1. PRINCIPLES AND PROCEDURES INVOLVEDPRINCIPLES AND PROCEDURES INVOLVED
IN THE ANALYSIS OF VITAMINS ININ THE ANALYSIS OF VITAMINS IN
PHARMACEUTICAL PREPARATIONS ANDPHARMACEUTICAL PREPARATIONS AND
DOSAGE FORMSDOSAGE FORMS.
Presented by:Presented by:
PRADIPKUMAR.L.GHORI
DEP.PHARMACEUTICSDEP.PHARMACEUTICS
M.M.C.P BELGAUMM.M.C.P BELGAUM

2. 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.

3. 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.

4.  Vitamin-A
 Vitamin-D
 Thiamine
 Niacin
 Vitamin-C

5. *VITAMIN-A* Chemistry:
 Retinol (Alcoholic)
 Retinal (Aldehydic form)
 Retinoic acid (Acidic form)
 β-Carotene (Provitamin-A)
RC
11
C
12
R
11-cis form
11-trans form
R -CH2OH, -CHO, -COOH.

6. 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.

7.  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

8. Sb Cl3+
Blue color
(λ 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

9.  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.

10.  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 550nm
Sb Cl3
+

11. 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

12.  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.

13. 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

14.  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.

15. *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.

16.  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
CH3
CH3CH2
OH
CH3

17. 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.

18. 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.

19.  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
CH3
CH3CH2
OH
CH3
CHO
OMe
OH
H2SO4/HCLO4
Green Color (625nm)+
CH3
CH3
CH3CH2
OH
CH3
H2SO4/HCLO4
Green Color (625nm)
OH
CHO
+
CH3
CH3
CH3CH2
OH
CH3
O CHO
H2SO4/HCLO4
Green Color (625
nm)
+

20.  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
CH3
CH3CH2
OH
CH3
Green Color (625nm)CH3COClSb Cl3 ++

21. VITAMIN DVITAMIN D
CONTD…CONTD…
 Vitamin D gives color with glycerol-1,3-
dichlorohydrin in presence of acetyl chloride
CH3
CH3
CH3CH2
OH
CH3
Green Color (625nm)CH3COCl
CH2 Cl
CH OH
CH2Cl
++

22. *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
NN
N SCH3
NH3+ CH2 CH2 O H
CH3
. 2 Cl
+
-
2

23.  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 SCH3
NH3+ CH2 CH2 O H
CH3
NN
N SCH3
CH2 CH2 O H
CH3
NK3 Fe (C N)6
+
2 Cl-

24.  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.

25.  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 SCH3
NH3 CH2 CH2 O H
CH3
N
C O C H3
N H
complex
diazotised p-amino acetophenone
+
520 nm

26.  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 SCH3
NH3 CH2 CH2 O H
CH3
OH
CH (CH3)2
CH3
N NH
color complex+

27. 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

28. *NIACIN (NICOTINIC ACID)*
Chemistry:Chemistry:
◦ It’s a heterocyclic. 3-pyridine carboxylic acid.
Functions:Functions:
◦ It functions as oxidising co-enzymes of many dehydrogenases
Deffiency 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
N
COOH

29.  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
λ,β− 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
COOH
SO3H
NH2
CNBr
cleavage of CN bond of pyridine by CNBr red color measured spectroscopically+ +

30.  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
+

31. 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
rearrangement
N
NH2
3-Amino Pyridine
NaNO2/HCl
HNO2 <8*C
N N Cl
diazotised compound
NH-CH2-CH2-NH2
. HCl
N N NH-(CH2)2NH3
red colored azo-compound
+
+ +

32. 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

33.  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
I2
O O
O O
HOHC
HOH2C
2
HI+ +
Dehydro ascorbic acidAscorbic acid

34.  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
CH2
OH
NaO N O
Cl
Cl
OO
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)
+ +

35.  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
CH2CO
N Br
OO
OO
CHOH
CH2OH
CH2-CO
CH2CO
NH H Br+ + +

36.  COLORIMETRIC METHODS
METHOD 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
+

37.  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
+

38. 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
N
N
NCH3
CH3
O
O
C
H
OH
C HOH
C HOH
CH2
OH

39. METHOD 1: FLUORIMETRIC METHOD
 There are three methods are in use in
fluorimetric determination of riboflavin;
 Direct determination
 Direct additive determination
 Adsorptive additive determination

40. 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.

41. 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.


42.  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.

43. 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.

44. 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.

45. 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.

46. 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.

47. 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.

48. 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

49. Thank You