PRINCIPLES & PROCEDURES

INVOLVED IN USAGE OF REAGENTS

IN PHARMACEUTICAL ANALYSIS

Presented ByY.DivyaM.Pharmacy First year-I semSPMVV

CONTENTS• Introduction to

reagents• Reagents

a) PDAB b)Folin Ciocalteauc)MBTH

• Reagent Profile• Principle & Mechanism• Procedure• Examples• Applications• Conclusion• References

oThe Reagents are mainly used for qualitative &

quantitative analysis.

oThere are a number of reagents which are mainly

used for determination of pharmaceuticals.•MBTH

•2,4,6-tripyridyl-S-thiazine(TPTZ)

•1,2-naphthaquinone-4-

sulphonate sodium(NQS)

•PDAB/PDAC

•Ninhydrin reagent

•Bratton marshall reagent

•Folin ciocalteau reagent

•Gibb’s reagent

INTRODUCTION

PDAB REAGENT•It is an aromatic compound and chromogenic

in nature.

•Used for colorimetric estimation of amine

group containing drugs.

Reagent Profile:

Synonym : Ehrlich’s reagentIUPAC Name : 4-(dimethyl amino)-benzaldehydeMolecular Formula : C9H11NOMolecular Weight : 149.9Color : White or offwhiteSolubility : soluble in alcohol, 0.1N HCl, water

The primary amine group which is present

in the structure of drug reacts with

carbonyl group in the PDAB reagent and

forms schiff base which can be measured

colorimetrically.

The principle in the formation of schiff’s

base is nucleophilic addition reaction.

Here the nucleophile is amine group.

PRINCIPLE INVOLVED

General Reaction

•More suitable for amines oximes.•NH2, NR2, NR3 are strong auxochromes.

•But in aromatic compounds it is 100% colored.

Example:

MECHANISM

Preparation Of PDAB Reagent:

To 400mg of PDAB

+2ml of conc H2

SO4

+10ml alcohol

Finally, make upto 100ml with distilled water

PROCEDURE:

1.Stability of color

2.Optimisation of reagent concentration

and volume

3.Quantification methods:

o Calibration plot by

Interpolation

Regression

y=mx + c

o Single point standardization

o Double point standardization

1. Stability of color:

The color formation depends upon: PH

Temperature Catalyst

In case of PDAB, color formation is more favourable in acidic PH

Conc of drug10-50mcg/mlX conc of reagen

t

Colored

compound.Mini

stability:

30minMax

stability: 1hr

Time(min)

Op

tical d

en

sity

2.Optimization Of Reagent Concentration And Volume:

•The point beyond which there is no change

in OD, corresponding to the volume of

reagent is optimized volume of reagent.

•Preferably it is below 3ml.

•Here conc of drug & % strength of reagent

is kept constant.

•Where as for optimized concentration of

reagent, volume of reagent and conc of

drug are kept constant

OD

Vol. of reagent (ml)

OD

Conc of reagent (mcg/ml)

3.Quantification Procedure:

a)Calibration Plot by:InterpolationRegressiony = mx + c

b) Single point standardization:

c) Double point standardization:

Applications Of PDAB ReagentPDAB reagent is used in:

•Micro determination of urea in urine

samples.

•Detection of sulphonamides

Amines

Ergot alkaloids

•Used as TLC stain.

•Used for spectrophotometric

determination of Ranitidine,

Chloramphenicol, Metronidazole,

Sulphamethoxazole.

Example:Estimation of Chloramphenicol in given pharmaceutical formulation:Principle:

The aromatic nitro group present in the chloramphenicol is reduced to aromatic amine group by refluxing with zinc dust in presence of HCl for 10mins.

The resultant amine group of chloramphenicol reacts with the carbonyl group of PDAB reagent and forms schiff base that is estimated colorimetrically at 441nm.

Reaction:

PPn of stock soln• To100 mg

ofdrug • +500mg of

zinc dust +2ml of 5N HCl, boil for 30mins

• Filterate was made upto 10ml with methanol

Determination of λmax • Pipette out

0.1ml from stock soln + 2ml of PDAB reagent

• Make upto 10ml with methanol

• Scan from 400-800nm, and λmax was found to be at 441nm

PPn of calibration std• From stock

pipette out 0.5, 1.5, 2.0, 2.5 ml

• +2ml of PDAB reagent

• Make upto 10ml with methanol

Assay Of Chrloramphenicol Capsules

To 100mg of capsule powder

+500mg of Zinc dust+2ml of 5N HCl, boil for 30mins

Cool & make to 100ml with methanol & filter

From filterate 1ml was pipetted out

+2ml of reagent

Make to 10ml with methanol, measure abs at 441nm

PPn of stock soln Ppn of test sample

FC REAGENT•Chemically it is Phosphomolybdotungistic acid

•Also known as hetero polyacids

•Used for colorimetris estimation of drugs containing phenols or amines

•It consists of:• Sodium tungstate• Sodium molybdate• 85% Phosphoric acid• Lithium sulphate• Bromine

Reagent Profile:

Synonym : Phosphomolybdotungistic

acid

Color : Clear bright yellow

Storage : Tightly capped at RT.

The can be diluted with

deionized water 1 Hexavalent phosphomolybdotungistic

acid complexes with the following

structures formed in the solution.3H2O.P2O5 13WO3. 5MoO3. 10H2O

3H2O xP2O5. 14WO3. 4MoO3. 10H2O

PREPARATION OF FC REAGENT

BASIC MECHANISMOrthophosph

oric acid

Converted to peiodic/

vanaldic acid, molybdic acid

Results in Heteroyl acid

mixture

Undergoes reduction if analyte has

oxidizing nature

Molybdenum Blue

Estimated colorimetricall

y at 400-800nm

APPLICATIONS OF FC REAGENT

•Used as spray reagent in many chromatographic procedures.•Used in Lowry method for determining protein concentration.•It is also reactive towards thiols and many vitamins.•Several drugs like:

AspirinPiroxicamAcetazolamideSparfloxacinPyridoxine HCl

are estimated using this FC reagent

Example:Estimation of Omeprazole in given pharmaceutical formulationPrinciple:

Omeprazole affects reduction of 1, 2 & 3 oxygen atoms from tungstate and

molybdate in Fc reagent, thereby producing one or more of possible reduced

species which have a characteristic blue color, that can be estimated

colorimetrically at 742nm

+3H2O.P2O5 13WO3. 5MoO3. 10H2O

3H2O xP2O5. 14WO3. 4MoO3. 10H2O

Blue colored complex

Preparation Of Standard Stock Solution

To 100mg of crude drug + 10ml of 0.1N NaOH, made upto

100ml with distilled water (1000mcg/ml) SOLUTION -1

From above solution, pippete out 10ml and make to 100ml

with distilled water(100mcg/ml) SOLUTION-2

From solution-2 pipette out 0.2, 0.8, 1.4, 2.0, 2.6 ml of

solution + 2ml of Fc reagent + 9ml of sod. Carbonate, make

to 25ml with distilled water

Keep aside for 10mins. Measure Abs at 742 nm

Assay Of Omeprazole Capsules:

Weight of powder equivalent to 10mg was dissolved in 10ml 0f sodium hydroxide solution

From above solution pipette out 2ml and add 2ml of FC reagent, 9ml of Sod. Carbonate, make to 25ml with distilled water

Keep aside for 10mins. Measure absorbance at 742nm

MBTH is a chromogenic reagent used for

colorimetric estimation of drug containing

phenolic, aromatic amines and active

methylene groups

MBTH REAGENT

Reagent Profile:

IUPAC name : 3-methyl-2-

benothiazolinone hydrazone

Molecular Formula : C8H10N3S. H2O

Molecular Weight : 233.72

Color :White to half white powder

Solubility : freely soluble in distilled water

PRINCIPLEMBTH first reacts with aldehydes to form azine. Only if there

is remaining MBTH, it is oxidized to another species which

combines with the azine to form formazan.

However if there is enough aldehyde, all the MBTH is

converted to azine and there is no formation of blue color.

Thus by using the limiting agent MBTH, the amount of

aldehyde can be tested.

Less aldehyde produce more blue color

More aldehyde produce less blue color

With Phenols

With phenols under suitable conditions

MBTH loses two electrons and one proton to

form the electrophilic intermediate.

This undergoes electrophilic substitution

with phenols and other groups to form the

colored product

Mechanism Involved

1. Oxidative coupling

2. Dehydration

Oxidative Coupling Reactions:Step-I

Formation of oxidation form of MBTH as an Intermediate

STEP-2Formation Of Colored Complexes:a) Reaction With Aldehydes:

b) Reaction With Phenols

CH3

c) Reaction with Amines:

DEHYDRATION REACTIONS

a) Reaction With aldehydes:

b) Reaction With Citrals:

•Applicable for analysis of drinking

surface and saline water, industrial

and dosmetic water

•Applicable for measuring Phenolic

material at 2mcg/ml.

•Used for determination of drugs like

acylovir, ceftazidine, cefadroxil,

nicorandil, methyldopa etc.,

•Used for estimation of samples that

contain high conc of aldehydes Eg:

Disinfectants

Applications of MBTH Reagent:

ExampleEstimation of Lamotrigine in given pharmaceutical formulation

Principle:

The principle involved in this method is Ion catalysed coupling

reaction of MBTH. In presence of FeCl3, MBTH loses 2 electrons

and one proton forming an electrophilic intermediate and which is

proton active coupling species.

The intermediate formed undergoes electrophilic substitution

with the drug to form green colored product, that is estimated

colorimetrically at 663nm.

REACTION

PPn of stock soln

•Dissolve 100mg of crude drug in 100ml of methanol(100mcg/ml)

Construction of calibration curve

•From std soln pipette out 0.2, 0.4, 0.6, 0.8 ml•+2ml of 0.2% MBTHreagent +2ml of 0.2% FeCl3•Keep aside for 20mins•Make upto 10ml with distilled water•Measure abs at 663nm

Procedure

Assay Of Lamotrigine Tablets

Dissolve equivalent to 10mg powder in 10ml methanol

From above solution 60mcg/ml of solution was

taken, to this +2ml of 0.2% MBTH +2ml of 0.2%

FeCl3. Allow to stand for 20mins

Make up the volume to 10ml with distilled water.

Measure absorbance at 663nm

Conclusion….

Here by it can be concluded

that when reagents are

used in optimized volume

and optimized

concentration they be

successfully used for the

estimation of drugs in

pharmaceutical

preparations both

quantitatively and

qualitatively

REFERENCES1. Textbook of Pharmaceutical analysis by P.D.Chaithanya Sudha

2. Peterson, G.L., Anal. Biochem. 100, 201-220 (1979).

3. Bray, H.G., and Thorpe, W.V., Meth. Biochem. Anal. 1, 27-52

(1954).

4. Lowry, O.H., et al., Protein measurement with the Folin phenol

reagent J. Biol. Chem. 193, 265-275 (1951)

5. Eberhardt et al. (1985) Marine Chemistry vol. 17: 199–212

Than

k

you…

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