PRESENTED BY VARUN DEV I K M.

PHARM(1ST YEAR) NCP

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.

ASSAY OF THIAMINE

NON-AQ EO S TIT ATION FO THIAMINE OW E S LIQ I H OMATOG A HY FO THIAMINE INJE TION & TA LETS

OLO IMET I METHO THIO H OME FLO IMET I METHO SILI OT NGSTI METHO

OFFICIAL METHOD:- NON-A UEOUS TITRATION

PRINCIPLE


PROCEDURE

Thiamine is determined by non-aqueous titration End point is determined by potentiometry

eigh sample + anhydrous formic acid Add mercuric acetate stir Titrate with 0.1 M perchloric acid end point potentiometrically

OFFICIAL METHOD FOR TABLETS/INJECTION

PRINCIPLE The official method for determination of thiamine tablets and injections are achieved by liquid chromatography  The principle here is the reverse phase chromatography


PROCEDURE- PREPARATION OF TEST SOLUTION FOR INJECTION

Volume of injection(0.1 thiamine hydrochloride) + 0.1 M HCl

FOR TABLETS
10 mg Sample + 0.1M HCl + water

5ml 100ml with water

Shake , dilute with water

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 6aminothymol is the basis for colorimetric determination of thiamine  Degradation product of thiamine does not give this reaction


PROCEDURE
EXTRACTION

PROCEDURE

Powdered tablet equivalent to 20-40 mg + mixture of water HCl tand our wit s a ing filter and was wit HCl 0ml aliquot to 00ml

6- aminot ymol cooled in ice bat + sodium nitrate+ NaOH after - minute Dilute add to ml sample measure absorbance
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THIOCHROME FLOURIMETRIC METHOD FOR THIAMINE PRINCIPLE



 

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
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SILICOTUNGSTIC CID
PRINCIPLE


ET OD

PROCEDURE
Sample+ water+ HCl HEAT silicotungstic acid drop by drop Filtered the precipitate wash the precipitate with hot HCl water acetone dry
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Thiamine may be determined by precipitation with silicotungstic acid Thermogravimetric analysis The precipitate is weighed to determine the equivalent weight

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

COLORIMETRIC METHOD FOR RIBOFLAVINE COLORIMETRIC METHOD FOR RIBOFLAVINE TABLETS

FLOURIMETRIC METHODS SPECTROPHOTOMETRIC METHOD

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COLORIMETRIC DETERMINATION OF RIBOFLAVIN

PRINCIPLE


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
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The assay method is based on Beers law. A= cl Absorbance is measured at 444nm

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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FLOURI ETRIC

ET OD-

DIRECT DETERMINATION
PRINCIPLE PROCEDURE


Based on flourimetric emission spectroscopy Used for mixtures which are free of interfering substances.

Sample+ boiled, distilled water heat agitate cool add water Filter centrifuge add water(0.2 mcg riboflavin)

FLOURIMETRIC METHOD DIRECT ADDITIVE DETERMINATION

PRINCIPLE


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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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

FLOURIMETRIC METHOD ADSORPTION ADDITIVE DETERMINATION

PRINCIPLE


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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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

SPECTROPHOTOMETRIC METHOD
PRINCIPLE


PROCEDURE
Sample+ water+ 1N NaOH shake gently+ 5N acetic acid+ water Aliquot+ chloroform separating funnel filter repeat Absorbance at 267 m

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

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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

Volumetric method for nicotinic acid Volumetric method for niacin tablets

Cyanogen bromide (colorimetric)method Spectrophotometric method for niacin

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VOLUMETRIC METHOD FOR NICOTINIC ACID

PRINCIPLE


PROCEDURE
Sample+ CO2 free water

Nicotinic acid content can be determined by acid-base titration method Nicotinic acid is titrated against sodium hydroxide

Titrate with 0.1 M NaOH phenol red indicator

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VOLUMETRIC METHOD FOR NIACIN TABLETS

PROCEDURE
eigh powder 0.25g add 40ml ethanol neutralized with phenolphthalein shake 15 mins swirl filter

Add 50ml CO2 free water titrate with 0.1 m NaOH

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CYANOGEN ROMIDE METHOD

PRINCIPLE


This method is based on the colour reaction for pyridine and and - 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 450 m
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PROCEDURE


Four solutions are prepared

Tube 1 1 Tube 2 1 1 0.5 6.5 2 1 drop 5 0.5 1.5 2 0.5 6.5 2 1 drop 5
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Solution(ml) Standard preparation Assay preparation Ammonia dilution ater Sulfanilic acid HCl Cyanogen bromide

Tube 3

Tube 4

1 0.5 1.5 2

SPECTROPHOTOMETRIC METHOD
PRINCIPLE


PROCEDURE

Niacin is measured spectrophotometrically at 262nm The principle is based on the Beers law A= cl

Sample+ monobasic phosphate-NaOH buffer

5ml solution+ buffer absorbance at 262nm specific absorbance 241

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PYRIDOXINEVITAMINB6
 

 

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

VOLUMETRIC METHOD FOR PYRIDOXINE HYDROCHLORIDE SPECTROPHOTOMETRIC METHOD FOR PYRIDOXINE TABLET

SPECTRPHOTOMETRIC METHOD COLORIMETRIC METHOD

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VOLUMETRIC METHOD FOR PYRIDOXINE HYDROCHLORIDE

PRINCIPLE


PROCEDURE

Official method of pyridoxine hydrochloride is based on volumetric method Pyridoxine is titrated against perchloric acid by non- aqueous titration method

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



PROCEDURE

Pyridoxine content in the fraction of tablet can be determined using spectrophotometry at 290nm Specific absorbance value is 430

Sample+ 0.1M HCl heat on water bath swirl cool Add0.1m HCl filter 5ml 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 325m in an aqueous solution of pH 6.75  Specific absorbance is 440


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COLORIMETRIC METHOD PRINCIPLE

 

PROCEDURE

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

Tablets/capsules powder add NaOH HEAT cool

Add water+ manganese dioxide shake 5ml+ isopropyl alcohol 5ml 3 tubes
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Blank Sample NH4OH-NH4Cl Sodium acetate Boric acid

Sample Sample NH4OH-NH4Cl Sodium acetate ater

Internal standard Sample NH4OH-NH4Cl Sodium acetate Dilute standard solution

1ml chlorimide solution is added to the solutions and the absorbance is measured at 650m
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ASCORBIC ACIDVITAMIN 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

VOLUMETRIC METHOD FOR ASCORBIC ACID VOLUMETRIC METHOD FOR ASCORBIC ACID INJECTION VOLUMETRIC METHOD FOR ASCORBIC ACID TABLETS

COLORIMETRIC METHODS
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VOLUMETRIC METHOD FOR ASCORBIC ACID I.P PRINCIPLE



PROCEDURE

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

sample+ freshly boiled cooled water+ sulphuric acid

Add starch solution Immediately titrate with 0.05M iodine solution blue violet colour 35

VOLUMETRIC METHOD FOR ASCORBIC ACID INJECTION I. P PRINCIPLE



PROCEDURE
Sample+ metaphosphoric acid- acetic acid solution+ water 10ml Erlenmeyer flask Add metaphosphoric acidacetic acid solution 2,6dichloro indophenol pink colour
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Ascorbic acid determination using a standardized solution of 2,6dichlorophenol-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

VOLUMETRIC METHOD FOR ASCORBIC ACID TABLETS PRINCIPLE



PROCEDURE

Ascorbic acid content in the tablet is determined by oxidation-reduction titration Ascorbic acid is titrated against 0.1M cerric ammonium sulphate

20 tablets 0.15 g sample

Add water+ sulphur ic acid+ ferroin

Titrate cerric ammo nium sulph ate

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COLORIMETRIC PROCEDURE- REA ENTS COLORIMETRIC METHOD:USED METHOD

PROCEDURE 4-methoxy-2PRINCIPLE
nitro aniline lacial acetic 10%w/v sulphuric acid Amino upon Erlenmeyer flask acid reagent coupling with a Based

2.Nitrite reagent

0.2% sodium nitrate in water

reagent swirl- orange colour disappear+ n-butyl compound alcohol 3.

+ nitrite diazonium

Sample added+ a . Oxalic acid separator 4.Standard diethyl ether shake separate + NaOH+ 5.Standard B A- 1mg In 0.5% aq. bottom layer sample solution nm 1.5mg Oxalic acid

Ascorbic acid reacts with diazotized 41.Amino USP methoxy-2-nitroaniline to form blue Ascorbic reagent ascorbic acid(0.5 acid compound standard mg/ml)

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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

Volumetric method (B.P) Volumetric method (USP)

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VOLUMETRIC METHOD( B. P) FOR BIOTIN

PRINCIPLE


PROCEDURE

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

Sample+ dimethyl formamide heat add ethanol

Titrate with 0.1M tetrabutyl ammonium hydroxide

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PRINCIPLE


PROCEDURE

Volumetric determination of biotin is based upon acid-base titration Biotin is titrated with sodium hydroxide

Sample+ water+ phenolphthalein

Titrate with 0.1M sodium hydroxide

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PANTOTHENIC ACID
Pantothenic acid is (D(+)-N-( , - , dimethylbutyryl)- - 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 PRINCIPLE



PROCEDURE

Pantothenic acid is determined by nonaqueous titration End point is determined potentiometricaly

Sample+ anhydrous glacial acetic acid

Titrate with 0.1M perchloric acid

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