Steroids and Alkaloids

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PRINCIPLE AND PROCEDURE INVOLVED IN THE METHOD OF ANALYSIS OF STEROIDS & ALKALOIDS:

PRINCIPLE AND PROCEDURE INVOLVED IN THE METHOD OF ANALYSIS OF STEROIDS & ALKALOIDS A.Solairajan , 1 st year M.Pharm (analysis)

PowerPoint Presentation:

Steroids:- Adrenocortical steroids Cortisone Hydrocortisone Progesterone Androgens chloesterol

PowerPoint Presentation:

Alkaloids:- Digitoxin Digoxin Strophanthin

Steroids:-:

Steroids:- Steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. The steroids of natural origin produced by adrenal cortex,corpus lutem,placenta , testis, and ovaries. Examples of steroids include:- The dietary fat cholesterol, The sex hormones estrogen and Testosterone

Adrenocortical steroids:-:

Adrenocortical steroids:- The adrenocortical steroids include substances influencing carbohydrate, fat, protein, electrolyte and water metabolism. It has C-21 steroid nucleus and the pregnene series. Two types of steroids include this class Cortisone Hydrocortisone

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Cortisone:- Hydrocortisone:-

Methods of analysis:-:

Methods of analysis:- There are two methods Basic methods, Other methods. Basic methods Reaction of the α -keto group with tetrazolium salts. Reaction of 17,21 dihydroxy 20 keto group with phenyl hydrazine

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Other methods:- Reaction of the α - ketol group Reaction of the 17,21-dihydroxy 20-keto group Reaction of the α , β -unsaturated carbonyl group in Ring A Reaction of the oxygen function at C-11 Reaction of steroid esters with hydroxylamine

Basic methods:-:

Basic methods:- Reaction of the α -keto group with tetrazolium salts α -ketol group (-CH OH-CO) present in the adrenocoricoids react with alkaline TPTZ and reduces into red formazon complex which is determined by spectrophotometrically at 490 nm. It is used to determine α -ketolic steroids in the structure.

Determination of Desoxy corticosterone acetate in oil solution:-:

Determination of Desoxy corticosterone acetate in oil solution:- Reagents:- Dil. Tetra methyl ammonium hydroxide (10% in100ml of 95% alcohol) TPTZ (500mg in100ml of 95% alcohol) Standard solution:-10 mg of Desoxy corticosterone in 100ml of alcohol.

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Preparation of sample:- 5mg of oil solution of steroid+50 ml of isooctane-alcohol Extracted with 6 times with 20 ml of isooctane-alcohol Combined extracts evaporate to dryness Residue dissolved in alcohol Diluted to 50 ml with alcohol

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Development of colour:- Sample Standard Blank 1 ml of sample + 9 ml of alcohol + 1 ml of TPTZ+1 ml of TMAH 1 ml of standard + 9 ml of alcohol + 1 ml of TPTZ+1 ml of TMAH 9 ml of alcohol + 1 ml of TPTZ + 1 ml of TMAH Wait for 20 mts Absorbance measured at 490 nm

Calculation:-:

Calculation:- Weight of Desoxy corticosterone = 50 × C s A I in oil solution A S C s = concentration of std.solution A I = absorbance of sample solution A S = absorbance of std.solution

Reaction of 17,21-dihydroxy 20-keto group with phenyl hydrazine:- :

Reaction of 17,21-dihydroxy 20-keto group with phenyl hydrazine:- 17,21-dihydroxy 20-keto group of steroids react with phenyl hydrazine and sulfuric acid Yellow chromogen Measured at 410 nm

Other methods:-:

Other methods:- Reaction of the α -ketol group α -ketol group of adrenal steroids+ Copper arsenic molybdate reagent Colour produced & measured at 660 nm

Reaction of the 17,21-dihydroxy 20-keto group:

Reaction of the 17,21-dihydroxy 20-keto group 17,21-dihydroxy 20-keto group of steroids oxidised by bismuthate and produce 17-keto steroids which is determined by zimmermann reaction

Reaction of the α,β-unsaturated carbonyl group in Ring A:

Reaction of the α , β -unsaturated carbonyl group in Ring A α , β -unsaturated carbonyl group + 2,4 dinitro Phenyl hydrazine 2,4 dinitro phenyl hydrazone (red colour) Measured absorbance at 390 nm

Reaction of the oxygen function at C-11:

Reaction of the oxygen function at C-11 Cortisone+ Diphenyl amine reagent Violet chromogens Measured at 530 nm

Reaction of steroid esters with hydroxylamine:

Reaction of steroid esters with hydroxylamine It is used for steroid ester determination Steroid ester+ Hydroxyl amine hydroxyl amine complex + Ferric chloride purple colour measured at 540 nm

Progesterone:-:

Progesterone:- Progesterone also known as P4 ( p regn- 4 -ene-3,20-dione) is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans and other species. Progesterone consists of four interconnected cyclic hydrocarbons. Progesterone contains ketone and oxygenated functional groups, as well as two methyl branches.

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Progesterone Methods of analysis:- Basic methods Reaction with 2,4-dinitro phenylhydrazine Direct gravimetry Other methods

Reaction with 2,4-dinitro phenylhydrazine:

Reaction with 2,4-dinitro phenylhydrazine This method is used to determine the presence of progesterone and anhydro hydroxy progesterone. α , β - unsaturated carbonyl group of steroids + 2,4-dinitro phenylhydrazine 2,4-dinitro phenylhydrazone

Determination of progesterone in oil solution:-:

Determination of progesterone in oil solution:- Preparation of sample:- oil solution of progesterone containing 20 mg steroid+40 ml of petroleum ether-alcohol. Extracted with five 20 ml of petroleum ether- alcohol Combined extracts evporated to dryness Residue obtained

Precipitation of progesterone bis 2,4-dinitro phenyl hydrazone:-:

Precipitation of progesterone bis 2,4-dinitro phenyl hydrazone:- 75mg of 2,4-DPH+30 ml of alcohol added to the residue reflux for 15 mts . 1ml of con.HCL reflex for 15 mts cooled,ppt formed Ppt washed with five 10 ml of petroleum ether Finally wash with 0.5N HCL Filter free from color Ppt dried at 105°c

Calculation:-:

Calculation:- Weight of progesterone in sample obtained by multiplying the weight of precipitate by 0.466

Direct Gravimetry:-:

Direct Gravimetry:- Determination of anhydro hydroxy progesterone in tablets:- Procedure:- Weigh 10 tablets and equivalent to 50 mg of anhydro hydroxy progesterone in a micro soxhlet extractor Extracted with petroleum ether for 4 hrs and discard the extract dry petroleum ether Again extract with chloroform for 4 hrs Extract is evaporated to dryness Residue dried at 105°C

Calculation:-:

Calculation:- Weight of anhydro hydroxy progesterone in tablet }= RW/AN R= Wt of extracted residue(mg) W= Wt of the tablets taken(mg) A= Wt of powder extracted (Mg) N= No.of tablets taken

Other methods:-:

Other methods :- α , β -unsaturated group detected by using phospho molybdic acid and it produce color measured at 241 nm. α , β -unsaturated keto steroids react with iso nicotinic acid hydrazide in presence of HCL produce Isonicotinyl hydrazone of unsaturated steroids, measured at 380 nm.

Androgens:

Androgens Testosterone is a member of the C-19 steroid class. It is one of the testicular or androgenic hormones responsible for the development and maintenance of male characteristics and organs.

Methods of analysis:-:

Methods of analysis:- Basic methods:- Reaction with semi carbazide Spectrophotometry Other methods

Basic methods:- :

Basic methods:- Reaction with Semicarbazide :- Esters of testosterone react with semicarbazide and produce semicarbazone determined by gravimetry.

Determination of testosterone propionate in oil solution containing less than 10 mg/ml:

Determination of testosterone propionate in oil solution containing less than 10 mg/ml Reagent:- Semicarbazide acetate solution :- 2.5 g of semi carbazide+ 2.5 g of anhydrous sod.acetate+30 ml of methanol-reflex for 2 hrs. Solution is then cooled,Nacl is formed and removed by filtration. The filterate is diluted to 100 ml with methanol.

Preparation of sample:-:

Preparation of sample:- 50 mg of oil solution of testosterone add 40 ml of petroleum ether-alcohol Extracted with eight 20 ml of ether-alcohol Combine extract evaporated Residue is transferred to RBF containing methanol Methanol removed by steam bath.

Precipitate of testosterone propionate semicarbazone:

Precipitate of testosterone propionate semicarbazone 3 ml of semicarbazide acetate solution added to the residue and reflex for 2 hrs cooled and add 10 ml of iso octane Contents are poured into 75ml ice water Rinse with two 5 ml portion of iso octane and added to water Refrigerate for 3 hrs Poured into glass filtering funnel Remove the layer by suction Crystals washed with 10 ml of iso octane Wash with 4-6, 5 ml portions of water Dry at 105 ̊ ̊c

Calculation:-:

Calculation:- The weight of testosterone in the sample is obtained by multiplying the weight of prcipitate by 0.8579

Spectrophotometry:-:

Spectrophotometry: - α , β -unsaturated carbonyl group in ring A absorbs strongly in the region of 240 nm Determination of methyl testosterone in tablets by UV Spectrophotometry:- Reagent:- Std.solution:- 50 mg methyl testosterone dissolved in alcohol and diluted to 100 ml with alcohol. 10 ml of this solution transfer to 500 ml flask with alcohol Resulting standard solution contains 0.01 mg of methyl testosterone/ml

Preparation of sample:-:

Preparation of sample:- Powder equivalent to 5-10 mg of methyl testosterone add 25 ml of water 25 ml of ether shaken for 1 minute Layers allowed to separate To the Aqueous layer add 25 ml of ether Again separate aq.layer add 25 ml of ether Aq.layer discarded Ether extracts washed with 5ml of saturated NaHC03 add 5 ml of water Aq.layer discarded Ether evaporated to dryness on steam bath Residue dissoled in alcohol Make upto 100 ml with alcohol

UV absorption measurement:-:

UV absorption measurement:- 10 ml of solution make upto 100 ml with alcohol. Absorbance of sample,Std & blank measured at 241 nm

Calculation:-:

Calculation:- Weight of methyl A I C S W testosterone per tablet = 100 A S AN A I = absorbance of sample A S = absorbance of standard C S = concentration of std W = weight of tablet taken A = weight of powder analysed N = No.of tablets

Other methods:-:

Other methods:- α , β -unsaturated carbonyl group react with iso nicotinyl acid hydrazide and produce hydrazone 2,4-dinitro phenyl hydrazone of testosterone measured at 460 nmin alcoholic KOH solution

Chloesterol:

Chloesterol Methods of analysis :- Basic methods The liebermann-burchard reaction Other method Fecl 3 colorimetry

Basic methods:-:

Basic methods:- Liebermann-burchard reaction:- sample+acetic anhydride+H 2 SO 4 chloroform Yellow colour is produced Absorbance measured at 630 nm

Determination of cholesterol in wool wax:-:

Determination of cholesterol in wool wax:- Reagent:- 1 volume of H 2 SO 4 + 4 volume of acetic anhydride in ice bath Diluted to 2.5 ml glacial acetic acid+ 1,4 dioxane kept at 0°c

Preparation of sample:-:

Preparation of sample :- 200mg of wool wax dissolved in ethanol,if it is not soluble add 20% 1,4-dioxane in ethanol and make upto 100 ml 5ml of above solution+0.1g of 50% aq.KOH+1 drop of Hph placed in 15 ml centrifuge tube, kept at 50-60°c for 3 hrs. Cooled and neutralise with 10% aq.HCL 2ml of above solution in 80% alcohol and boil gently +6 ml of water boil for 2-3 mts

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Cooled to 20°c Tube is centrifuged at 2400r.p.m for 5 mts Remove supernatant liquid Ppt is dissolved in 12.5 ml of acetic acid Lake upto 100 ml with chloroform Colour development:- 10 ml of above sample+5 ml of liebermann-burchard reagent-measure the absorbance at 630 nm

Calculation:-:

Calculation:- The percentage of C s A I =200× chloesterol in the sample A s w C s = concentration of std.solution A I = absorbance of sample A s = absorbance of std W = weight of sample

Other method:-:

Other method:- Colorimetric procedure:- Acetic acid solution chloesterol + acetyl chloride + Zncl 2 Absorbance at 528 nm

Fecl3 colorimetry :-:

Fecl 3 colorimetry :- Chloesterol + Fecl 3 + H 2 SO 4 Purple colour produced Measured at 560 nm

Alkaloids:

Alkaloids Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. Some of the alkaloids are, Cinchona alkaloids Ergot Opium Rauwolfia

Cinchona alkaloids:-:

Cinchona alkaloids:- Cinchona is the dried stem or root bark of cinchona succiruba,C.ledgeriana,C.calisaya Family:- Rubiaceae The alkaloids of cinchona are combined with quinine, cinchono tannic acids and other acids.

Quinine:-:

Quinine:- Identification test:- Dissolve 10 mg in sufficient water to produce 10 ml. To 5 ml of the solution add 0.2ml of bromine water and 1 ml of 2M ammonia Emerald green colour is produced

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Dissolve 5 mg in 5 ml of water add 1 ml of 2M ammonia and 5 ml of ether Shake and acidify with 2M nitric acid The aqueous layer yields reaction characteristics of chlorides.

Instrumental methods:-:

Instrumental methods:- Colorimetric method UV method Spectroflourimetric method TLC method HPLC method

Colorimetric method:-:

Colorimetric method:- To the 10 ml of solution add 20% trichloro acetic acid-shake and centrifuge Filter and adjust an aliquot of filterate to pH 11.7 with NaoH Add 2% rose bengal solution (1ml) and 3.5ml chloroform Allow to stand for several hours with shaking 5 or 6 times. Measure the colour in the chloroform layer at 550nm

UV method:-:

UV method:- 10 ml of test solution is mixed with 1 ml of 0.01M Ticl 4 in 0.8N Hcl,1 ml of 0.01M sod.salicylate & 10 ml of CHcl 3 pH is adjusted to 3 with 0.1 N NaoH The yellow ternary complex is extracted with CHcl 3 for 2 mts. The extract is filtered & measure the absorbance at 380 or 400 nm.

Spectroflourimetric method:-:

Spectroflourimetric method:- Pipette out 1 ml and make upto 10 ml with 0.1N H 2 SO 4 The excitation and emission filters are set at 365 & 459 nm. The F.I of the instrument are also set by using blank and higher concentration. Measure the F.I of different standard and also sample solution From the std.graph, we get the concentration of quinine

TLC method:-:

TLC method:- The mixture of cinchona alkaloids like quinine,quinidine,cinchonine are separated on silica gel G with mixture of 20 volumes of toluene,12 volumes of ether and 5 volumes of diethyl amine as mobile phase. The alkaloids are then identified by using Dragendroff’s reagent .

Ergot alkaloids:-:

Ergot alkaloids:- Ergot is the dried sclerotium of the parasite fungus claviceps purpurea developed in plants of rye secale cerate of the family Graminae . Ergotamine tartarate:- It is a salt of ergotamine alkaloid that comes under water insoluble alkaloids.

Instrumental methods:-:

Instrumental methods:- UV method Colorimetric method Flourimetric method TLC method HPLC method

UV method:-:

UV method:- The ergotamin sample solution is measured in the region of 310-320 nm dependent on the solvent used. Colorimetric method :- It is based on the reaction between sample and PDAB and it produces blue violet colour. The absorbance of the final sample is measured at 550 nm

Flourimetric method:-:

Flourimetric method:- The powdered sample is extracted with acidic solution, after being made alkaline was extracted with benzene. Evaporate the benzene, the residue is dissolved in ethanol. The fluorescence intensity was read with excitation wavelength of 319 nm and emission wavelength of 492 nm

TLC METHOD:-:

TLC METHOD:- Measurement is done by using UV or Colorimetry. Solvent system Stationary phase Rf Chloroform-methanol Silica gel 0.39 Chloroform-methanol-ammonia Silica gel 0.25 Benzene – chloroform Silicagel G 0.65

HPLC method:-:

HPLC method:- A reverse phase bondapak phenyl or Bondapak C18 column with acetonitrile-aqueous ammonium carbonate buffer as mobile phase. This permits the seperation of ergotamine from its degradation products .

OPIUM ALKALOIDS:-:

OPIUM ALKALOIDS:- Morphine:- It is dried latex obtained by incision from the unripe capsules of papaver somniferum. Category:- Narcotic analgesic

Methods of analysis:

Methods of analysis Colorimetric method UV method Spectrofluorimetry Tlc method

Colorimetric method:-:

Colorimetric method:- It is based on the colour of nitoso compound formed by the reaction of nitrous acid and morphine. 2mg of samples add 3ml of 1 volume of con.H 2 SO 4 and 4 ml of water add 2 ml of 1% sodium nitrite solution After 10 mts 4 ml of NaoH added and cool in ice water bath for 5 mts. The obtained colour is read in suitable colourimeter.

UV method:-:

UV method:- 10-20 tablets quantitatively extracted with 0.1 N H 2 SO 4 and filter in 100 ml volumetric flask and make upto 100 ml. 10 pipetteout and make upto 100 ml with 0.1 N H 2 SO 4 The absorbance of the solution is measured at 298 nm.

Spectroflourimetry:-:

Spectroflourimetry:- 40-50 mg dissolved in 0.1 N H 2 SO 4 and make upto 100ml Pipetteout 10 ml in 50 ml volumetric flask and make up with 0.1 N H 2 SO 4 –solution A Another 10 ml is pipetteout make upto 100 ml with 0.1N NaoH-solution B Fluorescence measured at 285 nm and concentration of morphine is calculated from calibration curve.

TLC method:-:

TLC method:- Stationary phase Solvent system Rf Silica gel G Methanol-ammonia(100:15) 0.34 Silica gel Benzene- dioxane -ethanol-ammonia(50:40:5:5) 0.11 Silica gel Acetic acid-ethanol-water 0.27

Rauwolfia Alkaloids:

Rauwolfia Alkaloids Reserpine:- It is the crude drug used as a hypotensive agent ,consists of dried roots of Rauwolfia serpentina, family apocyanaceae

Method of analysis:

Method of analysis TLC method UV spectroscopy Colorimetric method Flourimetric method

TLC METHOD:

TLC METHOD Thin layer chromatography is used for seperation of reserpine from other alkaloids. Visualising agent:- Dragendroff’s agent-brown spot PDAB in con. H 2 SO 4 -greenish black Solvent system Sorbent Rf methanol Silica gel G with 0.1 N NaoH impregnated 0.69

UV spectroscopy:-:

UV spectroscopy:- The seperation of reserpine from roots and crude preparation is done by extensive extraction by paper chromatography or by electrophoresis on paper using 5 N acetic acid as the eletrolyte. The absorbance of final sample is measured at 268 nm

Colorimetric method:-:

Colorimetric method:- The most commonly used colorimetric procedure for reserpine involves oxidation of the compound to 3,4 didehydro reserpine with nitrite and measurement of absorbance of oxidation product at 390 nm .

Flourimetric method::

Flourimetric method: 3,4 didehydro reserpine is strongly fluorescent and it acan be detected by flourimetric method Notrite oxidation and flourimetric determination have been used for analysis of tablets at ppm level

References:-:

References:- Pharmaceutical analysis – 2 nd edition, Higuchi,Hassan