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Pdac,ninhydrin,1,2 naphthoquinone reagents

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Pdac,ninhydrin,1,2 naphthoquinone reagents

  1. 1. 1 Principles and procedures involved in usage of reagents in pharmaceutical analysis PRESENTED BY: T. LAKSHMI BHAVANI (2015MPH40023) Pharmaceutical Analysis -1sem Under the guidance of: Dr. G.RAJITHA MADAM M.Pharm.,Ph.D SPMVV - TIRUPATHI
  3. 3. INTRODUCTION  The 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.
  4. 4. PARA DIMETHYLAMINO CINNAMALDEHYDE(PDAC) REAGENT • Para 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.
  5. 5. Reagent profile:  Synonym : Ehrlich’s reagent  IUPAC name: 4-(dimethyl amino) cinnamaldehyde  Structure:  Molecular formula : C11 H13 NO  Molecular weight: 175.23 g/mol H CHN CH3 CH3 CH C O
  6. 6.  Color : dark yellow powder  Solubility:  soluble in warm dioxane(500mg/ml- clear ,deep greenish yellow solution)  soluble in mixture of chloroform: ethanol (1:1).
  7. 7. CH N CH3 CH3 CH CH O + R NH2 N CH3 CH3 R N + HOH PDAC Amines Dehydration Schiff base General reaction
  8. 8.  Chromophore formed with PDAC is more intense than with PDAB because of additional double bond. EXAMPLE: CH N CH3 CH3 CH CH O + N CH3 CH3 N + HOH PDAC Dehydration Schiff base NH2 Aniline
  9. 9. PRINCIPLE  The reaction between primary amine and carbonyl compound form the condensation product are referred as schiff’s base.  The primary amine group which is present in the structure of drug react with the carbonyl group in PDAC reagent and forms schiff’s base (colored compound) which can be measured by using colorimetry.
  10. 10. MECHANISM OF PDAC REAGENT: CH N CH3 CH3 CH CH O + R NH2 N CH3 CH3 R N + HOH PDAC 10 Amines H OH N CH3 CH3 R NH Schiff’s base(colored compound)
  11. 11. PREPARATION OF REAGENT  Preparation 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.
  12. 12. APPLICATIONS OF PDAC REAGENT For 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.
  13. 13. EXAMPLES 1.ASSAY OF RANITIDINE PRINCIPLE: The primary amine group of ranitidine reacts with carbonyl group of PDAC reagent and forms schiff’s base which can be measured colorimetrically.
  15. 15. PROCEDURE: PREPARATION OF STOCK SOLUTION: A standard stock solution containing 1000µg/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.
  16. 16. 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.
  17. 17. ASSAY OF RANITIDINE HCl TABLETS 3 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.
  18. 18. 2.Estimation of Lenalidomide in pharmaceutical formulation by PDAC reagent PRINCIPLE:  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 schiff’s base which can be measured colorimetrically.
  19. 19.  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.
  20. 20. + N CH3 CH3 OHN H NH2 N O O O H2SO4 - H2O N H N N O O O N CH3 CH3 Lenalidomide PDAC Schiff's base(red colored compound)
  21. 21. ASSAY OF LENALIDOMIDE CAPSULES  Aliquots of LLD solution (100μg/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.
  22. 22. NINHYDRIN REAGENT  It 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 Ruhemann’s purple.
  23. 23. REAGENT PROFILE Synonym: Indanetrione IUPAC name: 2,2-dihydroxy indane-1,3-dione. Structure: Molecular formula:C9 H6 O4 Appearance/form : white crystalline powder Solubility : soluble in water and alcohol. slightly soluble in ether and chloroform. O O OH OH
  24. 24. PRINCIPLE  Ninhydrin 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, Ruhemann’s purple.
  25. 25. Reaction with Amino acids: O O OH OH R NH2O OH + O O N O O + C O H R + co2 H2O+ Ninhydrine Amino acid Purple coloured complex
  26. 26. O O OH OH + O O N O O + + co2 H2O+ Ninhydrine Phenyl alanine Purple coloured complex H NH2 O OH CH3 O REACTION
  27. 27. REACTION WITH IMINO ACIDS O O OH OH + Ninhydrine L- Proline Red coloured complex O NH OH O OO O OH N
  28. 28. REACTION WITH PRIMARY AMINE O O OH OH + Ninhydrin Primary amine R NH2 O O N R Colored compound
  30. 30. N R H O O - O H H N R H O O H R-CHO NH2 O O H N O O O O - H2O,H+ H+ Ruhemann’s purple
  31. 31. APPLICATIONS  Used 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.
  32. 32. 1. Estimation of ampicillin in pharmaceutical formulation by Ninhydrin reagent PRINCIPLE:  Ampicillin is an antibacteral drug which is chemically (6R)-6(α-phenyl)-O-glycyl amino pencillanic acid.  It contains primary amine group so it can be estimated by using ninhydrin. EXAMPLES
  33. 33.  Tautomerised form of ninhydrin react with ampicillin to form schiff’s 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 λ max.
  34. 34. PROCEDURE PREPARATION 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 1000µg/ml stock solution.
  35. 35. 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.
  36. 36. REACTION O O OH OH O O O + H NH2 O NH N S O OH CH3 O CH3 N O NH N S O OH CH3 O CH3 O O H O O NH2 H O OO O N Ninhydrin 1,2,3-indanetrione Ampicillin Schiff's base Intermediate amineColored complex
  37. 37. ANALYSIS OF AMPICILLIN CAPSULES  Two 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.
  38. 38. 2.Estimation of levodopa in pharmaceutical formulation by ninhydrin reagent PRINCIPLE  Levodopa {(-)-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.
  39. 39. REACTION OH OH NH2 O OH + O O OH OH OH OH O OH OH + O O OH OH O O O O N O O Levodopa Ninhydrin Hydrindatin Ruhemann's purple
  40. 40. ANALYSIS OF CAPSULES  Weigh accurately 100mg of levodopa , add 60ml of DMF and stirred.  It was filtered with whatmann filter paper size≠1 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.
  41. 41.  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 λmax of levodopa by using blank solution.
  42. 42. 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
  43. 43. REAGENT PROFILE  SYNONYM: Sullivan reagent Follin’s reagent  STRUCTURE:
  44. 44. PRINCIPLE  When 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.
  45. 45. GENERAL RECTIONNa O O SO O O + R NH2 - OH O O NH R - OH O O NH NQS Aliphatic amine colored chromogen NH2 + O O SO O O Na
  46. 46. APPLICATIONS  Sulpha drugs  Succinyl sulphathiazole  Chloramphenicol  Nimesulide  Nifedipine  Nitrendipine
  47. 47. EXAMPLES 1.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 λmax of 453nm.
  48. 48. REACTION Cefadroxil . H2O + OH NH2 O O O N N S O CH3 OOHOrange colored product NQS
  49. 49. ANALYSIS OF CEFADROXIL TABLETS  5 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.
  50. 50. 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.
  51. 51. N O CH3 NH CH3 NH2 + O O S OO ONa N O CH3 N CH3 NH O O O O 2 + 2 NaHSO4 Primaquine 1, 2 - Naphthoquinone - 4 sulfonate Brown coloured compound Sodium hydrogen sulphate REACTION
  52. 52. PROCEDURE: PREPARATION OF STANDARD AND SAMPLE SOLUTION  Stock standard solution of Primaquine diphosphate (PQ -200 μg/ 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.
  53. 53.  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.
  54. 54. Analysis of primaquine tablets  A 2.0 mL of 200 μg ∕ 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.
  55. 55. REFERENCES  Text 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 .
  56. 56.  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