classification and analysis

1. PREPARED BY :
DEVIP RIYA P V
M PHARM
DEPT. OF PHAMACEUTICAL ANALYSIS

2. DEFINITION
 The non energy yielding organic compounds, essential for
normal human metabolism, that must be supplied in small
quantities in diet.
2

3. CLASSIFICATION
VITAMINS
Fat soluble
VIT A, D, E,K
Water soluble
B COMPLEX
VITAMIN C
3

4. 4
FAT SOLUBLE VITAMINS
VITAMIN CHEMICAL FORM THERMO
STABILITY
DAILY
ALLOWANCE
DEFICIENCY
DISEASE
A Retinol(A1)
Dehydroretinol(A2)
β carotene(provit)
Stable in
absence of air
1000μg Night
blindness,
xerophthalmia
D Calciferol(D2)
Cholecalciferol(D3)
Calcitriol
stable 5μg
1μg
Rickets,
osteomalacia
E α- tocopherol stable;air and
UV light
decompose
10 -15 mg Pheripheral
neuropathy
K Phytonadione(K1)
Menaquinone (K2)
Menadione(K3)
Stable;
decomposed
by light
50- 100μg Increased
tendency to
bleed

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WATER SOLUBLE VITAMINS
VITAMIN CHEMICAL FORM THERMO
STABILITY
DAILY
ALLOWANCE
DEFICIENCY
DISEASE
B1 Thiamine Relatively
labile
1.5 mg Beriberi
B2 Riboflavin Relatively
labile
1.7 mg Cheilosis
B3 Niacin Stable 20 mg Pellagra
B6 Pyridoxine Stable in
absence of air
2mg Peripheral
neuritis
Panthothenic acid Labile 4-7 mg weakness
Biotin Stable 0.1-0.2 mg Dermatitis,
Alopecia
Folic acid Labile 0.2 mg Megaloblastic
anaemia
B12 cyanocobalamine stable 2μg anaemia
C Ascorbic acid Labile in
solution
60 mg Scurvy

6. METHODS OF ANALYSIS OF VITAMINS
 Analytical procedures based on chemical, physical,
biological and microbiological methods are used in the
assay.
 Fundamental analysis are based on biological response.
 Exhibit species specificity.
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7. VITAMIN A
 Three methods are used:
1. Biological assay based on rat or chicken growth or on liver
storage.
2. Carr- price colorimetric method.
3. UV spectrophotometric method.
7

8. Carr price method :
 Anhydrous antimony trichloride react with a dilute solution
of vitamin A to form a transient blue color.
 Reaction between antimony tri chloride and unsaturated
side chain of vitamin A.
 The color rapidly reaches a maximum intensity and fades
rapidly.
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9. Spectrophotometric method
 Based on UV absorption characters of vitamin A.
 Sample is saponified, Vit A is extracted with ether, the
ether is evaporated and vit A is dissolved in isopropanol.
 The absorbance of the resulting solution are determined at
the wavelengths 310,325,334nm
9

10. VITAMIN D
 UV Spectrophotometric method:
 Both Vit D2 and D3 have absorption maximum at 265 nm
in hexane.
 Colorimetric method:
 Glycerol dichlorohydrin method.
 Vit D gives green color .
 Ergosterol gives a pink orange color which turns to a
fluorescent green.
 7- dehydrocholesterol produce no color for several min ,
then gives a faint pink color.
10

11. VITAMIN E
 Assay:
 Dissolve sample in 95% ethanol, add ethanolic H2SO4.
 Reflux , cool and add 25 ml 0.02 M H2SO4 and 10 ml water.
 Titrate with ceric ammonium nitrate.
 Indicator used is diphenylamine.
 End point is appearance of blue color.
 Perform a blank determination.
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 Spectrophotometric method.
 α-tocopherol in 95% ethanol exhibit an absorption
maximum at 292 nm.
 α-tocopheryl acetate in 95% ethanol exhibit the
absorption maximum at 284 nm
 Colorimetric method.
 Based on the oxidation of xylene extracted tocopherols
of blood samples by FeCl3.
 Pink complex of ferrous ion with bathophenanthroline is
measured at 536 nm.

13. VITAMIN K
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 Titrimetric procedures based on oxidation reduction
are available for analysis.
 Assay for menadione involves reduction with zinc and HCl;
the reduced form is then titrated with ceric sulfate and
o-phenanthroline is used as indicator.
 Vit K1 , K2 and menadione react with 2,4-
dinitrophenylhydrazine to give colored products which are
suitable for colorimetric determination.

14. VITAMIN C
 Assay:
 To the sample solution add 1M H2SO4
 Titrate with 0.05 M iodine using starch indicator.
 Titration with 2,6 dichlorophenol indophenol:
 Extract the sample with metaphosphoric acid.
 Pink end point
 Read absorbance at 545 nm.
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15. VITAMIN B1
 Thiochrome fluorometric method:
Oxidation of thiamine with alkaline potassium
ferricyanide produce a blue flourescence.
 Colorimetric method
:p-aminoacetophenone method
: red pigment.
:absorption at 520 nm.
 silico tungstic acid method:
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16. VITAMIN B2
 Fluorometric methods.
 Direct determination(free of any interfering substances)
 High concentration of riboflavin.
 Spectrophotometric method.
 UV absorption spectrum at 267 nm
 % absorbance= 100(As/Ar)(Wr/Ws)
.
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 Assay:
 Dissolve sample in 2M NaOH.
 Add 100 ml water, 2.5 ml glacial acetic acid and dilute to
500 ml with water.
 To 20 ml of the solution, add 3.5 ml sodium acetate (1.4%
w/v).
 Dilute to 200 ml with water.
 Measure the absorbance at 444 nm.

18. VITAMIN B6
 Spectrophotometric method:
 The total concentration of vit B6 can be determined at 325
nm.
 Colorimetric method:
 Pyridoxine couples with 2,6 dichloroquinonechloroimide to
produce a blue color.
 Assay :
 Dissolve in glacial acetic acid and mercuric acetate.
 Titrate with 0.1 M perchloric acid using crystal violet as
indicator.
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19. VITAMIN B12
 Cobalt coordinated complex.
 Assay:
 Dissolve the sample in water to produce 1000ml and read
the absorbance at 361 nm.
 Cyanide colorimetric method:
 Based on the quantitative liberation of cyanide group.
 2-20 mcg of sample.
 Dicyanide colorimetric method:
 200 cg of sample
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20. NIACIN
 Nicotinic acid can be titrated with NaOH using
phenolphthalein as indicator.
 Determined spectrophotometrically in the UV region or by
colorimetric methods.
 Cyanogen bromide method:
 Cyanogen bromide breaks one C-N linkage and produce
colored compound on addition of NH3/amine.
 Nictotinamide , when boiled with NaOH ,release the nitrogen
of amido group as NH3 which is collected in H2SO4 and
determined by titration with 1N HCl.
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21. FOLIC ACID
 Colorimetric method:
 Based on reduction ,diazotization and coupling.
 Reductive cleavage gives 2-amino-4-hydroxy-6-methyl-
pteridine & p-aminobenzoyl glutamic acid.
 Diazotized and coupled with N-(1-napthyl)-
ethylenediamine to produce a colored azo compound.
 Polarographic method:
 Sample is dissolved in tetramethyl ammonium hydroxide
containing cadmium chloride.
 NH4Cl prevent precipitation of cadmium.
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22. 22
 Assay:
 To the sample add :
: 0.1 M NaOH
:2M HCl
:0.5 g zinc powder
:5ml sodium nitrate
:5 ml ammonium sulphamate.
:5 ml N –(1- naphthyl )-ethylenediamine
dihydrochloride solution
 Read absorbance at 550 nm

23. PANTOTHENIC ACID
 Based on the colorimetric determination of the cleavage
products.
 Acid hydrolysis of pantothenate yields β-alanine & α,γ-
dihydroxyβ, β-dimethylbutyrolactone (pantoyl lactone).
 Lactone reacts with 2,7-naphthaleindiol in conc H2SO4 to
form a greenish yellow colored complex which can be
estimated spectrophotometrically at 465nm.
 β- alanine reacts with 1,2-naphthoquinone-4-sulfonate to
form a yellowish orange solution at pH 9.3
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24. MICROBIAL ASSAY
 Applied to any substance which will influence the growth of
microorganism in a regular manner.
 Better than chemical methods.
 Based on the nutritional requirement of a microorganism
for a certain vitamin.
 The response (growth of microorganism) is proportional to
the dose added to the medium.
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25. CHARACTERISTICS OF ORGANISM
 Non pathogenic.
 Rapid growth cycle.
 Have a growth response not easily influenced by
neutralization salts or other substances.
 Genetically constant.
 Organisms used
:Lactobacilli ,saccharomyces, protozoa
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26. PROCEDURE
1. Preparation of stock culture and inoculum.
2. Preparation of sample.
3. Preparation of standard vessels.
4. Preparation of assay vessels.
5. Sterilization.
6. Inoculation and incubation.
7. Turbidimetry and acidimetry.
8. Analysis of data.
9. Calculations.
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27. Media for maintenance of stock culture
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 Malt agar medium:
 Liver tryptone agar medium.
 Yeast agar medium.
Malt extract 4g
Yeast extract 1g
Glucose 0.5 g
Peptone 1 g
Agar 1 g
Water to 100 ml
Final PH about 5.4

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30. 30
TURBIDIMETRY
 Turbidimetry or % transmission is measured against an un
inoculated control.
 Rapid method.
ACIDIMETRY:
 Transfer the contents of each tube to a 50 ml Erlenmeyer
flask.
 Rinse the tubes with water.
 Add bromothymol blue
 Titrate against o.1 N NaOH to green color(pH 6.8)

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ANALYSIS OF DATA
 Standard curve is prepared by plotting growth against mg
or μg of vitamin per tube.
 The amount of vitamin in the various levels of test
solution can be determined by interpolation.
CALULATIONS:
 Vitamin content = avg. μg/ml x volume x DF
weight of sample

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