**Determination of Vitamin B12 by UV-Vis Spectrophotometry**
Vitamin B12, also known as cyanocobalamin, is a cobalt-containing organic compound that appears as dark red crystals. It is the only vitamin containing a major mineral and is commonly referred to as the "red vitamin." The traditional method for determining the content of vitamin B12 in tablets involves using a UV-Vis spectrophotometer with the absorption coefficient method. However, this approach requires high precision from the instrument. In contrast, the standard curve method is one of the most widely used techniques in UV-Vis spectroscopy. When the instrument is calibrated and its operating conditions are fixed, this method can reduce some errors caused by instrumental variations, making it simple and efficient.
**Instrument and Reagents**
The experiment was conducted using a UV-1800PC UV-Vis spectrophotometer (China Instrument Co., Ltd.). The reference substance for vitamin B12 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products. The test samples were vitamin B12 tablets (Hengruida Pharmaceutical Co., Ltd.) with batch numbers 060801, 060701, 061002, 060902, and 070303. A 0.8 μm filter (Automatic Science (Tianjin) Instrument Co., Ltd.) was used for filtration.
**Methods and Results**
**Wavelength Selection:** The absorption spectrum of vitamin B12 shows three peaks at 278 nm, 361 nm, and 550 nm. The peak at 361 nm has minimal interference and the strongest absorption. According to the Chinese Pharmacopoeia, the specific absorption coefficient (E1%1 cm = 207) at 361 nm is used for calculation. In this study, 361 nm was selected as the measurement wavelength.
**Preparation of Solutions:**
- **Reference Solution:** Accurately weighed 0.0025 g of vitamin B12 reference substance and dissolved it in 25 mL of water. The solution was stored in a refrigerator for later use.
- **Test Solution:** Fifty sugar-coated tablets were de-coated, ground into a fine powder, and accurately weighed to obtain approximately 1.25 mg of vitamin B12. The sample was placed in a 50 mL volumetric flask, 35 mL of water was added, and the mixture was shaken for 30 minutes. After diluting to the mark, the solution was shaken vigorously for 10 minutes, allowed to settle, and filtered through a 0.8 μm membrane. The filtrate was stored in a refrigerator.
**Standard Curve Preparation:**
The reference solution was diluted to concentrations of 1.5, 2, 4, 6, 8, 10, 15, and 20 times. The absorbance at 361 nm was measured. A linear regression equation was established: Y = 0.0193X + 0.048, with a correlation coefficient of r = 0.9937. This indicated a good linear relationship between absorbance and concentration in the range of 5.00–100.00 μg/mL.
**Precision Test:**
A 100 μg/mL reference solution was measured five times at 361 nm. The average absorbance was 2.087, with an RSD of 0.7%. The low RSD value confirmed the method’s high precision.
**Stability Test:**
The same test solution was stored at 4°C for 2, 4, 8, 12, and 24 hours. The average absorbance was 0.468, with an RSD of 0.99%. The results showed that the solution remained stable within 24 hours under refrigeration.
**Repeatability Test:**
Five test solutions were prepared from the same batch of tablets (batch number 070303). The average vitamin B12 content was 22.6 μg/mL, with an RSD of 1.08%. This demonstrated the method's good repeatability.
**Content Determination:**
Using water as a blank, the filtrate was analyzed at 361 nm. The content was calculated using the standard curve method. The labeled amount of vitamin B12 per tablet was 25.0 μg. The measured content ranged from 90.0% to 110.0%, indicating that the product meets quality standards and is consistent across batches.
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