Accelerated Degradation Experiment
Conditions
Procedure A: |
10, 20 then 30 days exposure at 50 °C and 100% relative humidity. |
Procedure B: |
30 days exposure at 50 °C and 100% relative humidity followed by 7 days at 70 °C and 100% relative humidity. |
Appearance of the substance
The substance, originally in the form of dark red crystals or a red, crystalline powder, changes after procedures A and B into a dark red compact mass.
Gain of moisture
Before exposure to heat and humidity: |
18% |
After procedure A: |
27% |
After procedure B: |
43% |
Chromatographic test
Adsorbent: Silica gel F-254
Solution applied to the plate: Substance dissolved in a mixture of ethane 95%-water (1:1).
Solvent system:
n-butanol ........................... 50 volumes
ethane 95%........................ 25 volumes
concentrated ammonia ....... 10 volumes
water .................................. 25 volumes
Development conditions: The solvent is allowed to migrate until the front reaches a line 15 cm from the starting line.
Detection: Direct observation of the spots in daylight.
Results: A reference solution of hydroxocobalamin shows a main spot and a very weak, secondary spot. A solution of the sample subjected to procedure A shows that the secondary spot increases, whereas the main spot decreases. After procedure B, the degradation represents more than 50% inversion of the size of the spots.
Ultraviolet spectrophotometry
The absorbances are measured at about 350 nm.
A solution of the samples subjected to the test shows a regular decrease in absorbance during the course of procedure A and after 30 days the degradation reaches 50%. After procedure B a similar result is obtained.
Conclusion
Hydroxocobalamin is subject to strong decomposition under accelerated degradation conditions.
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