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Principles and procedures involved in usage of reagents in pharmaceutical analysisPRESENTED BY:T. LAKSHMI BHAVANI(2015MPH40023)Pharmaceutical Analysis -1semUnder the guidance of: Dr. G.RAJITHA MADAMM.Pharm.,Ph.D

SPMVV - TIRUPATHI

CONTENTSINTRODUCTIONREAGENTS PDACNINHYDRIN REAGENT1,2 NAPHTHOQUINONE - 4- SULFONATEREAGENT PROFILEREACTION AND MECHANISMPRINCIPLEPROCEDUREAPPLICATIONSEXAMPLESREFERENCE

INTRODUCTIONThe reagents are mainly used for quantitative and qualitative analysis of drugs. The reagents have a greater application in the pharmacy field.Reagents are also applicable to recent analytical techniques.

PARA DIMETHYL AMINO CINNAMALDEHYDE(PDAC) REAGENTPara dimethyl amino cinnamaldehyde(PDAC) is an aromatic compound and chromogenic in nature. which is chemically 4-(dimethyl amino) cinnamaldehyde containing amine and aldehyde moities. PDAC reagent is used for determination of hydrazine.

Reagent profile:Synonym : Ehrlichs reagentIUPAC name:4-(dimethyl amino) cinnamaldehydeStructure:

Molecular formula : C11 H13 NOMolecular weight: 175.23 g/mol

Color : dark yellow powderSolubility: soluble in warm dioxane(500mg/ml- clear ,deep greenish yellow solution) soluble in mixture of chloroform: ethanol (1:1).

General reaction

Chromophore formed with PDAC is more intense than with PDAB because of additional double bond.EXAMPLE:

PRINCIPLEThe reaction between primary amine and carbonyl compound form the condensation product are referred as schiffs base.The primary amine group which is present in the structure of drug react with the carbonyl group in PDAC reagent and forms schiffs base (colored compound) which can be measured by using colorimetry.

MECHANISM OF PDAC REAGENT:

Schiffs base(colored compound)

PREPARATION OF REAGENTPreparation of 2% PDAC reagent: weigh 200mg of PDAC reagent and transfer in to a 10ml volumetric flask add 0.4ml H2SO4 mix well and make up with distilled water.

APPLICATIONS OF PDAC REAGENTFor detection of indole derivatives.Determination of aromatic amines.Chromogenic reagent for the detection of benzodiazepine and flavanoids.Used for spectrophotometric determination of ranitidine, ganciclovir, sumatriptane, succinate, mesalamine, lenalidomide etc.

EXAMPLES 1.ASSAY OF RANITIDINE PRINCIPLE: The primary amine group of ranitidine reacts with carbonyl group of PDAC reagent and forms schiffs base which can be measured colorimetrically.

REACTION:SCHIFFS BASE(COLORED COMPOUND)

PROCEDURE:PREPARATION OF STOCK SOLUTION: A standard stock solution containing 1000g/ml of ranitidine drug in distilled water and diluting to volume of 10ml in volumetric flask.PREPARATION OF CALIBRATION STANDARD: From the above stock solution 0.2, 0.4, 0.6, 0.8 & 1ml of solutions was pipetted out in to 10ml volumetric flask.

After colour development cool the solution later diluted to volume with distilled water.The resulting solutions were measured for the absorbance at 632nm.The calibration curve was plotted using concentration on x-axis & absorbance on y-axis.

From the above stock solution , to each flask add 0.5 ml of PDAC reagent, mix well and heat on hot plate for 15min at 1000c.

ASSAY OF RANITIDINE HCl TABLETS3 tablets were weighed and powdered. A powder equivalent to 100mg of ranitidine HCl was weighed and transferred into 10ml volumetric flask.From this 10ml was pipette out.Now add 0.5ml of PDAC reagent and heated at 800c for 15mins.After development of color, cool the solution and later diluted with distilled water.The resulting solution was measured at 632nm.

2.Estimation of Lenalidomide in pharmaceutical formulation by PDAC reagentPRINCIPLE: Lenalidomide(LLD) is an immuno modulatory agent with anti angiogenic and anti-neoplastic properties.Lenalidomide is used to treat multiple myeloma in people who have received at least on other therapy. The A primary amine group of Lenalidomide reacts with carbonyl group of PDAC reagent and forms schiffs base which can be measured colorimetrically.

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LLD in combination with dextramethasone is indicated for the treatment of multiple myeloma patients.The chemical name of LLD is 3-[4-amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl] piperidine 2,6-dione.

ASSAY OF LENALIDOMIDE CAPSULESAliquots of LLD solution (100g/ml) ranging from 0.1 to 0.5 ml were transferred in to a series of 10ml volumetric flask.To each flask 1.0ml of PDAC(0.2%w/v) solution and 0.1ml of H2SO4 were added.After 5min. The volume was brought up to 10ml with methanol and the absorbance of red colored species was measured at 530nm against reagent blank.

NINHYDRIN REAGENTIt is a chromogenic reagent which is chemically 2,2-dihydroxy indane-1,3-dione.It is used in colorimetric estimation of drugs containing free amine group.It gives deep blue or purple color which is popularly known as Ruhemanns purple.

REAGENT PROFILESynonym: Indanetrione IUPAC name: 2,2-dihydroxy indane-1,3-dione.Structure:

Molecular formula:C9 H6 O4 Appearance/form : white crystalline powderSolubility : soluble in water and alcohol. slightly soluble in ether and chloroform.

PRINCIPLENinhydrin tautomerizes to 1,2,3-indane trione which forms a schiff base when react with amine group containing amino acids.The ketamine formed undergoes decarboxylation yielding the aldehyde and was isolated by ruhemann.Condensation of this intermediate amine with another molecule of ninhydrin follows to form the expected chromophore, Ruhemanns purple.

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Reaction with Amino acids:

REACTION

REACTION WITH IMINO ACIDS

REACTION WITH PRIMARY AMINE

Colored compound

MECHANISM OF NINHYDRIN REAGENTNinhydrine

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Ruhemanns purple

APPLICATIONSUsed for both qualitative and quantitative analysis of amino acids and drugs containing amine groups.Used as a stain to amino acids.It is mostly used to detect fingerprints, as the terminal amines of lysine residues reacts with ninhydrin.PREPARATION OF 0.1%W/V NINHYDRIN REAGENT: 0.1g of ninhydrin reagent was Dissolved in 100ml of distilled water.

Estimation of ampicillin in pharmaceutical formulation by Ninhydrin reagent PRINCIPLE:Ampicillin is an antibacteral drug which is chemically (6R)-6(-phenyl)-O-glycyl amino pencillanic acid.It contains primary amine group so it can be estimated by using ninhydrin.EXAMPLES

Tautomerised form of ninhydrin react with ampicillin to form schiffs base.The ketamine formed under goes decarboxylation and this on condensation, with another molecule of ninhydrin gives a colored complex which can be estimated at absorbance of max.

PROCEDUREPREPARATION OF 0.1N HCl: 0.85 ml of HCl was made up to 10ml with distilled water.PREPARATION OF STANDARD STOCK SOLUTION: 10mg of ampicillin was dissolved in 10ml of 0.1N HCl to give 1000g/ml stock solution.

CONSTRUCTION OF CALIBRATION CURVE:

From the standard stock solution 1,1.5,2,2.5 were pipetted out in to 10ml volumetric flask.To each flask add 2ml of 0.2% ninhydrin reagent,1.5 ml of 0.1N HCl ,2ml of methanol, heat on the water bath until color develops.After the color develops make up the volume to 10ml with 0.1N HCl, Measure the absorbance at 490nm.

REACTION

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ANALYSIS OF AMPICILLIN CAPSULESTwo capsules were weighed and powder equivalent to 10mg was dissolved in 10ml of 0.1N HCl.From the above solution pipette out 2ml and transfer to 10ml volumetric flask and then add 2ml of 0.1%w/v Ninhydrin reagent,1.5ml of 0.1N HCl, 2ml of CH3OH was added and heated on a water bath until color develops. Make up the volumetric flask to 10ml with 0.1N HCl and measure absorbance at 490nm.

2.Estimation of levodopa in pharmaceutical formulation by ninhydrin reagentPRINCIPLELevodopa {(-)-3-[3,4-dihydroxyl phenyl-1-alanine] a precursor of dopamine, is an important neurotransmitter which is used for medication of neural disorder such as Parkinson's disease.After administration levodopa is converted into dopamine through enzymatic reaction catalyzed by dopadecarboxylase.Different methods to determine levodopa have been employed such as high performance liquid chromatography, fluorescence spectroscopy, electrochemical method.

REACTION

ANALYSIS OF CAPSULESWeigh accurately 100mg of levodopa , add 60ml of DMF and stirred.It was filtered with whatmann filter paper size1 and washed with DMF.The filtrate was diluted to 100ml with DMF.To 5ml of this solution ,2ml of 0.2% ninhydrin solution was added.The mixture was heated on a water bath at 80 to 850C for 5mins.

The mixture was cooled to room temperature and then transferred to a 100ml measuring flask.Final volume was made upto 100ml with DMF and scanned in the UV- visible range at 400 to 800nm,for the determination max of levodopa by using blank solution.

1,2 NAPHTHOQUINONE 4 SULFONATE REAGENT (NQS)NQS is the most widely used chromogenic reagent for the estimation of drugs containing aromatic amine.It is a derivative of O-quinone

REAGENT PROFILE

SYNONYM: Sullivan reagent Follins reagent

STRUCTURE:

PRINCIPLEWhen the NQS reacted with any amine containing compound that will release hydroxyl group and the sodium sulphonate group is replaced with the aromatic amine group.

GENERAL RECTION

APPLICATIONSSulpha drugsSuccinyl sulphathiazoleChloramphenicolNimesulideNifedipineNitrendipine

EXAMPLES1.Estimation of cefadroxil in pharmaceutical formulation by NQS reagent

Cefadroxil is an antibacterial drug. As it contains amine group, it reacts with NQS reagent.The principle involved is nucleophilic substitution in alkaline condition.In the presence of 0.1NaOH ,NQS reagent reacts with amino group of cefadroxil to form a colored complex which is measured at max of 453nm.

REACTION

Cefadroxil.H2O+

Orange colored productNQS

ANALYSIS OF CEFADROXIL TABLETS5 Tablets were weighed and powdered.The powder equivalent to 50mg of cefadroxil was taken in to a 10 ml volumetric flask and make up with distilled water .From Above stock solution 0.1ml was pipetted out and again dilute to 10ml with distilled water , then add 1ml of 0.1N NaOH(0.4g of NaOH in 100ml d.w.)and add 1ml of 0.5%w/v NQS reagent. Measure the absorbance at 453nm.

2. Spectrophotometric Method for Determination of Primaquine in Pharmaceutical Formulations via Derivatization with 1,2-Naphthoquinone-4-Sulfonate PRINCIPLE:Primaquine is anti malarial drug. The nucleophilic substitution reaction proceeds quantitatively at PH 10 buffer solution with absorption maximum at 485 nm. The method is based on the formation of a brown color product from the reaction between PQ and NQS.

REACTION

PROCEDURE: PREPARATION OF STANDARD AND SAMPLE SOLUTIONStock standard solution of Primaquine diphosphate (PQ -200 g/ mL) An accurately 0.02 g of PQ standard was dissolved in distilled water and transferred in 100 mL volumetric flask diluted to mark. The solution was further diluted to obtain working solution.

Sample Solution Four tablets (PQ 15 mg tablet) were weighed and finely powdered. A portion of the powder equivalent to 0.09 g of the drug was dissolved in distilled water and transferred into 50 mL volumetric flask the solution was completed to mark, shaken well filtered and then analyzed by spectrophotometric method.1,2-napthoquinone-4-sulfonate Reagent{ NQS (0.4%, w/v) } It was prepared by dissolving 0.4 g in 100 mL volumetric flask and complete the volume with distilled water. The solution was freshly prepared.

Analysis of primaquine tabletsA 2.0 mL of 200 g mL primaquine diphosphate standard was transferred in 10 mL volumetric flask. 2.0 mL of NQS was added and followed by 1.5 mL of buffer solution PH 10.The flask was completed to volume with distilled water and the absorbance was measured at 485 nm against blank.

REFERENCESText book of pharmaceutical analysis by P.D.chaithanya sudha Austin journal of analytical and pharmaceutical chemistry by Aida Makram Nouralla Altigani, Abdalla Ahmed Elbashir* ,University of Khartoum, Faculty of Science, Chemistry Department, Sudan .

El-Kommos ME, Emara KM, Alexandria Spectrophotometric determination of certain pharmaceutical secondary aromatic amines using p-dimethylamino-cinnamaldehyde J. Pharm. Sci. 1988; 2: 171-176. Aida Makram Nouralla Altigani, Abdalla Ahmed Elbashir* University of Khartoum, Faculty of Science, Chemistry Department, Sudan