A: Infrared Absorption á197Kñ.

B: Ultraviolet Absorption á197Uñ—

C: The chromatogram of the Assay preparationobtained as directed in the Assayexhibits a peak for trenbolone acetate,the retention time of which corresponds to that exhibited by the Standard preparation.

Test solution: 5mg per mL,in methanol.

Diluent— Prepare a mixture of chloroform and methanol (9:1).

Standard solutions— Prepare four solutions in Diluentcontaining USP Trenbolone RSand USP Trenbolone Acetate RScontaining in each mL0.1mg of each,0.05mg of each,0.02mg of each,and 0.01mg of each,corresponding to 1.0%,0.5%,0.2%,and 0.1%of impurities,respectively.

Test solution— Prepare a solution of Trenbolone Acetate in Diluentcontaining 10mg per mL.

Procedure— Separately apply 10µLof each of the Standard solutionsand the Test solutionto a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture as follows.Develop the chromatograms in a solvent system consisting of a mixture of chloroform and acetone (98:2)in an unsaturated chromatographic chamber protected from light until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,dry it for about 15seconds in a stream of dry nitrogen,and immediately develop the chromatograms a second time until the solvent front has moved about three-fourths of the length of the plate.Examine the plate under short-wavelength UVlight.Spray the plate with phosphomolybdic acid TS,and heat the plate at 100for about 10minutes.Examine the plate under visible light,and compare the intensities of any secondary spots in the chromatogram of the Test solutionwith those of the principal spots in the chromatograms of the Standard solutions.No trenbolone spot from the chromatogram of the Test solutionis larger or more intense than the trenbolone spots from the Standard solutioncontaining 0.1mg of USP Trenbolone RSper mL(1%).Estimate the percentage of each other impurity observed in the chromatogram of the Test solutionby comparison with the trenbolone acetate spots in the chromatograms of the Standard solutions:No other impurity spot is greater than 0.5%,and the total of all other impurities,including that of the 17a-isomer obtained in the test for Limit of trenbolone acetate 17a-isomer,is not more than 1%.

Mobile phase ,Resolution solution,and Chromatographic system—Proceed as directed in the Assay.

Standard solution— Prepare a solution of USP Trenbolone Acetate RSin Mobile phasehaving a known concentration of 4µg per mL.

Test solution— Transfer about 20mg of Trenbolone Acetate,accurately weighed,to a 20-mLvolumetric flask,add about 10mLof Mobile phase,swirl to dissolve,dilute with Mobile phaseto volume,and mix.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.Trenbolone acetate 17a-isomer,if present,has a retention time of about 0.8relative to that of the trenbolone acetate peak.Calculate the percentage of 17a-isomer found in the Trenbolone Acetate taken by the formula: in which Cis the concentration,in µg per mL,of USP Trenbolone Acetate RSin the Standard solution,Wis the weight,in mg,of Trenbolone Acetate taken to prepare the Test solution,riis the response of any peak at a retention time of about 0.8in relation to that of the main trenbolone acetate peak in the chromatogram obtained from the Test solution,and rSis the peak area response of the trenbolone acetate peak in the chromatogram obtained from the Standard solution.Not more than 0.5%of the 17a-isomer is found.

Mobile phase— Prepare a mixture of acetonitrile and 1%ammonium acetate solution (55:45).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Resolution solution— Prepare a solution in Mobile phasecontaining about 0.2mg each of USP Trenbolone RSand USP Trenbolone Acetate RSper mL.

Standard preparation— Prepare a solution of USP Trenbolone Acetate RSin Mobile phasehaving a known concentration of about 0.2mg per mL.

Assay preparation— Transfer about 20mg of Trenbolone Acetate,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 344-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.4for trenbolone and 1.0for trenbolone acetate,and the resolution,R,between the trenbolone peak and the trenbolone acetate peak is not less than 25.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 14,000theoretical plates when calculated by the formula: the tailing factor is not more than 1.2when calculated by the formula: in which W0.1is the width of the peak at 10%height,and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of trenbolone acetate (C20H24O3)in the portion of Trenbolone Acetate taken by the formula: in which Cis the concentration,in mg per mL,of USP Trenbolone Acetate RSin the Standard preparation,and rUand rSare the trenbolone acetate peak area responses obtained from the Assay preparationand the Standard preparation,respectively.