section 11 -folic acid

Cyanocobalamin (vitamin B12) [USP]

Mol. formula C63H88CoN14O14P

M.W. 1355 Pharm. Use Treatment of vitamin B12 deficiency. Formulation Cyanocobalamin Tablets

Definition: Cyanocobalamin contains NLT 96.0% and NMT 100.5% of C63H88CoN14O14P,

calculated on the dried basis.

Identification:

Dissolve 100 μg of cyanocobalamin in 5 mL of water in a 50-mL distilling flask connected

with a short, water-cooled condenser. To the flask add 2.5 mL of hypophosphorous acid,

close, boil gently but short of distillation for 10 min, and then distill 1 mL into a test tube

containing 1 mL of 20 mg/mL sodium hydroxide solution. To the test tube add 4 drops of

cold saturated ferrous ammonium sulfate solution, shake gently, then add 30 mg of sodium

fluoride, and bring the contents to a boil. Immediately add, dropwise, 5 N sulfuric acid until

a clear solution results, then add 3–5 drops more of the acid.

Acceptance criteria: A blue or blue-green color develops within a few min.

Assay:

Procedure:

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Standard solution: 30 μg/mL of USP cyanocobalamin in water

Sample solution: 30 μg/mL of cyanocobalamin in water

Mode: spectrophotometer, Analytical wave length: 361 nm

Cell: 1 cm, Blank: water

Analysis: determine the absorbance of standard solution and sample solution at the wave

length of maximum absorbance and then calculate the percentage of the C63H88CoN14O14P In

the portion of cyanocobalamin taken.

Results = (At/ As) x (Cs/ Ct) x 100

At = absorbance of the sample solution

At = absorbance of the standard solution

Cs = concentration of the USP cyanocobalamin RS in the standard solution (μg/mL); A (1%,

1cm) equal to 207 at 361 nm.

Ct = concentration of the cyanocobalamin in the sample solution (μg/mL)

Acceptance criteria: 96-100.5%

Analytical problems A cyanocobalamin injection contain labeled value of 1000 μg/ml was dissolves in 5 ml

water, 1 ml of this solution was diluted in volumetric flask to 250 ml, if the absorbance of

this solution at 361 nm (A) = 0.01606, taking 207 as the value of A (1%, 1cm) Calculate

1. The intact amount of cyanocobalamin

2. The recovery %



Folic acid powder

N

NHN

N HN

HN COOH

COOHO

O

H2N

PTERIDINEBENZOYL L(+)-GLUTAMIC ACID

4[(2-Amino-4-hydroxy-pteridin-6-yl)methylamino]benzoyl-L-(+)-glutamic acid Mol. formula C19H19N7O6

M.W. 441.1396 g/mol

Pharm. Use Treatment of vitamin B12 deficiency. Formulation Cyanocobalamin Tablets

Activity:

1- For 1C fragment transfer.

2- Its deficiency stop metaphase during cell division thus inhibits cell division.

3- Necessary for normal development of the RBCs, its deficiency causes megaloplastic

anemia (prevent transfer of megaloplast into normal plast).

4- Important during pregnancy

5- Folic acid and heart disease:

6- Folic acid and cancer:

Assay principles: Colorimetric assay or purity indicating assay

Ø 0.1 M NaOH to solubilize the powder giving Na folate.

Ø Zn / HCl produce nascent hydrogen which causes a reductive cleavage of the

methylamino group.



N

NHN

CH3H2N

HN COOH

COOHO

O

H2N

FREE AMINO

+

The amino group of the pteridine group is a typical aromatic group so no diazotization will

occur (the lone pair of electrons are involved in tautomerism (amino ↔ imino).

N

NHN

CH3O

H2N

N

NHN

CH3

HN

OH Diazotization takes place only for the amino group of the p-amino benzoic acid

COH2N CONNCl

NaNO2 + HCl HNO2 + NaCl

+

diazonium salt Ammonium sulfamate: Remove the excess of HNO2 (brown colour, ---)

HNO2 + NH4SO3NH2 N2 + NH4HSO4 + H2O

Brotton B Marcheal reagent:

N-(1-naphthyl ethylene diamine) dihydrochloride

CONNCl

NHCH2CH2NH2 NHCH2CH2NH2

N N CO

+

2 HCl

+

2 HCl

Violet color (550) Why assay for unreduced and why impurity indicating assay During the preparation of folic acid, hydrolysis into p-amniobenzoic acid or p-amniobenzoic acid derivatives may occur, so impurity can be diazotized and give wrong



results. So we do colorimetric assay for the unreduced tablet to determine impurity and to another tablet after reductive cleavage these assay is called impurity indicating assay. Procedure: -In 100 ml measuring flask, dissolve a quantity equivalent to 50 mg of folic acid in 50 ml of 0.1 M NaOH and complete the volume with 0.1 M NaOH (solution a). -Take 3 ml + 20 ml HCl and complete to 100 ml with water. -In a beaker, take 50 ml + 0.5 gm Zn powder, shake frequently for 20 minutes away from light, filter, discard the first 10 ml -In 100 ml measuring flask, take 20 ml, complete with water to 50 ml + 10 ml dil HCl + 5 ml NaNO2 solution, mix for 2 minutes + 5 ml ammonium sulfamate solution, mix for 2 minutes + 5 ml Brotton-Marcheal reagent, mix for 10 minutes and complete for 100 ml with distilled water. -Measure the At reduced at 550 nm. Unreduced: -Take 30 ml of solution (a) + 20 ml dil HCl complete with water to 100 ml. -Take 20 ml and complete to 50 ml with water + 5 ml HCl + 5 ml NaNO2 solution and complete as above. Authentic solution

Reference (blank): Mix 25 ml H2O + 10 ml dil HCl + 5 ml NaNO2 and complete as above

A fo lic a cid (te st) = A t red -A T u n re d

10

A fo lic ac id (std ) = A s red -A T u n red

10

Test and authentic solution are treated by the same dilution so the dilution factor is cancelled. Completely reduced standard

0.1M NaOH 2 liters [Dissolve 8 g in 2 liters distilled water] Dil HCl 2 ---------------2 liters [350 ml conc HCl complete to 2 liters with distilled water] Sodium nitrite R ------------ 2 liters [1 g / liter[



Ammonium sulphamate R ------------- 2 liters [5 g / liter] Brotton B Marcheal reagent R1g of naphthyl ethylene diamide dihydrochloride R / liter]



section 11 -folic acid

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