PRINCIPLES AND PROCEDURES INVOLVED IN PRINCIPLES AND PROCEDURES INVOLVED IN THE ANALYSIS OF VITAMINS IN THE ANALYSIS OF VITAMINS IN

PHARMACEUTICAL PREPARATIONS AND PHARMACEUTICAL PREPARATIONS AND DOSAGE FORMSDOSAGE FORMS.

Presented by:Presented by: PRADIPKUMAR.L.GHORI DEP.PHARMACEUTICSDEP.PHARMACEUTICS

M.M.C.P BELGAUMM.M.C.P BELGAUM

INTRODUCTION TO VITAMINS:

Vital amines/growth factors/accessory factors.

Any group of organic compounds required in small amount to perform specific biological functions for normal maintenance of optimum growth and health of organism.

Classification: Fat soluble vitamins: vit-A, vit-D, vit-E, vit-K. Water soluble vitamins: vit-C, B-complex,

vit-H.

Need for study: Essential nutritional factors and potent organic compounds

require great care in analytical control and their formulations.

Analytical procedures based on chemical, physical, biological and microbiological methods are used in the assay.

The methods of analysis of vitamins and its formulations should be based on biological response.

Chemical, physical and microbiological methods are only valid if they correlate directly or indirectly with biological activity.

Care should be exercised as some vitamins exhibit species specificity in that they are required in certain species of animals and not in others.

Vitamin-A

Vitamin-D

Thiamine

Niacin

Vitamin-C

*VITAMIN-A* Chemistry: Retinol (Alcoholic)

Retinal (Aldehydic form) Retinoic acid (Acidic form) -Carotene (Provitamin-A)

RC11

C12

R11-cis form

11-trans form

R -CH2OH, -CHO, -COOH.

FUNCTIONS :FUNCTIONS :RHODOPSIN CYCLE.MAINTAINS NORMAL INTEGRITY OF EPITHELIAL

TISSUE.SYNTHESIS OF GLYCO PROTEINS AND

MEVALONATE.CAROTENOIDS FUNCTIONS AS ANTI-OXIDANTS

AND REDUCES RISK OF CANCER.

DEFICIENCY:DEFICIENCY: NIGHT BLINDNESS, XEROPHTHALMIA, KERATOMALACIA, MAY ALSO AFFECT ON REPRODUCTION AND EPITHELIAL CELLS.

PROPERTIES:PROPERTIES:1) THE FREE VITAMIN A ALCOHOL IS READILY OXIDIZED BY ATMOSPHERIC OXYGEN OR OXIDIZING AGENT. 2) VERY SENSITIVE TO LIGHT.3) VITAMINS OCCUR IN NATURE MAINLY IN THE FORM OF ESTERS WHICH ARE CONSIDERABLY STABLE TOWARDS OXIDATION.

Three methods: Bioassay based on RAT or CHICKEN growth or on liver storage. Colorimetric method/ Carr-price method. UV-spectrophotometric method.

COLORIMETRIC METHOD: COLORIMETRIC METHOD: CARR-PRICE METHOD: Anhydrous Antimony Tri chloride (SbCl3) in chloroform

reacts with a dilute solution of vitamin A to form a transient blue color.

Reaction occurs between Antimony tri chloride and unsaturated side chain of vitamin A. The carotene, polyene acids and various other material present in natural oils produce same color.

The color rapidly reaches maximum intensity and just as rapidly it fades. The reading must be taken within 10-15seconds

Sb Cl3+Blue color ( max- 550nm)

DisadvantagesDisadvantages:: Vitamin A is a recemic mixture containing 1/4th of cis-

form and 3/4th of Trans form in oils. While the biologically active form is the Trans form.

The Carr-price reaction does not differentiate between the two isomers, so it is not a specific test for all-trans compounds.

It does not give a stable blue color.

R

Applications:

This test can be used as an identification test for vitamin A

The blue color method can be used in determining Vitamin A in agricultural feeds and in oleomargarines.

The method extensively used in biochemical work because of its great sensitivity.

METHOD2: MODIFICATION OF CARR-PRICE :

Formation of blue color by reaction with vitamin A and activated Glycerol-1,3-dichlorohydrin.

Activation of reagent may be accomplished by distillation of glycerol 1,3-dichlorohydrin in presence of antimony tri chloride.

Maximum absorbance at 550nm. Color is more stable than the color produced in Carr-price

reaction. The intensity of solution reaches 2min. and the color is

stable for additional 3min.

Disadvantages: Disadvantages: Not as sensitive compared to carr-price method.

R

CH2Cl

CHOH

CH2Cl

blue color 550nmSb Cl3

+

SPECTROPHOTOMETRIC METHOD:

Method 3: DIRECT SPECTROPHOTOMETRIC METHOD (IP-1996 method):

Saponified sample is extracted with ether. Ether is evaporated. Residue

is dissolved in isopropanol.

Measure the absorbance at about 300, 310, 325 and 334 nm.

Determine the wavelength of maximum absorption.

If the wavelength of maximum absorption lies between 323

and 327 nm and the absorbance at about 300 nm relive to

that at about 325 nm does not exceed 0.73, a corrected

absorbance is derived from the equation.

A325 (CORR.) = 6.815 A325 – 2.555 A310 – 4.260 A334

Calculate the potency of the sample from the expression.

Vitamin A potency in Units per g = A325 1%, 1 CM) X 1830

If the corrected absorbance lies within +/- 3.0% of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance.

If the wavelength of maximum absorption lies outside the range 323 to 327 nm, or if the relative absorbance at about 300 nm exceeds 0.73, the unsaponifiable fraction of the sample must be further purified by chromatography.

Method 4: CONVERSION OF VITAMIN A TO ANHYDRO VITAMIN A:

Vitamin A may be easily converted to Anhydro vitamin A (II) in anhydrous solvents in the presence of traces of mineral acids or strong acids.

Anhydro-vitamin A results in a considerable displacement of absorption towards visible region with absorption maxima at 358nm, 377nm and 399nm in benzene. There is also an increase in absorptivity.

R mineral/organic acid

anhydro vit A. 1,3-dihydro vitamin

Formula:

USP units /ml. aliquot = __ A___

0.0122 A Increase in absorbance caused by

dehydration.

0.0122 Increase in absorbance corresponding to 1USP unit of vitamin A

This method can be applied for the vit-A concentrates, fish oils, oleomargarine, butter etc. this has been reported to be very specific.

*VITAMIN D**VITAMIN D* Steroidal vitamin/ anti-ricketic vitamin. Two forms:

◦ Ergocalciferol (vitamin D2)

◦ Cholecalciferol (vitamin D3)

Functions:Regulates plasma levels of calcium and phosphate.In osteoblasts of bone, vit D stimulates uptake of calcium

and deposition of calcium phosphate.1, 25 di-hydroxy cholecalciferol is active form also known as

calcitrol. Diffiency:

Rickets in children and osteomalacia in adults.

Properties:

1) It is not affected by dilute acids, alkalis or air

2) Vitamin D3 is stored in the skin as 7-dehydro cholestrol which is converted to activated vitamin D2 by sunshine or ultraviolet irradiation.

Ergosterol vitamin D2. (Activated form)

7-dehydro cholestrol vitamin D3(Activated form).

3) Biological assay methods uses rats and chicks are available for evaluation of vitamin D activity.

4) Physiochemical procedures employ the methods of uv- spectrophotometry / colorimetry.

CH3

CH3CH3CH2

OH

CH3

Method 1: UV-SPECTROPHOTOMETRY:

Both D2 and D3 have an absorption maximum at 265nm in

hexane. The A1%1cm of D2 is 459nm that of D3 is 474.

The determination involving uv-spectrophotometry suffers from lack of specificity.

The interference of interfering substances can be avoided by chromatographic separation.

TWO STEP ADSORPTION CHROMATOGRAPHIC METHOD: Passage of oil through an activated earth this removes vitamin A,

sterols and irradiation products of ergosterol other than Vitamin D.

The second adsorption step removes impurities due to first adsorbent, unsaturated compounds of squalene type and vitamin A decomposition products.

The eluate is collected and analyzed spectroscopically.

COLORIMETRIC METHOD Many colorimetric methods have been proposed for

the quantitative determination of vitamin D.  “MODIFIED CARR-PRICE METHOD”: The glycerol-1,3-dichloro hydrin reacts with vitamin

D producing blue color. The color is stable for several hours. The reaction is specific:

Ergosterol gives pink-orange color and slowly turns to a fluorescent green.

7-dehydro cholesterol produce no color but after hours produce faint pink color.

Cholesterol does not show reaction or absorption.

REACTION WITH ORGANIC ALDEHYDE: This is based on reaction between Vitamin D and

organic aldehyde (vanillin, furfural or anisaldehyde) and sulphuric acid or perchloric acid.

CH3

CH3CH3CH2

OH

CH3 CHO

OMe

OH

H2SO4/HCLO4 Green Color (625nm)+

CH3

CH3CH3CH2

OH

CH3

H2SO4/HCLO4 Green Color (625nm)

OH

CHO

+

CH3

CH3CH3CH2

OH

CH3

O CHO

H2SO4/HCLO4 Green Color (625nm)+

MODIFIED CARR-PRICE METHOD: The modified method has improved sensitivity and

reproducibility by addition of acetyl chloride to SbCl3. Mixture of vitamin A and vitamin D2 is dissolved in

benzene after passing through a column of bentonite activated with acid.

The treatment selectively destroys vitamin A and the eluate is then analyzed using antimony trichloride (SbCl3) reaction.

 

CH3

CH3CH3CH2

OH

CH3

Green Color (625nm)CH3COClSb Cl3 ++

VITAMIN D CONTD…VITAMIN D CONTD…

Vitamin D gives color with glycerol-1,3- dichlorohydrin in presence of acetyl chloride

CH3

CH3CH3CH2

OH

CH3

Green Color (625nm)CH3COCl

CH2 Cl

CH OH

CH2Cl

++

*THIAMINE* Chemistry:Chemistry:

Contains pyridine and thiazole ring structures. Functions: Functions:

Prosthetic group in decarboxylation. and transketolase reactions.

Deficiency disorders: Deficiency disorders: Beri beri.

Properties:Properties:1) White crystalline powder with slight characteristic odour.2) It is hygroscopic.

3) Soluble in water, glycerol, alcohol. Practically insoluble in ether, benzene,

hexane, and chloroform

Chemical, biological and microbiological methods are available for the

analysis of thiamine.

◦ The bio-assay is based on prevention and cure of polyneuritis and

weight gain in animals. Microbiological methods using Phycomyces

blakesleeanus and Lactobacillus ermenti are available. A yeast

fermentation method is also available.

NN

N SCH3NH3+ CH2 CH2 O H

CH3

. 2 Cl

+

-2

FLOURIMETRIC METHOD (Thiochrome)

1. Thiamine is quantitatively isolated from the foods, biologicals, and pharmaceuticals usually by boiling with dilute acids and treatment with enzyme preparations containing phosphatases which will free thiamine from its natural complexes.

2. Protein substances must be digested with proteolytic enzyme like papain.

3. Purification of extract is done by passing through zeolite, an inorganic ion exchanger and thiamine is retained in the zeolite.

4. Add acidified potassium chloride to elute thiamine.

5. Further it is oxidized with alkaline potassium ferricyanide to produce thiachrome. This compound is having blue fluorescence.

NN

N SCH3NH3+ CH2 CH2 O H

CH3NN

N SCH3CH2 CH2 O H

CH3

NK3 Fe (C N)6

+

2 Cl-

SILICO TUNGSTIC ACID METHOD (Gravimetry): {H4Si(W3O10)4 nH2O }

Prepared by One mole of Silicic acid for every twelve moles of Sodium Tungstate.

Thiamine in tablets and solutions may be determined by precipitation with silicotungstic acid.

The sample is dissolved in acidified water and heated to boiling.

The precipitate is filtered washed with acid and water finally with acetone. Then its weighed to constant weight.

Each gram is equivalent to 0.1936 g of thiamine hydrochloride.

 

COLORIMETRIC METHOD: Method 1: with p-Amino acetophenone:

 

The extraction is done as given in thiochrome method.

Thiamine couples with diazotized p-Amino Acetophenone which has an absorption maximum at 520nm.

This method can be used to determine thiamine in presence of phosphorylated thiamine and is useful in urine analysis.

This method is not recommended for assay of materials rich in protein content and low in thiamine.

Results of this method agrees with the biological method.

NN

N SCH3NH3 CH2 CH2 O H

CH3

N

C O C H3

N H

complex

diazotised p-amino acetophenone

+520 nm

Method 2: using 6-Aminothymol.

 

A color reaction between thiamine and diazotized 6-Amino thymol is seen. This method is simple fast and no interference is seen between the degraded thiamine and 6-Aminothymol.

NN

N SCH3NH3 CH2 CH2 O H

CH3

OH

CH (CH3)2

CH3N NH

color complex+

Miscellaneous Methods:Miscellaneous Methods:

Method 3: Non-aqueous titration: Thiamine HCl can be titrated with perchloric acid in glacial acetic acid solution,

if an excess of mercuric acetate is added. Both the nitrogen are titrated. p-Naphthol-benzein and quinaldine red are suitable indicators.

Method 4: Argentometric Method:. Total chlorine in thiamine HCl can be determined by dissolving in acidified

water (HNO3 ) added with excess of silver nitrate. The precipitate is filtered and washed. The filtrate is then titrated with 0.1N Ammonium Thiocyanate.

1ml of 0.1N AgNO3 is equivalent to 0.003546 g of chlorine. 

Method 5: Chloride as hydrochloride is determined by titration with 0.1 N sodium

hydroxide to pH7 using bromothymol blue as indicator.Each ml of 0.1 N NaOH is equivalent to 0.003546 g of chlorine

 Method 6

The nitrate in thiamine mononitrate is determined by precipitation with nitron (1,4-Diphenyl-3-phenylamino-1,2,4-triazolium hydroxide inner salt) from an acidified solution.

Nitron is a compound C20H16N4 used in the qualitative and quantitative determination of nitric acid with which it forms an insoluble nitrate

*NIACIN (NICOTINIC ACID)*Chemistry: Chemistry:

◦ It’s a heterocyclic. 3-pyridine carboxylic acid.

Functions: Functions:

◦ It functions as oxidising co-enzymes of many dehydrogenasesDeffiency disordersDeffiency disorders:

◦ Pellagra, characterised by dementia, dermatitis and diarrhoea.

PROPERTIES:PROPERTIES:

1. Solubility: Soluble in boiling water and in boiling ethanol (95%). Sparingly soluble in water. Very slightly soluble in chloroform. Practically insoluble in ether. It dissolves in dilute solutions of alkali hydroxides and carbonates.

2. Non-hygroscopic and stable in air.

3. It has absorption maximum in UV at 262nm.

4. Identification test:

Thiamine is dissolved in water, and neutralized to litmus paper with 0.1M sodium hydroxide, add 3 ml of copper sulphate solution; a blue precipitate is gradually produced.

N

COOH

ACID BASE TITRATION: 

Nicotinic acid can be titrated with NaOH using phenolphthalein solution as indicator.

  COLORIMETRIC METHOD USING CYANOGEN

BROMIDE:

 

This is based on a color reaction of pyridine and unsubstituted derivatives. Cyanogen bromide breaks

one carbon-nitrogen linkage and produces a color compound upon addition of amine or ammonia.

N

COOH

NaOH

N

COONa

H2 O+

+

N

COOHSO3H

NH2

CNBrcleavage of CN bond of pyridine by CNBr red color measured spectroscopically+ +

DETERMINATION OF NICOTINAMIDE ACID-BASE TITRATION:

◦ Nicotinamide when boiled with sodium hydroxide solution, it releases the nitrogen of the amido group in the form of ammonia, which can be collected in sulphuric acid and determined by titration.

  

The liberated ammonia collected in sulphuric acid and determined by titration with NaOH.

2NH3 + H2SO4----------- (NH4)2

(NH4)2 + 2 NaOH----- Na2SO4 + 2 H2O + 2NH3

End- point being determined using phenolphthalein indicator.

N

CONH2

alkaline hydrolysis

N

COOH

NH3

Nicotinic acid

+

HOFFMAN REARRANGEMENT REACTION:

Nicotinamide undergoes a Hoffmann rearrangement to form a 3-amino pyridine which reacts as an aromatic amine.

It can be diazotized and coupled with N-(1-naphthyl)- ethylene di-amine to produce red colored azo compound.

This is the basis of USP assay of nicotinamide in capsules, injectables and tablets.

 

N

CONH2 hoffman -co

rearrangementN

NH2

3-Amino Pyridine

NaNO2/HClHNO2 <8*C

N N Cl

diazotised compound

NH-CH2-CH2-NH2

. HCl

N N NH-(CH2)2NH3

red colored azo-compound

++ +

VITAMIN C (ASCORBIC ACID) Chemistry:Chemistry:

◦ It’s a heterocyclic furan-2-one derivative

Functions:Functions:◦ Anti-oxidants, helps in synthesis of collagen and

hydroxylation reactions.Defiency disorders:Defiency disorders:

◦ Scurvy, swollen joints, hemorrhages in various tissues and delayed wound healing.

Properties:Properties:1. It occurs as a white or slightly yellow crystal or powder.2. In dry state it is stable to air but degradation seen in presence of

some metals and it is unstable to light.3. Many chemical methods have been reported and this is based on

the reducing properties of ascorbic acid.

OO

OHOH

CHOH

CH2OH

VOLUMETRIC METHODS Iodometric method: Ascorbic acid content of pure solutions or the purity of

the substance can be determined by titration with 0.1 N Iodine solution.

Modifications of this method using instruments like potentiometric titration or polarized platinum-platinum electrodes and a dead-stop end point, have been applied to pharmaceutical products.

O O

OH OH

HOHC

HOH2C

I2O O

O O

HOHC

HOH2C

2 HI+ +

Dehydro ascorbic acidAscorbic acid

2) Titration with 2,6-DICHLORO PHENOL-INDOPHENOL:

 

A typical procedure for eliminating interfering substances consists of

a) The conversion of total ascorbic acid to dehydro ascorbic acid by passing it through Norit or by using ascorbic acid oxidase.

b) The reduction of dehydro ascorbic acid to ascorbic acid with hydrogen sulphide at pH 4 to 7

c) The titration of ascorbic acid with dichloro phenol –indophenol.

OO

OHOH

CHOH

CH2OH

NaO N O

Cl

ClOO

OO

CHOH

CH2OH

2,6- dichloro phenol indo phenol (pink or blue color) dehydro ascorbic acid

NaO N

Cl

OH

Cl

H

reduced form (color less)

+ +

METHOD 3: Titration With N-BROMO SUCCINIMIDE:

N-Bromo succinimide in aqueous solution readily oxidizes an aqueous solution of ascorbic acid to dehydro ascorbic acid, while N-Bromo succinimide is irreversibly reduced to succinimide with the formation of Hydrogen Bromide.

After all the ascorbic acid has been oxidized, the slightest excess of N-Bromo succinimide, In presence of potassium Iodide, liberates iodine.

The end point is being determined using starch solution added in the end.

OO

OHOH

CHOH

CH2OH

CH2-CO

CH2CON Br

OO

OO

CHOH

CH2OH

CH2-CO

CH2CONH H Br+ + +

COLORIMETRIC METHODSMETHOD 1: Using 2,4-DINITRO PHENYL

HYDRAZINE:

Dehydro ascorbic acid couples with 2,4-dinitro phenyl hydrazine to form an osazone which develops a red color in strong sulphuric acid.

In this reaction, de-hydro ascorbic acid is used so the titrimetric impurities like –SH, etc. are degraded previously, so pure ascorbic acid react with DNP and quantized the exact result.

     

OO

OO

CHOH

CH2OH

NH-NH2

NO2

O2N

H2SO4

OO

NN

CHOH

CH2OH

N

HN NO2

O2N

NO2

NO2

osazone (pale brown to red color)

H

+

METHOD 2: Using 4-METHOXY-2-NITROANILINE: This is based on ability of ascorbic acid to couple with

Diazonium compounds.

Ascorbic acid with diazotized 4-methoxy-2-nitro aniline forms a deep blue compound.

This method has an advantage as it is specific for ascorbic acid.

This method has been applied to pharmaceutical preparations, natural juices, powdered milk and fortified feed.

 OO CHOH

OHOH

CH2OH

HNO2< 8*, NaNO2/HCl

N

NO2

OMe

N-Cl

OO CHOH

OH OH

CH2OH

Deep blue color complex

N

NO2

OMe

N-Cl

diazotised 4-methoxy 2-nitroaniline

+

RIBOFLAVIN (VITAMIN B2) It is also called as lactoflavin. Properties: Yellow to orange-yellow, crystalline powder with slight. Odour Deffiency: Solubility:

Very slightly soluble in water; more soluble in saline solution than

in water; practically insoluble in chloroform, in ethanol (95%)

and in ether.

Aqueous solutions exhibit an intense yellow color-green fluorescence at pH6.

Specific rotation is Between –115o and –135o, determined in a 0.5% w/v solution in carbonate-free 0.05M sodium hydroxide .

Riboflavin shows absorption maxima at 224, 267, 373, 445, and 475nm.

Irradiation either with uv or visible light, of alkaline solutions, produces luminoflavin, irradiation of acid or alkaline solutions produces luminochrome, a blue fluorescent substance.

Reducing agents such as, sodium hydrosulphite, reduce riboflavin to a dihydro compound, leucoflavin which is not fluorescent. But this is reversible and leucoflavin is readily oxidized back to riboflavin by atmospheric oxygen.

N

CH2

NN

NCH3

CH3

O

O

CH

OH

C HOH

C HOH

CH2OH

METHOD 1: FLUORIMETRIC METHOD

There are three methods are in use in fluorimetric determination of riboflavin;

Direct determination Direct additive determination Adsorptive additive determination

FLUORIMETRIC METHOD: DIRECT DETERMINATION:

This method is employed for the mixtures which are free of interfering pigments or substances and contain relatively high concentration of riboflavin.

An appropriate quantity is weighed and add with boiling distilled water and shaken for few minutes, if required, boil the solution. It is then centrifuged.

Sample solution: a suitable aliquot of clear liquid is diluted appropriately to yield concentration of 0.2mcg/ml.

Blank solution: add a few granules of sodium hydrosulphite to the aliquot of sample solution. This shows indication of purity of solution.

Measurement and calculation: Readings must be taken as rapidly as possible.

Mcg of riboflavin = A – C * dilution factor

B – C wt of sample (g.)

A reading of unknown concentration.

C unknown blank

B reading of standard

D reading of standard blank.

FLUORIMETRIC METHOD: DIRECT ADDITIVE METHOD

The interference of other substances can be avoided by this method. In this method the addition of the known quantity of riboflavin to the assay solution is used to compensate for interfering substance which may absorb the incident or fluorescent light.

Sample treatment: samples of natural origin should be subjected for enzymatic hydrolysis to digest starchy substances and to “free” any “bound” riboflavin.

Standard solution I: 40mcg/ml of USP reference standard in 20% of ethanol.

Standard solution II: dilute solution I such that it contains 1.6mcg of riboflavinin distilled water.

Measurement and calculation:

Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factor

B- (0.94A) 16 wt of sample (g)

A fluorescence of sample solution

B fluorescence of standard II

C blank solution ( obtained by adding HYDROGEN SULPHITE to standard solution II and this should be repeated until successive additions shows no deflections)

1.07 and 0.94 constants due to change in volume of measurement.

 

USP method: in this an additional purification step is involved by treating with potassium permanganate and this produces oxidation of interfering substances. Excess permanganate is removed by treating with hydrogen peroxide.

Applications: This method can be used with vitamin mixtures of

wafers, flour enrichment mixtures and simple pharmaceutical preparations, provided they are substantially free from coloring or fluorescencing matter.

FLUORIMETRIC METHOD: ADSORPTIVE ADDITIVE DETERMINATION:

 In this method most of the interfering substances are eliminated by an adsorption step and those not eliminated are compensated by addition of a known quantity of riboflavin to the assay solution. . this method can be applied to universally to all samples.

Extraction: the extraction procedure is same as the direct additive determination.

Adsorption and elution:

An aliquot of sample solution or suitable dilution of the sample extract is measured accurately and passed through an adsorption column. The column is prepared by using fluorosil. After elution the column is washed with hot distilled water and the excess water is drained using vacuum.

The riboflavin is being retained in the column is then eluted using hot acetic acid-pyridine eluent is collected and mixed well.

FLUORIMETRIC METHOD: ADSORPTIVE ADDITIVE DETERMINATION:

Standard solution I: 40mcg/ml of usp reference standard in 20% of ethanol.

Standard solution III: dilute solution I such that it contains 1.6mcg of riboflavinin in acetic acid pyridine mixture.

  Measurement and calculation:

Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factor

B- (0.94A) 16 wt of sample (g)

A fluorescence of sample solution

B fluorescence of standard III

C blank solution ( obtained by adding hydrogen sulphite to standard solution II and this should be repeated until successive additions shows no deflections)

1.07 and 0.94 constants due to change in volume of measurement.

METHOD 2: SPECTROPHOTOMETRIC METHOD (IP 1996):

Procedure: Carry out the procedure in subdued light. Weigh accurately about 65 mg and transfer to an amber-glass 500-

ml volumetric flask, suspend in 5 ml of water, ensuring that it is completely wetted.

Dissolve in 5 ml of 2M sodium hydroxide. As soon as dissolution is complete add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water.

To 20.0 ml of this solution add 3.5 ml of a 1.4% w/v solution of sodium acetate and dilute to 200.0 ml with water.

Measure the absorbance of the resulting solution at the maximum at about 444 nm.

Calculate the content of C17H20N4O6 taking 328 as the value of A(1%, 1 cm) at the maximum at about 444 nm.

METHOD 2:

Riboflavin has a characteristic absorption spectrum in water with a maximum at 267nm. This is the basis of the method. This method is based on the assumption that extraction with chloroform will remove impurities from an aqueous solution.

Procedure: Carry out in subdued light. Weighed about 20mg of riboflavin transferred to a 1000ml volumetric

flask, dilute the solution and add few drops of 1N NaOH. Shake gently until the solution is complete, then add few drops of 5N

acetic acid and dilute upto the mark. A 20ml of aliquot of solution is taken and shaken with 25ml of chloroform

for 1min. separate the chloroform layer and discard. The extraction is repeated twice.

METHOD 2 CONTD…

The absorbance of clear aqueous layer is determined at 267nm.with water as a reference.

Repeat the procedure with reference sample.

% Riboflavin= 100 (As/Ar) . (Wr/Ws) Ws and As weight in mg and absorbance

of sample respectively. Wr and Ar corresponding values of

standard and reference sample of riboflavin.

REFERENCES:

Pharmaceutical analysis by Takeru Higuchi et.al. page no. 649-707.

Bentley and Drivers textbook of pharmaceutical chemistry. 8th edition.

Vogel’s textbook of quantitative chemical analysis.

IP-1996.

Instrumental methods of chemical analysis by G R Chatwal and Sham K

Anand. Enlarged edition. 2005. p no. 2.413-2.414

Guyton textbook of medical physiology. 11th edition.

Biochemistry by Leninger 4th edition 2005.

Biochemistry By Jeremy M Berg, John L Tymoczko And Lubert Stryler. 5th

Edition

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