A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

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

Test solution: 5 mg per mL, in methanol.

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

Standard solutions— Prepare four solutions in Diluent containing USP Trenbolone RS and USP Trenbolone Acetate RS containing in each mL 0.1 mg of each, 0.05 mg of each, 0.02 mg of each, and 0.01 mg 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 Diluent containing 10 mg per mL.

Procedure— Separately apply 10 µL of each of the Standard solutions and the Test solution to a thin-layer chromatographic plate (see Chromatography 621) 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 15 seconds 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 UV light. Spray the plate with phosphomolybdic acid TS, and heat the plate at 100 for about 10 minutes. Examine the plate under visible light, and compare the intensities of any secondary spots in the chromatogram of the Test solution with those of the principal spots in the chromatograms of the Standard solutions. No trenbolone spot from the chromatogram of the Test solution is larger or more intense than the trenbolone spots from the Standard solution containing 0.1 mg of USP Trenbolone RS per mL (1%). Estimate the percentage of each other impurity observed in the chromatogram of the Test solution by 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 17-isomer obtained in the test for Limit of trenbolone acetate 17-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 RS in Mobile phase having a known concentration of 4 µg per mL.

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

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

Mobile phase— Prepare a mixture of acetonitrile and 1% ammonium acetate solution (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Resolution solution— Prepare a solution in Mobile phase containing about 0.2 mg each of USP Trenbolone RS and USP Trenbolone Acetate RS per mL.

Standard preparation— Prepare a solution of USP Trenbolone Acetate RS in Mobile phase having a known concentration of about 0.2 mg per mL.

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

Chromatographic system (see Chromatography 621)—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 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.4 for trenbolone and 1.0 for 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 14,000 theoretical plates when calculated by the formula: the tailing factor is not more than 1.2 when calculated by the formula: in which W0.1 is 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 20 µL) of the Standard preparation and the Assay preparation into 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 C is the concentration, in mg per mL, of USP Trenbolone Acetate RS in the Standard preparation, and rU and rS are the trenbolone acetate peak area responses obtained from the Assay preparation and the Standard preparation, respectively.