PRINCIPLES & PROCEDURES INVOLVED IN USAGE OF REAGENTS

IN PHARMACEUTICAL ANALYSIS• KAPIL KUMAR PATIDAR• M.PHARM (ANALYSIS)

• 2015-2016

• GT Road (NH-95), Ghal Kalan, Moga(142001), Punjab, India

• Introduction • Classification based on their

reaction mechanism • Various types of reagents• Applications • Conclusion

Contents

Introduction • Most of the pharmaceuticals are colorless in nature and can be estimated by UV

spectrophotometry easily.

• But there are some drugs which do not show good results in UV region due to

following reasons

weak absorption in UV region

effect of interferences

• Hence to estimate these type of drugs a technique called chemical derivatization is

employed.

• In this technique, various spectrophotometric reagents are used.

• These reagents on reacting with drugs produces a colored complex depending

upon their concentration which can be easily estimated by visible

spectrophotometry. eg: Folins reagent, gibbs reagent etc.

Classification of reagents based on their mechanism of reaction

• Oxidation eg : Folin-ciocalteau reagent tetrazolium salts

• Oxidative coupling eg : MBTH

• Condensation: eg : P- dimethyl amino benzaldehyde (PDAB) p- dimethyl amino cinnamaldehyde (PDAC) 2,4-dinitro phenyl hydrazine (2,4 DNP)Folins reagent (1,2-naphthoquinon-4-sulfonate) Gibbs reagent (2,6-dichloro-4-chlorimide)

• Diazotisation :

eg : sodium nitrite & HCl / α - naphthol

sodium nitrite & HCl / Bratton Marshall Reagent

sodium nitrite & HCl / Resorcinol

sodium nitrite & HCl / p-aminophenol

sodium nitrite & HCl / aniline

• Oxidation followed by complexation :

eg : Ferric chloride and 1,10 phenanthroline

Ferric chloride and 2,2 1 bipyridine

Ferric chloride and potassium ferricyanide

• Complexation due to charge transfer:

eg : 2,3-dichloro-5,6-dicyano- 1,4-benzoquinone (DDQ),

2,4-dinitrophenol (DNP)

2,4,6-trinitrophenol (picric acid)

p – chloranilic acid Chloranil

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

PRINCIPLE

MBTH 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 coupling2. 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:

ApplicationsApplications It is used to estimate various drugs like phenols, aromatic amines,

heterocyclic bases, polyhydroxy compounds, aldehydes, amino hetero aromatic

compounds like indoles, carbazones, phenothiazines, glycerides, glyco

aminoglycones

Drug name Colour λ max Beers range

Ethacridine Violet 535nm 2-8µg/ml

Gancyclovir Bluish green 611.8nm 50-250µg/ml

Montelukast Green 610nm 2-10µg/ml

Moprolol Green 629nm 10-35µg/ml

Pregabalin Green 668nm 50-350µg/ml

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

Reagent compile with following test:

• 2ml aldehyde free methanol

• Add 60 ml of 0.01%w/v soln of propionaldehyde

• 5ml of 0.4 w/v soln of reagent MBTH

• Allow to stand for 30 min.

• 25ml of 0.2% w/v soln of Fecl3.6H2O

• Dilute to 100 ml with acetone

• Absorbance at 660 nm (0.60)•

Folin ciocalteau Reagent

• Also known as Folin -phenol / Folin denis reagent

• It is a mixture of phosphomolybdic acid and phosphotungstic acid

3H 2 O.P 2 O 5 .13WO 3 .5MoO 3 .10H 2 O

3H 2 O.P 2 O 5 .14WO 3 .4MoO 3 .10H 2 O

• Physical properties

Appearance: Clear bright yellow solution Boiling point:101.43°C

Solubility: Easily soluble in cold water, hot water. Soluble in methanol

Method of Preparation• Dissolve 10g of sodium tungstate and 2.5g sodium molybdate in 70 ml water.

• Add 5 ml 85% phosphoric acid and 10 ml concentrated hydrochloric acid, reflux

for 10 hr.

• Add 15g lithium sulfate, 5 ml water and 1 drop bromine. Again reflux for 5mins.

• Cool to room temperature and bring to 100 ml with water.

Reaction mechanism :

FC reagent is an oxidizing agent.

It oxidizes the drug and itself gets reduced.

This reduced form of FC reagent is blue colored in nature (initially

yellow colored) and can be estimated by colorimetrically.

The reaction is carried out in alkaline medium.

Applications

• It is used to estimate phenolic drugs and non phenolic drugs which can be

reduced.

• It can also be used as a spraying reagent for chromatographic techniques.

• The color development is due to the transfer of electrons at basic pH and hence

reducing the FC reagent.

Drug name Color λ max Beers range

Hydralazine hydrochloride

Blue 640nm o.1-1µg/ml

Ganciclovir Blue 764.7nm 50-250µg/ml

Ritodrine Blue 760nm 0.8-12µg/ml

Metolazone Blue 725nm 50-250µg/ml

Memantine Blue 760nm 4-12µg/ml

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. 10H2O3H2O 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

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

Physical properties:

Melting point: 72-75 °C

Boiling point: 176-177 °C

Density: 1.10 g/ mL

Solubility: soluble in water (0.3 g/L)

Reaction mechanism:

The reaction is carried out in acidic medium.

On reaction with amines, it results in the formation of a condensed product

called Schiffs base along with a water molecule.

This schiffs base is colored in nature.

Depending upon the reactants, it produces the following products

Amines - imines

Hydroximes - oximes

Hydrazines - hydrazones

General Reaction

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

•But in aromatic compounds it is 100% colored.

MECHANISM

Example:

Preparation Of PDAB Reagent:

To 400mg of PDAB

+2ml of conc H2SO4

+10ml alcohol

Finally, make upto 100ml with distilled water

Applications Of PDAB Reagent

PDAB 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.

Applications

• It is mainly used to determine primary amines

• Used to estimate various drugs like reserpine , ergot alkaloids, atropine, tetracycline,

antipyrine , paracetmol , isoniazid , oxyphenbutazone , terbutaline etc.,

Drug name Color λ max Beers limit

Abacavir sulfate Yellow 455nm 50-250 µg/ml

Ganciclovir Greenish yellow 401nm 80-200 µg/ml

Mesalamine Yellow 450nm 10-50 µg/ml

Acenocoumarol Yellow 441.5nm 5-25 µg/ml

Ornidazole Orange 390nm 25-162.5 µg/ml

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

PDAC

It is chemically para dimethyl amino cinnamaldehyde .

Also known as DMACA reagent.

Molecular structure:

Molecular formula:C 11 H 13 NO

Molecular weight: 175.23

Physical properties:

Melting point: 138-140 °C

Appearance: Dark yellow powder

Solubility: Soluble in ethanol, dioxane , insolube in water

Reaction mechanism:

The reaction is carried out in acidic medium.

On reaction with amines, it results in the formation of a condensed product

called Schiffs base along with a water molecule.

This schiffs base is colored in nature.

Depending upon the reactants, it produces the following products

Amines - imines

Hydroximes - oximes

Hydrazines - hydrazones

Applications

It is used for estimation of aromatic amines, isocarboxazid , benzodiazepines,

flavanoids , cysteine , cystine , methyl dopa , ranitidine, mefenamic acid,

indomethacin, nimesulide , nimodipine etc .,

Drug name Color λ max Beers range

Dapsone Red 548nm 4-12 µg/ml

Metoclopramide .HCl

Red 553nm 4-24 µg/ml

Modafinil Reddish brown 443nm 6-14 µg/ml

Sumatriptan succinate

Purple red 560nm 20-60 µg/ml

Nateglinide - 420nm 20-140 µg/ml

2,4-DNP

It is chemically 2,4 dinitro phenyl hydrazine. Also known as Brady's reagent Molecular structure: Molecular formula: C 6 H 6 O 4 N 4 Molecular weight: 198.14g

Physical properties

Melting point: 197-200 °C

Density: 0.843 g/ mL

Solubility: Soluble in 50% sulfuric acid & slightly soluble in water

Reaction mechanism:

2,4-DNP first attaches at the carbon-oxygen double bond (the addition

stage) to give an intermediate compound which then loses a molecule of water

(the elimination stage) and results in formation of a condensed chromogen

It is mainly used to determine aldehydes and ketones . Used to determine drugs like corticosteroids, flavanones , atorvastatin , ezetimibe , isoxsuprine , valproic acid etc.,

Applications

Drug name Color λ max Beers range

Tolperisone Pink 520nm 2.5-15 µg/ml

Didanosine Orange red 424nm 20-80 µg/ml

Salbutamol Red 540nm 2.5-17 µg/ml

Ritodrine Red 510nm 2-29 µg/ml

Amoxicillin Red 520nm 4-33 µg/ml

• Preparation of reagent:• 4gm ninhydrin• in 100 ml of 2methoxy

ethanol• Shake with 1gm cation

exchanger region filter it• Add 100ml 0.16%w/v of

(PH-5.5) stanous chloride in acetate buffer

Ninhydrin• Ninhydrin is a chemical used to detect

ammonia or primary and secondary amines.

• When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced.

Formula: C9H6O4

Molar mass: 178.14 g/mol

Density: 862.00 kg/m³

Melting point: 250 °C

IUPAC ID: 2,2-Dihydroxyindane-1,3-dione

Mechanism

Application1. Ninhydrin is primarly used to detect ammonia 1&2 amines.

Ninhydrine react with free amines to give a deep blue colour. 2. Growth of peptide chain is maintained by ninhydrin

A deproted peptide chain yield blue colour where as a coupled peptide chain yield yellow colour

3. Amine Carbamates &amides can be detect by ninhydrine in TLC

Carr-Price reagent (sbcl3)Sbcl3 in Hcl (22.5 gm /100ml cHcl3)Mol. wt. 228.13Reaction of antimony trichloride with Vit.A to yield a brillant Blue colour.Carotenoid can also be estimated Carotenoids accumulate in the golgi app. Of neuron can be estimatedCarotenoids –( Tetraterpenoids i.e. contain 40 C atom ) carotenoids are isoprenoids.They are polyenes.Generally yellow colour pigments.Vit.A –Retinol i.e. vit. A when react with sbcl3 a lewis acid in CHCL3 a blue colour is developed which may detected spectrometrically with ɻ max 620 nm.Carotenoids & vit, A can be estimated by calorimetric assay using carr- price reagent.

Mechanism of action The polyene system of a Retinoid & carotenoid is protonated by strong a line sbcl3 in organic solvent to give a characteristic blue colour of high intensity. Short coming of carr-price reagent: 1. Age of the reagent affects development & fading of blue colour. 2. Increase temprature affect stability of colour 3. Different batches of sbcl3 give discontent results.

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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

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