ANALYSIS OF STEROIDS

ANALYSIS OF STEROIDS:

ANALYSIS OF STEROIDS BY CH.ARJUN M.PHARM FIRST YEAR PHARMACEUTICAL ANALYSIS

STEROIDS:

STEROIDS STEROIDS: It is mainly contains cyclopentanoperhydrophenanthrene In steroids the substitution mainly takes palce in C 17 position At position (C 18 &C 19 ) methyl groups are present

DIFFERENT TYPES OF STEROIDS:

DIFFERENT TYPES OF STEROIDS Adrenalcortical steroids Progesterone & Related steroids Androgens Estrogens Bile acids & bile salts Cholesterol

ADRENALCORTICAL STEROIDS:

ADRENALCORTICAL STEROIDS Adrenalcorticalsteroids: They are produced from the adrenal cortex They are two types: 1.Gluco corticoids Ex:Hydro cortisone 2.Mineral corticoids Ex:Aldosterone

METHODS:

METHODS BASIC METHODS: Reaction of the α -Ketol groups with Tetrazonium salt Reaction of the 17,21-Dihydroxy-20-Keto group with phenyl hydrazine OTHER METHODS: Reaction of 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 the steriod Ester with Hydroxyl amine

BASIC METHODS:

BASIC METHODS REACTION OF THE α -KETOL GROUP WITH TETRAZONIUM SALTS: α -ketol(-CHOH-CO-) group posses reducing properties.Its reduction of alkaline tri phenyl tetrazonium chloride to form the red formazen for detection of α -ketolic sterols

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DETERMINATION OF DESOXYCORTISONE ACETATE IN OIL SOLUTION Reagents: Dilute tetra methyl ammonium hydroxide :By diluting 10ml of 10% aqueous solution to 100ml with 95% alcohol Triphenyltetrazonium chloride :By dissolving 5mg in 100ml of 95%alcohol. Standard solution :10mg of desoxycorticosterone acetate,previously dried for 3hrs over H 2 SO 4 in a vaccum desicator, is dissolved sufficient alcohol to make 100ml of solution and throghly mixed

PROCEDURE:

PROCEDURE Sample preparation : 5mg of desoxycorticisterone acetate oil solution is taken in a125ml of funnel which contain 50ml of iso octane alcohol(iso-octane saturated with 90%alcohol) This solution is extracted with 20ml of alcoholic iso-octane (90% of alcohol saturated with iso-octane) The combined alcoholic iso octane extracts are evapoorated to dryness on a steam bath The residue is taken up in 20ml and stirred with alcohol The solution is quantitavely taken in a 50ml volumetric flask

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Development of colour: 1ml of sample &standard solutions are pipetted out in 25ml glass stopperd cylinder. To each solution add 9ml of alcohol+1ml of dilute tetramethyl ammonium hydroxide+1ml of triphenyl tetrazolium chloride Solution are allowed to stand for 20min in strong light Absorbance measured at 490nm

CALCULATION:

CALCULATION The weight of the desoxycortisone acetate is taken as =50×CsAi As Cs=concentration of standard sample Ai=absorbance of sample solution As=absorbance of standard solution

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2.REACTION OF THE 17,21-DIHYDROXY-20-KETO GROUP WITH PHENYL HYDRAZINE The reaction of 17,21-dihydroxy-20-keto group of steroid with phenyl hydrazine in presence of sulphuric acid to the production of yellow chromogen. Ex:DETERMINATION OF CORTISONE ACETATE: Reagents: Dilute sulphuric acid :Mixing 310ml of conc H 2 SO 4 +190ml of H 2 O Phenyl hydrazine-sulphuric acid solution :65mg of phenyl hydrazine hydro chloride +absolute alcohol in dried over calcium chloride in 100ml of dil sulphuric acid Standard solution :10mg of cortisone dried for 3hrs over H 2 SO 4 in vaccum desicator and disssolved 100ml of absolute alcohol and mixed

PROCEDURE:

PROCEDURE Preparation of sample : Sample preparation is same as above method. Development of colour: 1ml of sample and solution are taken in separate test tubes To each test tubes add 8ml of phenyl hydrazine-sulphuric acid reagant Then take another two test tubes add 1ml of sample and standard solution to that add 8ml of dil H 2 SO 4. The test tubes are shaken and heated in water bath at 60 ⁰c for 20min and then cooled. Absorbance is taken at 410nm.

CALCULATION:

CALCULATION The weight of the cortisone acetate is taken as =100×Cs(Air-Aia) (Asr-Asa) Air&Asr=Absorbance of sample and standard with phenyl hydrazine-sulphuric acid reaagent Aia&Asa=Absorbance of sample standard solution with sulphuric acid

OTHER METHODS:

OTHER METHODS Reaction of α -ketol group: Reduction of various reagents (copper areseno molybdate,phospho molybdate)by α -ketol side chain of adrenal steroids. The reduction of copper aresenomolybdate reagent in spectro photometric measurement of adrenal steroids.The reaction involved in α -ketol function. 2.Reaction of the 17,21-dihydroxy-20-keto group : Here formaldehyde estimation by following periodate or bismuth oxidation. Periodate oxidation does not proceed beyond the α -hydroxy acid Bismuthate cleaves α -hydroxy acid to 17-keto steroids.

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3.Reaction of the α , β -unsturated carbonyl group in ring A: Absorption of ultra voilet by α , β -unsturated carbonyl group inn ring A provides an excellent means of determination of steroids. A double bond in conjugation with carbonyl group produces chromophore system with maximum 230-270nm. 4.Reaction of the oxygen function at C-11 : The oxygen function at C-11 are relatively non-reactive.This group may be useful in determination of cortisone or hydrocortisone.

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5.Reaction with steroid esters with hydroxylamine: The reaction of esters with alkaline hydroxylamine a purple colour develops when HCl+FeCl 3 are added RCOOR+H 2 NOH RCONHOH+ROH 3RCONH+FeCl 3 (RCONHO 3 )Fe+3HCl The resulting colour is proportional to steroid ester concentration and this procedure applied of determination of desoxycortisone acetate.

PROGESTERONE AND RELATED STEROIDS:

PROGESTERONE AND RELATED STEROIDS The harmone progesterone is produced from corpus lutem in normal menstrual cycle. Anhydroxy progesterone is produced synthetically in tablet form

METHODS:

METHODS BASIC METHODS Reaction with 2,4-Dinitrophenyl hydrazine Direct Gravimetry 1.REACTION WITH 2,4-DINITROPHENYL HYDRAZINE : When the steroid reacts with 2,4-dinitrophenylhydrazine which yeilds 2,4-dinitrophenylhydrazones. Thisb method is applicable to determination of progesterone and anhydrohydroxy progesterone

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DETERMINATION OF PROGESTERONE IN OIL SOLUTION Preparation of sample: 5mg of oil solution of progesterone is transferred in 125ml funnel followed by petroleum ether alcohol (petroleum ether saturated with 90%alcohol) and mixed The resulting solution is extracted with five 20ml portions of alcohol-petroleum ether(90%alcohol saturated with petroleum ether) The combined extracts are then evaporated to dryness in an Erlenmeyer flask on a steam bath

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Precipitation of progesterone bis 2,4-dinitro phenyl hydrazone: 75mg of 2,4-dinitrophenyl hydrazine+30ml of alcohol The mixture is refluxed for 15 min and then add 1ml of conc HCl The above mixture is again refluxed for 15 min The mixture is cooled and precipitate is transferred to a crucible Precipitate is finally washed with 10ml portions of petroleum ether and 5ml portions of alcohol Finally add 0.5M HCl untill filtrate is free from colour The precipitate is dried at 105°c and weighed

CALCULATION:

CALCULATION The weight of progesterone (mg) in sample taken =wt of precipitate × 0.466

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2.DIRECT GRAVIMETRY : This technique is completely non-specific and depends on the ability to separate the desired compound from the other constituents of the preparation. It is applied to aqueous suspension of steroids where sample is filtered and residue washed,dried and weighed

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DETERMINATION OF ANHYDROHYDROXY PROGESTERONE IN TABLETS: Procedure: Extraction: 10 tablets are weighed,finely grounded and 50mg of equivalent weight of powder is weighed into thimble of micro soxhlet extractor. The powder is extracted with petroleum ether for 4hrs extract is discarded. The thimble is removed and petroleum ether is allowed to evaporate The thimble is returned to soxhlet extractor and anhydrohydroxy progesterone is extracted with CHCl 3 for 4 hrs. Then extract is removed and dried to constant weight at 105°c

CALCULATION:

CALCULATION Weight of anhydrohydroxy progesterone(mg) = RW AN R=weight of extracted residue W=weight of the tablet A=weight of powder extracted N=number of tablets taken

ANDROGENS:

ANDROGENS Testosterone is the main androgenic harmone and it is a member of C-19 class. Responsible for maintainence of acessory male charecteristics & organs

METHODS:

METHODS BASIC METHODS : Reaction with semicarbizide Spectro photometry

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REACTION WITH SEMICARBAZIDE: The reaction of testosterone propionate with semicarbazide followed by gravimetric determination. Determination of Testosterone Propionate in oil solutions containing less than 10mg per ml: Reagent: Semicarbazide acetate solution : Prepared by mixing 2.5g of semicarbazide hydrochloride+2.5g of anhydrous is sodium acetate+30ml of methanol Reflux for 2 hrs The solution is cooled and sodium chloride which forms is removed by filtration Make up the filtrate to 100ml with methanol and store in refrigerator

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PROCEDURE: Preparation of sample : 50mg of testosterone propionate+40ml of petroleum ether-alcohol The testosterone propionate is extracted by shaking with eight 20ml portions of petroleum alcohol The combined extracts are evaporated and residue is transferred to 50 ml round bottom flask with help of methanol Methanol is removed on a steam bath until oily residue remains

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Preparation of testosterone propionate semicarbazone : Few mg of residue +3ml of semicarbazide acetate solution Reflux for two hrs Mixture is cooled and 10ml of iso-octane is added The contents of flask are poured into 75ml of ice water These are rinsed with two 5ml portions of iso-octane and added to water Mix the contents and placed the beaker in refrigerator for 3 hrs Contents are dried at 105°c for 1hr Formed crystals are washed with 10ml iso-octane and dried to constant weight at 105°c

CALCULATION:

CALCULATION Weight of testosterone propionate(mg) = wt of precipitate ×0.8579

SPECTROPHOTOMETRY:

SPECTROPHOTOMETRY Determination of methyltestosterone in tablets by UV: Reagent: Standard solution: 50mg of methyltestosterone is diluted 100ml with alcohol 10ml of above solution is diluted to 500ml with alcohol The resulting solution contains 0.01mg per ml conc

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Procedure: Preparation of sample : 5-10mg of powder tablets of methyltestosterone+25ml of water+25ml of ether are taken in a seperating funnel The aqueous layer formed is again transferred into another separating funnel containing 25ml of ether The ether extracts are washed with 5ml of saturated sod bi carbonate and two 5ml portions of water The obtained ether solutions are evaporated and the residue is dissolved in alcohol and is diluted to 100ml with alcohol

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UV absorption measurement: 10ml of sample prepared is diluted to 100ml with alcohol The absorbance of this solution and standard methyltestosterone solution are measured at 241nm CALCULATION: Weight of methyl testosterone(mg)=1000× A 1 C s W A s AN A1=absorbance of sample As=absorbance of standard Cs=conc of standard solution W=weight of tablets taken A=weight of powder analysed N=no of tablets taken

CHOLESTEROL:

CHOLESTEROL Cholesterol is the most important of all sterols

METHODS:

METHODS BASIC METHODS: The Liebermann-Burchard Reaction OTHER METHODS: Colorometric Procedures Miscellaneous

REFERENCES:

REFERENCES Higuchi,Bechmann and Hassan:Pharmaceutical Analysis,2 nd Edition(Page no:70-125)

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