THIAMINE, RIBOFLAVIN, NIACIN, PYRIDOXINE, ASCORBIC ACID, BIOTIN, PANTOTHENIC ACID

PRESENTED BYVARUN DEV I KM. PHARM(1ST YEAR)NCP

ASSAY OF VITAMINS

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

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

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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