Pradip vitamin
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  • 1. PRINCIPLES AND PROCEDURES INVOLVEDPRINCIPLES AND PROCEDURES INVOLVEDIN THE ANALYSIS OF VITAMINS ININ THE ANALYSIS OF VITAMINS INPHARMACEUTICAL PREPARATIONS ANDPHARMACEUTICAL PREPARATIONS ANDDOSAGE FORMSDOSAGE FORMS.Presented by:Presented by:PRADIPKUMAR.L.GHORIDEP.PHARMACEUTICSDEP.PHARMACEUTICSM.M.C.P BELGAUMM.M.C.P BELGAUM
  • 2. INTRODUCTION TO VITAMINS: Vital amines/growth factors/accessory factors. Any group of organic compounds required insmall amount to perform specific biologicalfunctions for normal maintenance of optimumgrowth and health of organism. Classification: Fat soluble vitamins: vit-A, vit-D, vit-E, vit-K. Water soluble vitamins: vit-C, B-complex, vit-H.
  • 3. Need for study: Essential nutritional factors and potent organic compoundsrequire great care in analytical control and their formulations. Analytical procedures based on chemical, physical, biological andmicrobiological methods are used in the assay. The methods of analysis of vitamins and its formulations should bebased on biological response. Chemical, physical and microbiological methods are only valid ifthey correlate directly or indirectly with biological activity. Care should be exercised as some vitamins exhibit speciesspecificity in that they are required in certain species of animalsand not in others.
  • 4.  Vitamin-A Vitamin-D Thiamine Niacin Vitamin-C
  • 5. *VITAMIN-A* Chemistry: Retinol (Alcoholic) Retinal (Aldehydic form) Retinoic acid (Acidic form) β-Carotene (Provitamin-A)RC11C12R11-cis form11-trans formR -CH2OH, -CHO, -COOH.
  • 6. TISSUE.SYNTHESIS OF GLYCO PROTEINS ANDMEVALONATE.CAROTENOIDS FUNCTIONS AS ANTI-OXIDANTSAND REDUCES RISK OF CANCER.DEFICIENCY:DEFICIENCY: NIGHT BLINDNESS, XEROPHTHALMIA,KERATOMALACIA, MAY ALSO AFFECT ONREPRODUCTION AND EPITHELIAL CELLS.PROPERTIES:PROPERTIES:1) THE FREE VITAMIN A ALCOHOL IS READILYOXIDIZED BY ATMOSPHERIC OXYGEN OR OXIDIZINGAGENT.2) VERY SENSITIVE TO LIGHT.3) VITAMINS OCCUR IN NATURE MAINLY IN THE FORMOF ESTERS WHICH ARE CONSIDERABLY STABLETOWARDS OXIDATION.
  • 7.  Three methods: Bioassay based on RAT or CHICKEN growth or on liverstorage. Colorimetric method/ Carr-price method. UV-spectrophotometric method.COLORIMETRIC METHOD:COLORIMETRIC METHOD:CARR-PRICE METHOD: Anhydrous Antimony Tri chloride (SbCl3) in chloroformreacts with a dilute solution of vitamin A to form a transientblue color. Reaction occurs between Antimony tri chloride andunsaturated side chain of vitamin A. The carotene, polyeneacids and various other material present in natural oilsproduce same color. The color rapidly reaches maximum intensity and just asrapidly it fades. The reading must be taken within 10-15seconds
  • 8. Sb Cl3+Blue color(λ max-550nm)DisadvantagesDisadvantages:: Vitamin A is a recemic mixture containing 1/4thof cis-formand 3/4thof Trans form in oils. While the biologically activeform is the Trans form. The Carr-price reaction does not differentiate between thetwo isomers, so it is not a specific test for all-transcompounds. It does not give a stable blue color.R
  • 9.  Applications: This test can be used as an identification test forvitamin A The blue color method can be used in determiningVitamin A in agricultural feeds and in oleomargarines. The method extensively used in biochemical workbecause of its great sensitivity.
  • 10.  METHOD2: MODIFICATION OF CARR-PRICE : Formation of blue color by reaction with vitamin A and activatedGlycerol-1,3-dichlorohydrin. Activation of reagent may be accomplished by distillation ofglycerol 1,3-dichlorohydrin in presence of antimony tri chloride. Maximum absorbance at 550nm. Color is more stable than the color produced in Carr-pricereaction. The intensity of solution reaches 2min. and the color is stable foradditional 3min.Disadvantages:Disadvantages: Not as sensitive compared to carr-price method.RCH2ClCHOHCH2Clblue color 550nmSb Cl3+
  • 11. SPECTROPHOTOMETRIC METHOD:Method 3: DIRECT SPECTROPHOTOMETRIC METHOD (IP-1996 method): Saponified sample is extracted with ether. Ether is evaporated. Residue isdissolved in isopropanol. Measure the absorbance at about 300, 310, 325 and 334 nm. Determine thewavelength of maximum absorption. If the wavelength of maximum absorption lies between 323 and327 nm and the absorbance at about 300 nm relive to that atabout 325 nm does not exceed 0.73, a corrected absorbance isderived from the equation. A325 (CORR.) = 6.815 A325 – 2.555 A310 – 4.260 A334 Calculate the potency of the sample from the expression. Vitamin A potency in Units per g = A3251%,1 CM) X 1830
  • 12.  If the corrected absorbance lies within +/- 3.0% of theuncorrected absorbance, ignore the corrected absorbanceand calculate the potency from the uncorrectedabsorbance. If the wavelength of maximum absorption lies outside therange 323 to 327 nm, or if the relative absorbance atabout 300 nm exceeds 0.73, the unsaponifiable fraction ofthe sample must be further purified by chromatography.
  • 13. Method 4: CONVERSION OF VITAMIN A TOANHYDRO VITAMIN A: Vitamin A may be easily converted toAnhydro vitamin A (II) in anhydrous solvents in the presenceof traces of mineral acids or strong acids. Anhydro-vitamin A results in a considerable displacement ofabsorption towards visible region with absorption maxima at358nm, 377nm and 399nm in benzene. There is also anincrease in absorptivity.R mineral/organic acidanhydro vit A. 1,3-dihydro vitamin
  • 14.  Formula:USP units /ml. aliquot = __ A___0.0122 A  Increase in absorbance caused bydehydration.0.0122 Increase in absorbance corresponding to1USP unit of vitamin A This method can be applied for the vit-A concentrates, fishoils, oleomargarine, butter etc. this has been reported to bevery specific.
  • 15. *VITAMIN D**VITAMIN D* Steroidal vitamin/ anti-ricketic vitamin. Two forms:◦ Ergocalciferol (vitamin D2)◦ Cholecalciferol (vitamin D3)Functions:Regulates plasma levels of calcium and phosphate.In osteoblasts of bone, vit D stimulates uptake of calcium anddeposition of calcium phosphate.1, 25 di-hydroxy cholecalciferol is active form also known ascalcitrol. Diffiency:Rickets in children and osteomalacia in adults.
  • 16.  Properties:1) It is not affected by dilute acids, alkalis or air2) Vitamin D3 is stored in the skin as 7-dehydro cholestrolwhich is converted to activated vitamin D2 by sunshine orultraviolet irradiation.Ergosterol vitamin D2. (Activated form)7-dehydro cholestrol vitamin D3(Activated form).3) Biological assay methods uses rats and chicks areavailable for evaluation of vitamin D activity.4) Physiochemical procedures employ the methods of uv-spectrophotometry / colorimetry.CH3CH3CH3CH2OHCH3
  • 17. Method 1: UV-SPECTROPHOTOMETRY: Both D2 and D3 have an absorption maximum at 265nm in hexane. TheA1%1cm of D2 is 459nm that of D3 is 474. The determination involving uv-spectrophotometry suffers from lack ofspecificity. The interference of interfering substances can be avoided bychromatographic separation. TWO STEP ADSORPTION CHROMATOGRAPHIC METHOD: Passage of oil through an activated earth this removes vitamin A,sterols and irradiation products of ergosterol other than Vitamin D. The second adsorption step removes impurities due to first adsorbent,unsaturated compounds of squalene type and vitamin A decompositionproducts. The eluate is collected and analyzed spectroscopically.
  • 18. COLORIMETRIC METHOD Many colorimetric methods have been proposed for thequantitative determination of vitamin D.  “MODIFIED CARR-PRICE METHOD”: The glycerol-1,3-dichloro hydrin reacts with vitamin Dproducing blue color. The color is stable for several hours. The reaction is specific: Ergosterol gives pink-orange color and slowly turns to afluorescent green. 7-dehydro cholesterol produce no color but after hours producefaint pink color. Cholesterol does not show reaction or absorption.
  • 19.  REACTION WITH ORGANIC ALDEHYDE: This is based on reaction between Vitamin D and organicaldehyde (vanillin, furfural or anisaldehyde) andsulphuric acid or perchloric acid.CH3CH3CH3CH2OHCH3CHOOMeOHH2SO4/HCLO4Green Color (625nm)+CH3CH3CH3CH2OHCH3H2SO4/HCLO4Green Color (625nm)OHCHO+CH3CH3CH3CH2OHCH3O CHOH2SO4/HCLO4Green Color (625nm)+
  • 20.  MODIFIED CARR-PRICE METHOD: The modified method has improved sensitivity andreproducibility by addition of acetyl chloride to SbCl3. Mixture of vitamin A and vitamin D2 is dissolved in benzeneafter passing through a column of bentonite activated withacid. The treatment selectively destroys vitamin A and the eluateis then analyzed using antimony trichloride (SbCl3) reaction. CH3CH3CH3CH2OHCH3Green Color (625nm)CH3COClSb Cl3 ++
  • 21. VITAMIN DVITAMIN DCONTD…CONTD… Vitamin D gives color with glycerol-1,3-dichlorohydrin in presence of acetyl chlorideCH3CH3CH3CH2OHCH3Green Color (625nm)CH3COClCH2 ClCH OHCH2Cl++
  • 22. *THIAMINE* Chemistry:Chemistry:Contains pyridine and thiazole ring structures. Functions:Functions: Prosthetic group in decarboxylation. and transketolase reactions. Deficiency disorders:Deficiency disorders: Beri beri. Properties:Properties:1) White crystalline powder with slight characteristic odour.2) It is hygroscopic.3) Soluble in water, glycerol, alcohol. Practically insoluble in ether, benzene,hexane, and chloroform Chemical, biological and microbiological methods are available for the analysis ofthiamine.◦ The bio-assay is based on prevention and cure of polyneuritis and weight gainin animals. Microbiological methods using Phycomyces blakesleeanus andLactobacillus ermenti are available. A yeast fermentation method is alsoNNN SCH3NH3+ CH2 CH2 O HCH3. 2 Cl+-2
  • 23.  FLOURIMETRIC METHOD (Thiochrome)1. Thiamine is quantitatively isolated from the foods, biologicals,and pharmaceuticals usually by boiling with dilute acids andtreatment with enzyme preparations containing phosphataseswhich will free thiamine from its natural complexes.2. Protein substances must be digested with proteolytic enzyme likepapain.3. Purification of extract is done by passing through zeolite, aninorganic ion exchanger and thiamine is retained in the zeolite.4. Add acidified potassium chloride to elute thiamine.5. Further it is oxidized with alkaline potassium ferricyanide toproduce thiachrome. This compound is having blue fluorescence.NNN SCH3NH3+ CH2 CH2 O HCH3NNN SCH3CH2 CH2 O HCH3NK3 Fe (C N)6+2 Cl-
  • 24.  SILICO TUNGSTIC ACID METHOD (Gravimetry):{H4Si(W3O10)4 nH2O } Prepared by One mole of Silicic acid for every twelve moles ofSodium Tungstate. Thiamine in tablets and solutions may be determined byprecipitation with silicotungstic acid. The sample is dissolved in acidified water and heated to boiling. The precipitate is filtered washed with acid and water finallywith acetone. Then its weighed to constant weight. Each gram is equivalent to 0.1936 g of thiamine hydrochloride. 
  • 25.  COLORIMETRIC METHOD: Method 1: with p-Amino acetophenone:  The extraction is done as given in thiochrome method. Thiamine couples with diazotized p-Amino Acetophenonewhich has an absorption maximum at 520nm. This method can be used to determine thiamine in presenceof phosphorylated thiamine and is useful in urine analysis. This method is not recommended for assay of materials richin protein content and low in thiamine. Results of this method agrees with the biological method.NNN SCH3NH3 CH2 CH2 O HCH3NC O C H3N Hcomplexdiazotised p-amino acetophenone+520 nm
  • 26.  Method 2: using 6-Aminothymol.  A color reaction between thiamine and diazotized 6-Amino thymol is seen. This method is simple fast andno interference is seen between the degraded thiamineand 6-Aminothymol.NNN SCH3NH3 CH2 CH2 O HCH3OHCH (CH3)2CH3N NHcolor complex+
  • 27. Miscellaneous Methods:Miscellaneous Methods: Method 3: Non-aqueous titration: Thiamine HCl can be titrated with perchloric acid in glacial acetic acid solution, if anexcess of mercuric acetate is added. Both the nitrogen are titrated. p-Naphthol-benzein and quinaldine red are suitable indicators.Method 4: Argentometric Method:. Total chlorine in thiamine HCl can be determined by dissolving in acidified water(HNO3 ) added with excess of silver nitrate. The precipitate is filtered and washed.The filtrate is then titrated with 0.1N Ammonium Thiocyanate.1ml of 0.1N AgNO3 is equivalent to 0.003546 g of chlorine. Method 5: Chloride as hydrochloride is determined by titration with 0.1 N sodium hydroxide topH7 using bromothymol blue as indicator.Each ml of 0.1 N NaOH is equivalent to 0.003546 g of chlorine Method 6 The nitrate in thiamine mononitrate is determined by precipitation with nitron(1,4-Diphenyl-3-phenylamino-1,2,4-triazolium hydroxide inner salt)from an acidified solution. Nitron is a compound C20H16N4 used in the qualitative and quantitative determinationof nitric acid with which it forms an insoluble nitrate
  • 28. *NIACIN (NICOTINIC ACID)*Chemistry:Chemistry:◦ It’s a heterocyclic. 3-pyridine carboxylic acid.Functions:Functions:◦ It functions as oxidising co-enzymes of many dehydrogenasesDeffiency disordersDeffiency disorders:◦ Pellagra, characterised by dementia, dermatitis and diarrhoea.PROPERTIES:PROPERTIES:1. Solubility: Soluble in boiling water and in boiling ethanol (95%). Sparinglysoluble in water. Very slightly soluble in chloroform. Practically insoluble inether. It dissolves in dilute solutions of alkali hydroxides and carbonates.2. Non-hygroscopic and stable in air.3. It has absorption maximum in UV at 262nm.4. Identification test:Thiamine is dissolved in water, and neutralized to litmus paper with 0.1Msodium hydroxide, add 3 ml of copper sulphate solution; a blue precipitate isNCOOH
  • 29.  ACID BASE TITRATION:  Nicotinic acid can be titrated with NaOH usingphenolphthalein solution as indicator.  COLORIMETRIC METHOD USING CYANOGENBROMIDE:  This is based on a color reaction of pyridine andλ,β− unsubstituted derivatives. Cyanogen bromide breaks onecarbon-nitrogen linkage and produces a color compound uponaddition of amine or ammonia.NCOOHNaOHNCOONaH2 O++NCOOHSO3HNH2CNBrcleavage of CN bond of pyridine by CNBr red color measured spectroscopically+ +
  • 30.  DETERMINATION OF NICOTINAMIDE ACID-BASE TITRATION:◦ Nicotinamide when boiled with sodium hydroxide solution, itreleases the nitrogen of the amido group in the form of ammonia,which can be collected in sulphuric acid and determined bytitration.   The liberated ammonia collected in sulphuric acid anddetermined by titration with NaOH.2NH3 + H2SO4----------- (NH4)2(NH4)2 + 2 NaOH----- Na2SO4 + 2 H2O + 2NH3 End- point being determined using phenolphthaleinindicator.NCONH2alkaline hydrolysisNCOOHNH3Nicotinic acid+
  • 31. HOFFMAN REARRANGEMENT REACTION: Nicotinamide undergoes a Hoffmann rearrangement to form a 3-amino pyridinewhich reacts as an aromatic amine. It can be diazotized and coupled with N-(1-naphthyl)- ethylene di-amine to producered colored azo compound. This is the basis of USP assay of nicotinamide in capsules, injectables and tablets. NCONH2 hoffman -corearrangementNNH23-Amino PyridineNaNO2/HClHNO2 <8*CN N Cldiazotised compoundNH-CH2-CH2-NH2. HClN N NH-(CH2)2NH3red colored azo-compound++ +
  • 32. VITAMIN C (ASCORBIC ACID) Chemistry:Chemistry:◦ It’s a heterocyclic furan-2-onederivative Functions:Functions:◦ Anti-oxidants, helps in synthesis of collagen andhydroxylation reactions. Defiency disorders:Defiency disorders:◦ Scurvy, swollen joints, hemorrhages in various tissuesand delayed wound healing. Properties:Properties:1. It occurs as a white or slightly yellow crystal or powder.2. In dry state it is stable to air but degradation seen in presence of somemetals and it is unstable to light.3. Many chemical methods have been reported and this is based on thereducing properties of ascorbic acid.OOOHOHCHOHCH2OH
  • 33.  VOLUMETRIC METHODS Iodometric method: Ascorbic acid content of pure solutions or the purity of thesubstance can be determined by titration with 0.1 N Iodinesolution. Modifications of this method using instruments likepotentiometric titration or polarized platinum-platinumelectrodes and a dead-stop end point, have been applied topharmaceutical products.O OOH OHHOHCHOH2CI2O OO OHOHCHOH2C2HI+ +Dehydro ascorbic acidAscorbic acid
  • 34.  2) Titration with 2,6-DICHLORO PHENOL-INDOPHENOL:  A typical procedure for eliminating interfering substancesconsists ofa) The conversion of total ascorbic acid to dehydro ascorbic acid bypassing it through Norit or by using ascorbic acid oxidase.b) The reduction of dehydro ascorbic acid to ascorbic acid withhydrogen sulphide at pH 4 to 7c) The titration of ascorbic acid with dichloro phenol –indophenol.OOOHOHCHOHCH2OHNaO N OClClOOOOCHOHCH2OH2,6- dichloro phenol indo phenol (pink or blue color) dehydro ascorbic acidNaO NClOHClHreduced form (color less)+ +
  • 35.  METHOD 3: Titration With N-BROMOSUCCINIMIDE: N-Bromo succinimide in aqueous solution readilyoxidizes an aqueous solution of ascorbic acid to dehydroascorbic acid, while N-Bromo succinimide is irreversiblyreduced to succinimide with the formation of HydrogenBromide. After all the ascorbic acid has been oxidized, theslightest excess of N-Bromo succinimide, In presence ofpotassium Iodide, liberates iodine. The end point is being determined using starch solutionadded in the end.OOOHOHCHOHCH2OHCH2-COCH2CON BrOOOOCHOHCH2OHCH2-COCH2CONH H Br+ + +
  • 36.  COLORIMETRIC METHODSMETHOD 1: Using 2,4-DINITRO PHENYLHYDRAZINE: Dehydro ascorbic acid couples with 2,4-dinitro phenylhydrazine to form an osazone which develops a red color instrong sulphuric acid. In this reaction, de-hydro ascorbic acid is used so thetitrimetric impurities like –SH, etc. are degraded previously,so pure ascorbic acid react with DNP and quantized theexact result.     OOOOCHOHCH2OHNH-NH2NO2O2NH2SO4OONNCHOHCH2OHNHN NO2O2NNO2NO2osazone (pale brown to red color)H+
  • 37.  METHOD 2: Using 4-METHOXY-2-NITROANILINE: This is based on ability of ascorbic acid to couple withDiazonium compounds. Ascorbic acid with diazotized 4-methoxy-2-nitro aniline formsa deep blue compound. This method has an advantage as it is specific for ascorbic acid. This method has been applied to pharmaceutical preparations,natural juices, powdered milk and fortified feed. OO CHOHOHOHCH2OHHNO2< 8*, NaNO2/HClNNO2OMeN-ClOO CHOHOH OHCH2OHDeep blue color complexNNO2OMeN-Cldiazotised 4-methoxy 2-nitroaniline+
  • 38. RIBOFLAVIN (VITAMIN B2) It is also called as lactoflavin. Properties: Yellow to orange-yellow, crystalline powder with slight. Odour Deffiency: Solubility: Very slightly soluble in water; more soluble in saline solution thanin water; practically insoluble in chloroform, in ethanol (95%)and in ether. Aqueous solutions exhibit an intense yellow color-green fluorescence at pH6. Specific rotation is Between –115oand –135o, determined in a 0.5% w/v solution incarbonate-free 0.05M sodium hydroxide . Riboflavin shows absorption maxima at 224, 267, 373, 445, and 475nm. Irradiation either with uv or visible light, of alkaline solutions, produces luminoflavin,irradiation of acid or alkaline solutions produces luminochrome, a blue fluorescentsubstance. Reducing agents such as, sodium hydrosulphite, reduce riboflavin to a dihydrocompound, leucoflavin which is not fluorescent. But this is reversible and leucoflavin isreadily oxidized back to riboflavin by atmospheric oxygen.NCH2NNNCH3CH3OOCHOHC HOHC HOHCH2OH
  • 39. METHOD 1: FLUORIMETRIC METHOD There are three methods are in use influorimetric determination of riboflavin; Direct determination Direct additive determination Adsorptive additive determination
  • 40. FLUORIMETRIC METHOD: DIRECTDETERMINATION: This method is employed for the mixtures which are free of interfering pigmentsor substances and contain relatively high concentration of riboflavin. An appropriate quantity is weighed and add with boiling distilled water andshaken for few minutes, if required, boil the solution. It is then centrifuged. Sample solution: a suitable aliquot of clear liquid is diluted appropriately toyield concentration of 0.2mcg/ml. Blank solution: add a few granules of sodium hydrosulphite to the aliquot ofsample solution. This shows indication of purity of solution. Measurement and calculation: Readings must be taken as rapidly as possible. Mcg of riboflavin = A – C * dilution factorB – C wt of sample (g.) A reading of unknown concentration. C unknown blank B reading of standard D reading of standard blank.
  • 41. FLUORIMETRIC METHOD: DIRECT ADDITIVEMETHOD The interference of other substances can be avoided by this method. In this method theaddition of the known quantity of riboflavin to the assay solution is used to compensatefor interfering substance which may absorb the incident or fluorescent light. Sample treatment: samples of natural origin should be subjected for enzymatichydrolysis to digest starchy substances and to “free” any “bound” riboflavin. Standard solution I: 40mcg/ml of USP reference standard in 20% of ethanol. Standard solution II: dilute solution I such that it contains 1.6mcg of riboflavinindistilled water. Measurement and calculation: Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factorB- (0.94A) 16 wt of sample (g) A fluorescence of sample solution B fluorescence of standard II C blank solution ( obtained by adding HYDROGEN SULPHITE to standard solution II andthis should be repeated until successive additions shows no deflections) 1.07 and 0.94 constants due to change in volume of measurement.
  • 42.  USP method: in this an additional purification step isinvolved by treating with potassium permanganateand this produces oxidation of interferingsubstances. Excess permanganate is removed bytreating with hydrogen peroxide.Applications: This method can be used with vitamin mixtures ofwafers, flour enrichment mixtures and simplepharmaceutical preparations, provided they aresubstantially free from coloring or fluorescencingmatter.
  • 43. FLUORIMETRIC METHOD: ADSORPTIVE ADDITIVEDETERMINATION: In this method most of the interfering substances are eliminated by anadsorption step and those not eliminated are compensated by addition of aknown quantity of riboflavin to the assay solution. . this method can beapplied to universally to all samples. Extraction: the extraction procedure is same as the direct additivedetermination. Adsorption and elution: An aliquot of sample solution or suitable dilution of the sample extract ismeasured accurately and passed through an adsorption column. The columnis prepared by using fluorosil. After elution the column is washed with hotdistilled water and the excess water is drained using vacuum. The riboflavin is being retained in the column is then eluted using hot aceticacid-pyridine eluent is collected and mixed well.
  • 44. FLUORIMETRIC METHOD: ADSORPTIVEADDITIVE DETERMINATION: Standard solution I: 40mcg/ml of usp reference standard in 20% ofethanol. Standard solution III: dilute solution I such that it contains 1.6mcg ofriboflavinin in acetic acid pyridine mixture. Measurement and calculation:Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factorB- (0.94A) 16 wt of sample (g)A fluorescence of sample solutionB fluorescence of standard IIIC blank solution ( obtained by adding hydrogen sulphite to standardsolution II and this should be repeated until successive additions shows nodeflections)1.07 and 0.94 constants due to change in volume of measurement.
  • 45. METHOD 2: SPECTROPHOTOMETRIC METHOD (IP 1996): Procedure: Carry out the procedure in subdued light. Weigh accurately about 65 mg and transfer to an amber-glass 500-ml volumetric flask, suspend in 5 ml of water, ensuring that it iscompletely wetted. Dissolve in 5 ml of 2M sodium hydroxide. As soon as dissolution iscomplete add 100 ml of water and 2.5 ml of glacial acetic acid anddilute to 500.0 ml with water. To 20.0 ml of this solution add 3.5 ml of a 1.4% w/v solution ofsodium acetate and dilute to 200.0 ml with water. Measure the absorbance of the resulting solution at the maximum atabout 444 nm. Calculate the content of C17H20N4O6 taking 328 as the value of A(1%, 1cm) at the maximum at about 444 nm.
  • 46. METHOD 2: Riboflavin has a characteristic absorption spectrum in water with amaximum at 267nm. This is the basis of the method. This method isbased on the assumption that extraction with chloroform will removeimpurities from an aqueous solution. Procedure: Carry out in subdued light. Weighed about 20mg of riboflavin transferred to a 1000ml volumetricflask, dilute the solution and add few drops of 1N NaOH. Shake gently until the solution is complete, then add few drops of 5Nacetic acid and dilute upto the mark. A 20ml of aliquot of solution is taken and shaken with 25ml of chloroformfor 1min. separate the chloroform layer and discard. The extraction isrepeated twice.
  • 47. METHOD 2 CONTD… The absorbance of clear aqueous layer isdetermined at 267nm.with water as areference. Repeat the procedure with referencesample.% Riboflavin= 100 (As/Ar) . (Wr/Ws) Ws and As weight in mg and absorbanceof sample respectively. Wr and Ar corresponding values ofstandard and reference sample of riboflavin.
  • 48. REFERENCES: Pharmaceutical analysis by Takeru Higuchi et.al. page no. 649-707. Bentley and Drivers textbook of pharmaceutical chemistry. 8thedition. Vogel’s textbook of quantitative chemical analysis. IP-1996. Instrumental methods of chemical analysis by G R Chatwal and Sham KAnand. Enlarged edition. 2005. p no. 2.413-2.414 Guyton textbook of medical physiology. 11thedition. Biochemistry by Leninger 4thedition 2005. Biochemistry By Jeremy M Berg, John L Tymoczko And Lubert Stryler. 5thEdition
  • 49. Thank You