalt determination. The reagent itself does not absorb at 500 nm and this offers a considerable advantage over the group of nitrosonaphtholes and nitroso-R-salts. In the work presented here, the application of the DPPH method for the indirect determination of cyanocobalamin in pharmaceutical preparation was studied. EXPERIMENTAL Reagents
2,2’-Dipyridyl-2-pyridylhydrazone (DPPH) was prepared as described previously (12). Solutions of DPPH were prepared by dissolving the required weight in ethanol. The ethanolic solution of DPPH is stable and could be kept for several weeks in an amber glass bottle. Standard solutions of cobalt(II) were prepared by dissolving the appropriate amount of cobaltous chloride hexahydrate in distilled water. The solutions obtained were standardized by EDTA titration. Cyanocobalamin (Merck) was dried for 4 hr over silica gel at 50°C before its use. Various amounts of cyanocobalamin were accurately weighed and transferred to a 50-ml beaker for the wet ashing. A standard solution of cyanocobalamin containing 4.35 ppm Co was prepared by dissolving 100 mg of cyanocobalamin in 1000 ml of distilled water. These solutions were tested either by the recommended procedure or by atomic absorption spectroscopy. All other reagents used were of analytical grade. Apparatus
Absorbance measurements were made in lo-mm quartz cells with a Zeiss M4QII spectrophotometer. A Perkin-Elmer Model 403 atomic absorption spectrophotometer equipped with an air-acetylene burner head was also used in the experimental work. Recommended
Procedure
The appropriate amount of the pharmaceutical preparation (so as to contain 7 to 100 pg of cobalt) is weighed and transferred to a 50-ml beaker. Then 5 to 20 ml of concentrated sulfuric acid and about 0.2 g of potassium perchlorate are added. The solution is heated slowly with constant swirling of the beaker and is then boiled. If the hot digest is not colorless, a few crystals of potassium perchlorate are added, and the solution is boiled again. Finally the bulk of sulfuric acid is volatilized. At this stage all organic matter should have been completely decomposed. After cooling the digest, about 10 ml of distilled water are added followed by a concentrated solution of ammonium hydroxide until the pH exceeds 5. The solution is transferred to a 50-ml volumetric flask and 2 ml of 10m2M
DETERMINATION
481
OF RI2
TABLE I CoB.4L.r DE~ERMISATION IS CYANOCORLAMIS B,, taken
SL~SDARDS Cobalt found
Cobalt calculated
(mg)
(I*g)
(/a)
0.200 0.400 0.800 1.384 1.809 2.191
8.7 14.4 34.8 60.2 78.7 9.5.3
7.9 IS.8 32.4 60.8 76.5 94.4
ethanolic solution of DPPH are added. Then 10 ml of concentrated sulfuric acid are added and the flask is filled with distilled water. The absorbance of the resulting solution is measured at 500 nm and compared with the calibration curve which is obtained using standard solutions of cobalt. In the case of injectable pharmaceutical preparations, an appropriate volume is transferred into the beaker, boiled nearly to dryness, and then treated as described above. RESULTS AND DISCUSSION
The method developed has been applied to the determination of cobalt in cyanocobalamin standard samples. The results obtained are summarized in Table 1. For the first three samples listed in the table the appropriate volumes of the standard cyanocobalamin solution were taken and transferred to the beaker for the wet ashing. The other three samples were taken by weighing cyanocobalamin. The results show that the recovery of cobalt from the wet ashing is complete. The method has also been applied to the determination of cyanocobalamin (as cobalt) in two pharmaceutical preparations coded as A tablets and B injections. The analysis of these two preparations has also been performed by A.A.S. The results obtained are summarized in Table 2. TABLE 2 CYANOCOBALAMIN DETERMINATIOS (AS COBALT) IN PHARMACEVTICAL PRwAu.rtoNs Cobalt found (ppm)” Sample
Cobalt calculated
(ppm)
Present method
A.A.S
A 1251pg B,Jtablet
17.6
16.2 t 1.2
16.5 t 1.4b
B I mg B,,/ml
43.5
42.4 k 2.3
45.7 T 1.8r
ClFive determinations * After wet ashing. c Direct aspiration.
482
VASILIKIOTIS,
KOUIMTZIS,
AND
VOULGAROPOULOS
These results confirm the recovery of cobalt and the precision of the method developed. The procedure described is simple to use for routine testing in pharmaceutical preparations, mainly when these products are not dissolved in water or the solutions obtained produce cloudiness. It offers advantages over the other reagents used for this purpose because of the lack of interferences. Since the present method determines the total cobalt present, the presence of cobalt compounds other than cyanocobalamin will give higher values. This is applied to all methods that determine cyanocobalamin as cobalt. In this case, ionic cobalt, if present, could be extracted into a CHCl, solution of 8-hydroxyquinoline prior to the wet ashing (9). The method developed could also be applied to the determination of cobalt in various biological materials or food after the appropriate decomposition of the samples. SUMMARY A spectrophotometric method for the determination of cyanocobalamin (as cobalt) in pharmaceutical preparations has been developed. The sample is first decomposed with sulfuric acid and potassium perchlorate. The liberated cobalt is then determined using 2,2’dipyridyl-2-pyridylhydrazone as reagent in solutions containing 20% sulfuric acid. The presence of other metal cations does not interfere with the determination of cobalt. The method has been applied to the determination of cobalt in pure cyanocobalamin and in two pharmaceutical preparations.
REFERENCES 1. Bayer, J., Spectrophotometric examination of cyano- and hydroxocobalamines. Pharmazie 19, 602-605 (1964). 2. Bruening, C., Hall, W., and Kline, O., Rapid determination of the relative purity of vitamin B,, (cyanocobalamin) in pharmaceutical products. J. Amer. Pharma. Assoc. Sci. Ed. 47, 15-20 (1958); Anal. Abstr. 5, 3124 (1958). S. Tortolani, G., Bianchini, P., and Mantovani, V., Separation and determination of cobalamins on an SP-Sephadex column. J. Chromatogr. 53, 577-579 (1970). 4. Marini-Scotti, M., Determination of vitamin B,, and uridine-5’-triphosphate associated in lyophilised preparations. Farmaco Ed. Praf. 18, 332-334 (1963); Anal. Absrr. 11, 4509 (1964). 5. Monnier, D., Chaliounghi, V., and Saba, R., Determination of traces of vitamin B,,. Anal. Chim. Acta 28, 30-40 (1963). 6. Craciuneanu, R., and Florean. E., Photometric procedure for determining cyanocobalamin [as cobalt]. Pharmazie 24, 462-464 (1969). 7. El Raheem, Abd, and Dokhama, M., Calorimetric assay of cobalt and vitamin B,, with fast navy 2R. Z. Anal. Chew. 189, 389-396 (1962). 8. Rosenblum, C., Analytical application of radioactive vitamin B,,. Talanra 11, 255-270 (1964). 9. Mandrou, B., and Bres, J., Determination of vitamin B,, [cyanocobalamin] in some pharmaceutical preparations by atomic-absorption spectroscopy. J. Pharm. Eefg. 25, 3-25 (1970); Anal. Abstr. 20, 1272 (1971).
DETEKXIISAI-ION
OF R,.’
483
10. Diaz, F. J., Determination of cyanocobalamin by atomic-absorption spectrophotometry with a pre-mix air-acetylene flame. Am/. Chin?. AC/U 58, 455-458 (1972). 11. Charlot, G., “Calorimetric Determination of Elements.” p. 235. Elsevier, New York, 1964. 12. Vasilikiotis, G. S., Kouimtzis, Th. A., Apostolopoulou, C. and Voulgaropoulos, A.. Spectrophotometric determination of cobalt(H) with 2,2’-dipyridyl-2-pyridylhydrazone. Ancrl. Chim. Acta 70, 319-326 (1974).