DEFINITION
Vitamins B and C Injection is a sterile solution of Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin Sodium Phosphate, Nicotinamide and Ascorbic Acid (as the sodium salt) in Water for Injections, containing Glucose for the intravenous injection or Benzyl Alcohol for the intramuscular injection.
Vitamins B and C Injection is prepared immediately before use by mixing the contents of two ampoules, (1) and (2). Ampoule (1) contains Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin Sodium Phosphate and, where appropriate, Benzyl Alcohol. Ampoule (2) contains Nicotinamide and Ascorbic Acid and, where appropriate, Glucose. The air in ampoule (2) is replaced by nitrogen or other suitable inert gas.
The injection complies with the requirements stated under Parenteral Preparations and the contents of ampoules (1) and (2) comply with the following requirements, as appropriate.
Content of thiamine hydrochloride, C12H17ClN4OS,HCl
92.0 to 106.0% of the stated amount.
Content of pyridoxine hydrochloride, C8H11NO3,HCl
90.0 to 110.0% of the stated amount.
Content of riboflavin, C17H20N4O6
90.0 to 110.0% of the stated amount.
Content of nicotinamide, C6H6N2O
95.0 to 105.0% of the stated amount.
Content of ascorbic acid, C6H8O6
95.0 to 105.0% of the stated amount.
Content of glucose, C6H12O6
Where appropriate, 90.0 to 110.0% of the stated amount.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and a mixture of 60 volumes of methanol and 40 volumes of a solution containing 0.14% w/v of potassium dihydrogen orthophosphate and 0.5% w/v of disodium edetate as the mobile phase. Apply separately to the plate 2 μL of each of the following solutions in water. For solution (1) use a volume of the contents of ampoule (1) diluted, if necessary, with water to contain 0.2% w/v of Pyridoxine Hydrochloride. For solution (2) use a volume of the injection in ampoule (2) diluted, if necessary, with water to contain 2% w/v of Ascorbic Acid. Solutions (3) and (4) contain 1% w/v and 0.4% w/v respectively of thiamine mononitrate BPCRS. Solution (5) contains 0.2% w/v of pyridoxine hydrochloride BPCRS. Solution (6) contains 0.022% w/v of riboflavin sodium phosphate BPCRS. Solution (7) contains 0.64% w/v of nicotinamide BPCRS. Solution (8) contains 2% w/v of ascorbic acid BPCRS. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm and 365 nm). The principal spots in the chromatogram obtained with solution (1) correspond to those in the chromatograms obtained with either solutions (3), (5) and (6) or solutions (4), (5) and (6). The principal spots in the chromatogram obtained with solution (2) correspond to those in the chromatograms obtained with solutions (7) and (8).
B. For injections containing Glucose, heat 1 mL of the contents of ampoule (2) with cupri-tartaric solution R1. A copious precipitate of copper(I) oxide is produced.
ASSAY
For thiamine hydrochloride, pyridoxine hydrochloride and nicotinamide
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in water. Solution (1) contains 0.01% w/v of thiamine mononitrate BPCRS and 0.002% w/v of pyridoxine hydrochloride BPCRS. Solution (2) contains 0.01% w/v of thiamine mononitrate BPCRS and 0.005% w/v of pyridoxine hydrochloride BPCRS. Solution (3) contains 0.016% w/v of nicotinamide BPCRS. For solution (4) dilute a suitable volume of the contents of ampoule (1) with water to contain 0.01% w/v of Thiamine Hydrochloride. For solution (5) dilute a suitable volume of the contents of ampoule (2) to
contain 0.016% w/v of Nicotinamide.
The chromatographic procedure may be carried out using (a) a stainless steel column (30 cm × 3.9 mm) packed with end- capped octadecylsilyl silica gel for chromatography (10 μm) (μBondapak C18 is suitable), (b) as the mobile phase with a flow rate of 1 mL per minute, 0.22% w/v of sodium heptanesulfonate in a mixture of 75 volumes of a 1.36% w/v solution of potassium dihydrogen orthophosphate and 25 volumes of methanol, the pH of the final mixture being adjusted to 3.0 with orthophosphoric acid, and (c) a detection wavelength of 280 nm.
Calculate the content of C12H17ClN4OS,HCl using the declared content of C12H17ClN4OS in thiamine mononitrate BPCRS. Each mg of C12H17ClN4OS is equivalent to 1.030 mg of C12H17ClN4OS,HCl. Calculate the content of C8H11NO3,HCl using the declared content of C8H11NO3,HCl in pyridoxine hydrochloride BPCRS. Calculate the content of C6H6N2O using the declared content of C6H6N2O in nicotinamide BPCRS.
For riboflavin
Dilute a volume of the contents of ampoule (1) containing the equivalent of 4 mg of riboflavine with sufficient phthalate buffer pH 4.0 to produce 200 mL and measure the absorbance of the resulting solution at the maximum at 446 nm, Appendix II B. Calculate the content of C17H20N4O6 taking 323 as the value of A(1%, 1 cm) at the maximum at 446 nm.
For ascorbic acid
Dilute a volume of the contents of ampoule (2) with water to produce a solution containing 1% w/v of Ascorbic Acid. To 20 mL of the resulting solution add 5 mL of 1M sulfuric acid and 50 mL of 0.05M iodine VS and titrate the excess iodine with 0.1M sodium thiosulfate VS using starch mucilage as indicator. Each mL of 0.05M iodine VS is equivalent to 8.806 mg of C6H8O6.
For glucose
For injections containing Glucose, dilute a volume of the contents of ampoule (2) containing 0.8 g of Glucose to 500 mL with water and dilute 2 mL of the resulting solution to 100 mL with water. Transfer 3 mL of this solution to a boiling tube previously cleaned with chromic-sulfuric acid mixture and rinsed with water. In two similar tubes place separately 3 mL of glucose standard solution and 3 mL of water. Add 6 mL of anthrone reagent in such a manner as to ensure rapid mixing, allow to stand for 10 minutes, cool quickly and measure the absorbance, Appendix II B, of the test solution and of the standard solution at the maximum at 625 nm using the solution prepared with water in the reference cell. Calculate the concentration of C6H12O6 in the test solution.
STORAGE
Ampoules (1) and (2) for Vitamins B and C Injection should be protected from light.
LABELLING
For each ampoule the label states (1) the directions for the preparation of the injection; (2) whether the final injection is for intravenous or intramuscular use; (3) that the final injection is a high potency injection.
For ampoule (1) the label states the quantity of Riboflavin Sodium Phosphate in terms of the equivalent amount of riboflavin.
