ANALYTICAL PRINCIPLE

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Reaction with Semicarbazide. Determination of Testosterone Propionate in oil solutions containing less than 10mg per ml. Determination of Testosterone Propionate in oil solutions containing 10mg per ml. Spectrophotometry Determination of Methyl testosterone in Tablets by ultraviolet Spectrophotometry. Determination of Methyl testosterone in Tablets by Infrared Spectrophotometry ddd dfffxca Analysis of Androgen can be carried out by the following methods

REACTION WITH SEMICARBAZIDE:

The esters of testosterone are frequently encountered in oil solution . The reaction of testosterone Propionate with semicabazide followed by gravimetric determination of the resulting semicarbazone has been described by Madigan, Zenno, and Pheasant. Other esters of testosterone as well as methyl testosterone react similarly. Since the reaction is not dependent on the unsaturation in ring, androstanolone should also be determinable by either this procedure or a modification of it. Any carbonyl compound precipitable under the conditions of the reaction constitutes interference. REACTION WITH SEMICARBAZIDE

Determination of Testosterone Propionate in oil solutions containing less than10mg per ml:

Reagent: Semicarbazide acetate solution: Prepared by mixing 2.5g of semicarbazide Hcl , 2.5g of anhydrous sodium acetate, and 30 ml of methanol and refluxing this mixture for 2 hours. The solution is then cooled, and the sodium chloride which forms is removed by filteration . The filtrate is diluted to 100 ml with methanol, mixed, and stored in the refrigerator. Determination of Testosterone Propionate in oil solutions containing less than10mg per ml

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Preparation of sample : An accurately measured volume of oil solution containing about 50 mg of testosterone propionate is transferred to a 125 ml separator funnel containing 40 ml of petroleum ether-alcohol (petroleum ether saturated with 90% alcohol). The stopcock should not be greased. The testosterone propionate is extracted by shaking with eight 20 ml portions of alcohol-petroleum ether (90% alcohol saturated with petroleum ether). The combined extracts are evaporated to dryness and the residue is transferred to a 50 ml round bottom flask with the aid of small portions of methanol. The methanol is then removed on a steam bath until only an oily residue remains PROCEDURE

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PROCEDURE

PRECIPITATION OF TESTOSTERONE PROPIONATE SEMICARBAZONE: :

PRECIPITATION OF TESTOSTERONE PROPIONATE SEMICARBAZONE : 3 ml of the semicarbazide acetate solution are added to the residue, the flask is attached to a reflux condenser, without the use of grease, and the contents are heated under reflux for 2 hours. The mixture is cooled, and 10 ml of iso-octane are added with mixing. The contents of the flask are then poured, with stirring, into 75 ml. of ice water in a 150 ml beaker. The flask is rinsed with two 5 ml portions of iso-octane ,and the rinsing are added to the water. If any material still adheres to a flask it is washed out into the water with two 2 ml portions of methanol. After a thorough mixing of its contents, the beaker the beaker is placed in the refrigerator for 3 hours.

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The contents of the beaker are then poured, in several portions, onto a tared , medium porosity, glass filtering funnel previously dried at 105o c for 1 hour. Care is taken not to mix the layers, and suction is applied only long enough to pull through most of the liquid but without matting the crystals on the disk of the funnel. Crystals remaining in the beaker are transferred to the funnel with the aid of several 10ml portions of isooctane. The crystals are washed with 10ml of isooctane, sucked dry, washed with four to six 5ml, again sucked dry, and dried to constant weight at 105 C.

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CALCULATION The weight of testosterone propionate (mg) in the in the sample is obtained by multiplying the weight of the precipitate by 0.8579.

Determination of Testosterone Propionate in oil solutions containing 10mg per ml:

Determination of Testosterone Propionate in oil solutions containing 10mg per ml In the case of oil solutions containing at least 10 mg of testosterone propionate per millilitre, no extraction is required. An accurate measured volume of oil solution containing about 50 mg of testosterone propionate is transferred to a 50 ml round bottom flask suitable for attachment for a reflux condenser. To this sample are added 3 ml of Semicarbazide acetate solution.From this point on the procedure and the calculations are exactly the same as those described in section

Spectrophotometry :

The alpha, beta-unsaturated carbonyl group in the ring A absorbs strongly in the region of 240mu. Carol (52) has described a procedure for the determination of methyl testosterone based on measurement at its 241mu absorption maximum. Testosterone and its esters may be similarly determined. Infrared Spectrophotometry provides highly specific analytical methods. The application of infrared methods to steroid analysis has been confined largely to the estrogens. Carol (52) has developed a procedure for the determination of methyl testosterone based on measurements at 935 cm , in the so called “fingerprint” region. Spectrophotometry

Determination of Methyl testosterone in Tablets by ultraviolet Spectrophotometry:

REAGENT: Standard solution : Fifty mg of methyl testosterone is dissolved in alcohol, transferred to a 100ml volumetric flask and diluted to 100ml with alcohol. The resulting standard solution contains 0.01 mg of methyl testosterone per millilitre. Determination of Methyl testosterone in Tablets by ultraviolet Spectrophotometry

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Preparation of sample : Accounted number of testosterone tablets are weighed and grounded to a fine powder. A portion of the powder, equivalent to 5 to 10mg of methyl testosterone, is weighed and quantitatively transferred to a 125 ml seperating funnel containing 25ml of water. 25ml of ether is added and the mixture is shaken for 1 min. The layers are allowed to separate, and the aqueous layer is transferred to a second 125ml separating funnel containing 25ml of ether. The extraction is repeated, the aqueous layer is transferred to a third 125ml separating funnel containing 25ml of ether, the extraction is repeated again. PROCEDURE

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The aqueous layer is then discarded The ether extracts are washed in succession with 5ml of saturated sodium bicarbonate solution and two 5ml portions of water. The aqueous washes are discarded. The ether solutions are combined in a 150ml beaker and evaporated to dryness on a steam bath with the aid of current of air. The residue is dissolved in alcohol, transferred quantitatively to a 100ml volumetric flask and diluted to 100ml with alcohol.

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ULTRAVIOLET ABSORPTION MEASUREMENT A 10ml aliquot of the solution above is transferred to 100ml volumetric flask and diluted to 100ml with alcohol. The absorbance’s of this sample solution and the standard methytestosterone solutions are measured at 241nm against alcohol as a blank, by using matched 1cm quartz cells. CALCULATION : 100 * A I Cs W A s A N A is the absorbance of the sample; A s is the absorbance of the standard solution; C s is the concentration of the standard solution (mg per ml); W is the weight of tablets taken; A I is the weight of the aliquot of powder analyzed; and N is the number of tablets taken. The wt of methyl testosterone (mg) per tablet is

Determination of methyl Testosterone in tablets by infrared Spectrophotometry:

REAGENT Standard solution: Fifty mg of methyl testosterone are transferred to a 5ml volumetric flask and diluted to 5ml with carbon disulfide immediately before use to avoid loss of solvent by volatilization. Determination of methyl Testosterone in tablets by infrared Spectrophotometry

PROCEDURE:

Preparation of sample: Extraction of pulverized tablets is carried out as described in section . The methyl testosterone residue is dissolved in a small amount of chloroform and quantitatively transferred to a 15 x 50 mm glass- stoppered weighing bottle. The solvent is then evaporated on a steam bath in a stream of air. PROCEDURE

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INFRARED ABSORPTION MEASUREMENT One ml of carbon disulfide is added to the residue, the bottle immediately stoppered, and the residue dissolved by gentle mixing. The absorbance of the sample solution and of the standard solutions are measured against a carbon disulfide blank at 935 cm by using 1.0mm cells

CALCULATION::

CALCULATION : 100 * A I Cs W A s A N A is the absorbance of the sample; A s is the absorbance of the standard solution; C s is the concentration of the standard solution (mg per ml); W is the weight of tablets taken; A I is the weight of the aliquot of powder analyzed; and N is the number of tablets taken. The wt of methyl testosterone (mg) per tablet is

References::

References: Pharmaceutical analysis by Takeru Higuchi. Pg.no-89-95

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