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Pharmaceutical reagents, PDAB, FC, MBTH

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pharmaceutical reagents

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Pharmaceutical reagents, PDAB, FC, MBTH

  1. 1. PRINCIPLES & PROCEDURES INVOLVED IN USAGE OF REAGENTS IN PHARMACEUTICAL ANALYSIS Presented By Y.Divya M.Pharmacy First year-I sem SPMVV
  2. 2. CONTENTS • Introduction to reagents • Reagents a) PDAB b)Folin Ciocalteau c)MBTH • Reagent Profile • Principle & Mechanism • Procedure • Examples • Applications • Conclusion • References
  3. 3. 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
  4. 4. •It is an aromatic compound and chromogenic in nature. •Used for colorimetric estimation of amine group containing drugs. Reagent Profile: Synonym : Ehrlich’s reagent IUPAC Name : 4-(dimethyl amino)-benzaldehyde Molecular Formula : C9H11NO Molecular Weight : 149.9 Color : White or offwhite Solubility : soluble in alcohol, 0.1N HCl, water
  5. 5. 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.
  6. 6. General Reaction •More suitable for amines oximes. •NH2, NR2, NR3 are strong auxochromes. •But in aromatic compounds it is 100% colored.
  7. 7. Example:
  8. 8. Preparation Of PDAB Reagent: To 400mg of PDAB +2ml of conc H2SO4 +10ml alcohol Finally, make upto 100ml with distilled water
  9. 9. 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
  10. 10. 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 drug 10-50mcg/ml X conc of reagent Colored compound. Mini stability: 30min Max stability: 1hr Time(min) Opticaldensity
  11. 11. 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)
  12. 12. 3.Quantification Procedure: a) Calibration Plot by: Interpolation Regression y = mx + c b) Single point standardization: c) Double point standardization:
  13. 13. 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.
  14. 14. 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.
  15. 15. Reaction:
  16. 16. 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
  17. 17. 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
  18. 18. •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
  19. 19. 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
  20. 20. Cool to RT, make to 100ml with water +15g Lithium sulphate +5ml water + 1drop Bromine, reflux for 15min +5ml 85% Phosphoric acid + 10ml Conc HCl, Reflux for 1ohr. Dissolve 10g Sodium tungstate and 2.5g sodium molybdate in 70ml water
  21. 21. Orthophosphoric acid Converted to peiodic/ vanaldic acid, molybdic acid Results in Heteroyl acid mixture Undergoes reduction if analyte has oxidizing nature Molybdenum Blue Estimated colorimetrically at 400-800nm
  22. 22. 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: Aspirin Piroxicam Acetazolamide Sparfloxacin Pyridoxine HCl are estimated using this FC reagent
  23. 23. Example: Estimation of Omeprazole in given pharmaceutical formulation Principle: 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
  24. 24. 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
  25. 25. 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
  26. 26. MBTH is a chromogenic reagent used for colorimetric estimation of drug containing phenolic, aromatic amines and active methylene groups
  27. 27. 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
  28. 28. 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
  29. 29. 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
  30. 30. Oxidative Coupling Reactions: Step-I Formation of oxidation form of MBTH as an Intermediate
  31. 31. STEP-2 Formation Of ColoredComplexes: a) Reaction With Aldehydes:
  32. 32. b) Reaction With Phenols CH3
  33. 33. c) Reaction with Amines:
  34. 34. a) Reaction With aldehydes:
  35. 35. b) Reaction With Citrals:
  36. 36. •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:
  37. 37. Example Estimation of Lamotriginein givenpharmaceutical 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.
  38. 38. 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
  39. 39. 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
  40. 40. 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
  41. 41. 1. 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

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