Download - Assay of Vitamins
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THIAMINE, RIBOFLAVIN, NIACIN, PYRIDOXINE, ASCORBIC ACID, BIOTIN, PANTOTHENIC ACID
PRESENTED BYVARUN DEV I KM. PHARM(1ST YEAR)NCP
ASSAY OF VITAMINS
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THIAMINE- VITAMIN B1 Thiamine hydrochloride is 3-[(4-amino-2-
methyl pyrimidine-5-yl)methyl]-5-(2-hydroxy ethyl)-4-methyl thiazolium chloride hydrochloride.
Thiamine hydrochloride is a white crystalline solid having a slight characteristic odour.
The compound is hygroscopic in nature.
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ASSAY OF THIAMINE
OFFICIAL METHODS
• NON-AQUEOUS TITRATION FOR THIAMINE POWDERS• LIQUID CHROMATOGRAPHY FOR THIAMINE
INJECTION & TABLETS
OTHER METHODS
• COLORIMETRIC METHOD• THIOCHROME FLOURIMETRIC METHOD• SILICOTUNGSTIC METHOD
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OFFICIAL METHOD:- NON-AQUEOUS TITRATION
PRINCIPLE PROCEDURE
Thiamine is determined by non-aqueous titration
End point is determined by potentiometry
Weigh sample + anhydrous formic acid
Add mercuric acetate stir
Titrate with 0.1 M perchloric acid end point potentiometrically
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OFFICIAL METHOD FOR TABLETS/INJECTION
The official method for determination of thiamine tablets and injections are achieved by liquid chromatography
The principle here is the reverse phase chromatography
PRINCIPLE
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PROCEDURE- PREPARATION OF TEST SOLUTION
FOR INJECTION
Volume of injection(0.1 thiamine
hydrochloride) + 0.1 M HCl
5ml – 100ml with water
FOR TABLETS
10 mg Sample + 0.1M HCl + water
Shake , dilute with water
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PROCEDURE
Reference solution:- solution of thiamine mononitrite in hydrochloric acid
Stationary phase :- octadecyl silane bonded to porous silica
Mobile phase:- sodium heptane sulphonate dissolved in a mixture of methanol and triethylamine and pH adjusted to 3.2 with orthophosphoric acid
Flow rate :- 2ml per minute Spectrophotometer at 244nm
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COLORIMETRIC METHOD- PRINCIPLE
Color reaction between thiamine and 6-aminothymol is the basis for colorimetric determination of thiamine
Degradation product of thiamine does not give this reaction
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PROCEDURE
EXTRACTION
Powdered tablet equivalent to 20-40 mg + mixture of water & HCl
Stand 1 hour with shaking filter and wash with HCl 10ml aliquot to 100ml
PROCEDURE
6- aminothymol cooled in ice bath+ sodium nitrate+ NaOH after ½-1 minute
Dilute & add to 1ml sample measure absorbance
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THIOCHROME FLOURIMETRIC METHOD FOR THIAMINE
Thiamine is oxidized to thiochrome using potassium ferricyanide
A blue fluorescence is obtained
Fluorescence is determined using galvanometer
PROCEDURE
10ml extract+ water at pH 3+ methyl alcohol
centrifuge remove precipitate
Add methyl alcohol 30% NaOH + iso propyl
alcohol extract upper layer flourimeter
PRINCIPLE
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SILICOTUNGSTIC ACID METHOD
PRINCIPLE
Thiamine may be determined by precipitation with silicotungstic acid
Thermogravimetric analysis
The precipitate is weighed to determine the equivalent weight
Sample+ water+ HCl HEAT silicotungstic acid drop by drop
Filtered the precipitate
PROCEDURE
wash the precipitate with hot HCl water acetone dry
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RIBOFLAVIN- VITAMIN B2
Riboflavin is 6,7-dimethyl-9-(d,1’- ribityl) isoalloxazine
It is yellow to orange crystalline powder
Have slight odour Slightly soluble in
water, insoluble in chloroform and ethanol
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METHODS OF ASSAY
OFFICIAL METHODS
• COLORIMETRIC METHOD FOR RIBOFLAVINE
• COLORIMETRIC METHOD FOR RIBOFLAVINE TABLETS
OTHER METHODS
• FLOURIMETRIC METHODS• SPECTROPHOTOMETRIC METHOD
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COLORIMETRIC DETERMINATION OF RIBOFLAVIN
The assay method is based on Beer’s law.
A= Єcl Absorbance is
measured at 444nm
PROCEDURE
65 mg sample+ water completely wetted
Add to 2M NaOH+ water+ glacial acetic acid
20 ml solution+ 1.4%w/v sodium acetate+ water measure
PRINCIPLE
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COLORIMETRIC METHOD FOR RIBOFLAVIN TABLETS
PROCEDURE
Sample+ glacial acetic acid+ water HEAT for 1 hour
Add water COOL add 1M NaOH + water mix and filter
Measure the absorbance at 444nm
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FLOURIMETRIC METHOD- DIRECT DETERMINATION
PRINCIPLE
Based on flourimetric emission spectroscopy
Used for mixtures which are free of interfering substances.
PROCEDURE
Sample+ boiled, distilled water heat & agitate cool add water
Filter centrifuge add water(0.2 mcg riboflavin)
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FLOURIMETRIC METHOD - DIRECT ADDITIVE DETERMINATION
PRINCIPLE
In the cases where interferences are present this method is used
A known quantity of riboflavin is added to compensate interference
The sample is first subjected to enzymatic hydrolysis
PROCEDURE
Sample+ 0.05N sulphuric acid digest cool add 2N sodium acetate(adjust pH
to 4- 4.5)Add enzyme solution incubate at 500c for
overnight dilute with water centrifuge and filter
Dilute to 0.02- 0.20 mcg and determine
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FLOURIMETRIC METHOD - ADSORPTION ADDITIVE DETERMINATION
PRINCIPLE
Used in the presence of interference substances
Interfering substances eliminated by an adsorption step
This method can applied universally to all samples
The results obtained are accurate and reproducible
PROCEDURE
Sample+ 0.05N sulphuric acid digest cool add 2N sodium acetate(adjust pH
to 4- 4.5)Add enzyme solution incubate at 500c for
overnight dilute with water centrifuge and filter
Aliquot adsorption column of FLOROSIL elute using hot acetic acid- pyridine
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SPECTROPHOTOMETRIC METHOD
PRINCIPLE
Riboflavin has a characteristic UV spectrum in water with a maximum absorption at 267nm
This method is based on the assumption that extraction with chloroform will remove interfering impurities from an aqueous solution
PROCEDURE
Sample+ water+ 1N NaOH shake gently+ 5N
acetic acid+ water
Aliquot+ chloroform separating funnel
filterrepeat
Absorbance at 267 mµ
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NIACIN- VITAMIN B3
Niacin or nicotinic acid is pyridine-3-carboxylic acid
Niacin crystallizes as white needles from water or alcohol
It is non-hygroscopic and stable in air
OFFICIAL METHODS• Volumetric method for
nicotinic acid• Volumetric method for
niacin tablets
OTHER METHODS
• Cyanogen bromide (colorimetric)method
• Spectrophotometric method for niacin
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VOLUMETRIC METHOD FOR NICOTINIC ACID
PRINCIPLE
Nicotinic acid content can be determined by acid-base titration method
Nicotinic acid is titrated against sodium hydroxide
PROCEDURE
Sample+ CO2 free water
Titrate with 0.1 M NaOH phenol red
indicator
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VOLUMETRIC METHOD FOR NIACIN TABLETS
PROCEDURE
Add 50ml CO2
free water titrate with 0.1 m NaOH
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CYANOGEN BROMIDE METHOD
PRINCIPLE
This method is based on the colour reaction for pyridine and α and γ- substituted pyridine derivatives
Cyanogen bromide breaks one carbon-nitrogen linkage and provides a coloured compound upon addition of ammonia or an amine
Spectrophotometer is used to measure the absorbance at 450µm
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Four solutions are preparedSolution(ml) Tube
1Tube 2 Tube
3Tube 4
Standard preparation
1 1
Assay preparation
1 1
Ammonia dilution
0.5 0.5 0.5 0.5
Water 6.5 1.5 6.5 1.5
Sulfanilic acid 2 2 2 2
HCl 1 drop 1 drop
Cyanogen bromide
5 5
PROCEDURE
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SPECTROPHOTOMETRIC METHOD
PRINCIPLE
Niacin is measured spectrophotometrically at 262nm
The principle is based on the Beer’s law
A= Єcl
PROCEDURE
Sample+ monobasic phosphate-NaOH buffer
5ml solution+ buffer absorbance at 262nm specific absorbance 241
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PYRIDOXINE- VITAMINB6
Molecular formula:- C8H11NO3
Vitamin B6 is a water-soluble vitamin and is part of the vitamin B complex group. Several forms of the vitamin are known, but pyridoxal phosphate(PLP) is the active form.
Other forms are pyridoxine and pyridoxamine. A white or almost white, crystalline powder,
freely soluble in water, slightly soluble in alcohol.
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METHODS OF ASSAY
OFFICIAL METHODS• VOLUMETRIC METHOD FOR PYRIDOXINE
HYDROCHLORIDE• SPECTROPHOTOMETRIC METHOD FOR
PYRIDOXINE TABLET
OTHER METHODS
• SPECTRPHOTOMETRIC METHOD• COLORIMETRIC METHOD
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VOLUMETRIC METHOD FOR PYRIDOXINE HYDROCHLORIDE
PRINCIPLE
Official method of pyridoxine hydrochloride is based on volumetric method
Pyridoxine is titrated against perchloric acid by non- aqueous titration method
PROCEDURE
Sample+ mixture of anhydrous glacial acetic acid+ mercuric acetate
Add crystal violet indicator Titrate with 0.1 M perchloric acid green colour
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SPECTROPHOTOMETRIC DETERMINATION OF PYRIDOXINE TABLET
PRINCIPLE
Pyridoxine content in the fraction of tablet can be determined using spectrophotometry at 290nm
Specific absorbance value is 430
PROCEDURE
Sample+ 0.1M HCl heat on water
bath swirlcool
Add0.1m HCl filter5ml
filterate+ 0.1M HCl measure at
290 nm
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SPECTROPHOTOMETRIC METHOD
Three forms of vitamin B6 pyridoxine, pyridoxal and pyridoxamine possess characteristic absorption in UV region
Total concentration of vitamin B6 can be determined at 325mµ in an aqueous solution of pH 6.75
Specific absorbance is 440
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COLORIMETRIC METHOD
Coupling reaction Pyridoxine couples
with diazo compounds such as sulfanilic acid, diazotized p-amino acetophenone and 2,6 dichloro quinone chloride in a buffer to produce blue colour
PRINCIPLE PROCEDURE
Tablets/capsules powder add NaOH
HEAT cool
Add water+ manganese dioxide
shake 5ml+ isopropyl alcohol
5ml 3 tubes
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Blank Sample Internal standard
Sample Sample Sample
NH4OH-NH4Cl NH4OH-NH4Cl NH4OH-NH4Cl
Sodium acetate
Sodium acetate
Sodium acetate
Boric acid Water Dilute standard solution
1ml chlorimide solution is added to the solutions and the absorbance is measured at 650mµ
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ASCORBIC ACID- VITAMIN C
It occurs as a white slightly yellow powder or crystal
In dry state it is stable to air, but in solution it is rapidly oxidised in presence of air
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METHODS OF ASSAY
OFFICIAL METHODS
• VOLUMETRIC METHOD FOR ASCORBIC ACID• VOLUMETRIC METHOD FOR ASCORBIC ACID INJECTION• VOLUMETRIC METHOD FOR ASCORBIC ACID TABLETS
OTHER METHODS
• COLORIMETRIC METHODS
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VOLUMETRIC METHOD FOR ASCORBIC ACID I.P
PRINCIPLE
The official method for the determination of ascorbic acid is volumetric method
Ascorbic acid determined by iodimetry in which it is directly titrated against standard iodine solution
Ascorbic acid readily reduces iodine to iodide
PROCEDURE
sample+ freshly boiled & cooled water+
sulphuric acid
Add starch solution Immediately titrate with 0.05M iodine
solution blue violet colour
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VOLUMETRIC METHOD FOR ASCORBIC ACID INJECTION I. P
PRINCIPLE
Ascorbic acid determination using a standardized solution of 2,6-dichlorophenol-indophenol is widely used
The end point is determined visually, photometricaly and electrometrically
The best solvents for extracting ascorbic acid are metaphosphoric acid and oxalic acid
PROCEDURE
Sample+ metaphosphoric acid- acetic acid solution+
water10ml Erlenmeyer flask
Add metaphosphoric acid- acetic acid
solution 2,6- dichloro indophenol pink
colour
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VOLUMETRIC METHOD FOR ASCORBIC ACID TABLETS
PRINCIPLE
Ascorbic acid content in the tablet is determined by oxidation-reduction titration
Ascorbic acid is titrated against 0.1M cerric ammonium sulphate
20 tablet
s 0.15
g sampl
e
Add water
+ sulphuric
acid+ ferroi
n
Titrate
cerric ammonium
sulphate
PROCEDURE
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COLORIMETRIC METHOD
Based upon coupling with a diazonium compound
Ascorbic acid reacts with diazotized 4-methoxy-2-nitroaniline to form blue compound
PRINCIPLE
COLORIMETRIC METHOD:- PROCEDURE- REAGENTS USED
1.Amino
reagent
Glacial
acetic
acid
4-methoxy-2-
nitro aniline
10%w/v sulphuri
c acid
2.Nitrite reagent
0.2% sodium
nitrate in water
3.Ascorbi
c acid(0.5 mg/ml)
In 0.5% aq. Oxalic
acid
USP ascorbic
acid standard4.Standard A- 1mg5.Standard B 1.5mg
PROCEDURE
Amino reagent Erlenmeyer flask + nitrite reagent swirl- orange colour
disappear+ n-butyl alcohol
Sample added+ aq. Oxalic acid separator + 10%NaOH+
diethyl ether shake separate bottom layersample solution 270nm
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BIOTIN Molecular formula:- C10H16N2O3S A white or almost white crystalline powder or
colourless crystals, very slightly soluble in water and in alcohol, practically insoluble in acetone.
It dissolves in dilute solutions of alkali hydroxides.
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METHODS OF ASSAY
OFFICIAL METHODS
• Volumetric method (B.P)• Volumetric method (USP)
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VOLUMETRIC METHOD( B. P) FOR BIOTIN
Biotin is determined by volumetric method
Non- aqueous titration is the method
Biotin is titrated with 0.1M tetrabutyl ammonium hydroxide
End point is determined potentiometricaly
PROCEDURE
Sample+ dimethyl formamide heat add
ethanol
Titrate with 0.1M tetrabutyl ammonium
hydroxide
PRINCIPLE
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VOLUMETRIC METHOD(USP) FOR BIOTIN
Volumetric determination of biotin is based upon acid-base titration
Biotin is titrated with sodium hydroxide
PROCEDURE
Sample+ water+ phenolphthalein
Titrate with 0.1M sodium hydroxide
PRINCIPLE
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PANTOTHENIC ACID
Pantothenic acid is (D(+)-N-(α,γ- β,β- dimethylbutyryl)-β- aniline).
Molecular formula:- C18H32CaN2O10
Calcium pantothenic occurs as an odourless, slightly hygroscopic powder, with a bitter taste.
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VOLUMETRIC METHOD FOR PANTOTHENIC ACID
Pantothenic acid is determined by non-aqueous titration
End point is determined potentiometricaly
PROCEDURE
Sample+ anhydrous glacial acetic acid
Titrate with 0.1M perchloric acid
PRINCIPLE
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