Test solution: 10 mg per mL, in water.

Anhydrous methanol— Wash about 150 g of 8- to 17-mesh type 3A molecular sieve with several 100-mL portions of methanol to remove the fine particles. Place the washed molecular sieve in a shallow glass dish, heat in an oven at 350 for 2 hours, and cool in a desiccator. Transfer the dry molecular sieve to a 1-L glass container, add about 700 mL of methanol, insert a stopper, mix, and allow to stand in a desiccator for not less than 48 hours before using.

Standard solutions— Prepare solutions in Anhydrous methanol containing 0.4, 0.8, and 1.2 mg of distilled water per mL.

Test solution— [note—Prepare immediately prior to use.] Transfer about 20 mg of Gonadorelin Hydrochloride, accurately weighed, to a vial, place a cap on the vial, add 800 µL of Anhydrous methanol by means of a 1000-µL gas-tight syringe, and swirl to mix.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a thermal conductivity detector and a 2-mm × 180-cm glass column packed with 80- to 100-mesh support S3. The column temperature is maintained at about 100, and the injection port and detector temperatures are maintained at 130. Helium is used as the carrier gas at a flow rate of about 30 mL per minute. Chromatograph the Standard solution containing 1.2 mg per mL, record the chromatograms, and measure the peak responses as directed for Procedure: the elution order is water, followed by a broad methanol peak; the retention time of the water peak is between 0.5 and 3 minutes; and the relative standard deviation for not less than three replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes (1 to 3 µL) of each of the Standard solutions, Test solution, and Anhydrous methanol into the chromatograph, and measure the responses for the first (water) and second (methanol) major peaks, correcting the peak areas obtained from the Test solution and the Standard solutions against the Anhydrous methanol blank. Plot the responses of the water peaks versus concentration, in mg per mL, of water in each of the Standard solutions, and determine the regression line using the least-squares method. The coefficient of variation from the regression line is not more than 3.0%. From the graph so obtained, determine the concentration, C, in mg per mL, of water in the Test solution. Calculate the percentage of water in the portion of Gonadorelin Hydrochloride taken by the formula: in which W is the weight, in mg, of Gonadorelin Hydrochloride in the Test solution: not more than 7.0% is found.

Mobile phase— To 500 mL of water in a 1-L volumetric flask, add 1 mL of sulfuric acid. Dilute with water to volume, mix, and pass through a membrane filter having a 0.45-µm or finer porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).

Acetate standard solutions— Dissolve an accurately weighed quantity of sodium acetate trihydrate in Mobile phase to obtain a stock solution having a known concentration of about 0.5 mg per mL. Quantitatively dilute accurately measured volumes of this stock solution with Mobile phase to obtain Standard solutions having known concentrations of about 100, 10, and 1 µg of sodium acetate trihydrate per mL.

Test solution— Transfer about 50 mg of Gonadorelin Hydrochloride, accurately weighed, to a 50-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 205-nm detector and a 6.5-mm × 30-cm column that contains packing L17. The flow rate is about 0.5 mL per minute. [note—Do not allow the flow rate to exceed 0.6 mL per minute. Condition the column for about 60 minutes until a stable baseline is obtained.] Chromatograph the Acetate standard solution containing 100 µg of sodium acetate trihydrate per mL, and record the peak responses as directed for Procedure: the retention time of the acetate peak is between 10 and 16 minutes; the column efficiency is not less than 2000 theoretical plates; the tailing factor is not more than 2; and the relative standard deviation for not less than three replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100 µL) of each Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. [note—If more than five specimens are analyzed, reinject the Standard solutions before injecting further specimens of the Test solution.] Plot the responses of the Standard solutions versus concentration, in µg of sodium acetate trihydrate per mL, and determine the regression line, using the least-squares method. The coefficient of variation from the regression line is not more than 3.0%. From the graph so obtained, determine the concentration, C, in µg per mL, of sodium acetate trihydrate in the Test solution. Calculate the percentage of acetate (C2H3O2) in the portion of Gonadorelin Hydrochloride taken by the formula: in which 59.03 and 136.08 are the molecular weights of acetate and sodium acetate trihydrate, respectively; and W is the weight, in mg, of Gonadorelin Hydrochloride in the Test solution: not more than 1.0% is found.

Solution A— Dissolve 13.6 g of monobasic potassium phosphate in water, dilute with water to 2000 mL, and mix. Filter and degas. Adjust with 1 N potassium hydroxide to a pH of 6.5. Make adjustments if necessary (see System Suitability under Chromatography 621).

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.

Standard solutions— Dissolve an accurately weighed quantity of USP Gonadorelin Hydrochloride RS in and dilute quantitatively with Solution A to obtain a stock solution having a known concentration of about 1 mg per mL. Quantitatively dilute accurately measured volumes of this stock solution with Solution A to obtain Standard solutions having known concentrations of about 40, 5, and 1.5 µg per mL.

Test solution— Dissolve an accurately weighed quantity of Gonadorelin Hydrochloride in Solution A to obtain a solution having a concentration of 1 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm UV detector and a 4.6-mm × 15-cm column containing 5-µm packing L1, and is programmed to provide variable mixtures of Solution A and Solution B, beginning with 100% of Solution A, changing after 3 minutes to a mixture of 82% Solution A and 18% Solution B, maintained at that composition for the next 17 minutes, then changed linearly over the next 10 minutes so that it consists of a mixture of 30% Solution A and 70% Solution B at 30 minutes, and maintained at that composition for the next 5 minutes, then changed linearly over the next 3 minutes so that the composition at 38 minutes is again 100% Solution A. Pump Solution A through the column at a flow rate of about 1 mL per minute for about 30 minutes or until a stable baseline is obtained, then inject 100 µL of Solution A, and run the gradient elution program to completion to condition the column. Again inject 100 µL of Solution A, and run the gradient elution program to completion. Chromatograph the Standard solution containing 40 µg per mL, and record the peak responses as directed for Procedure: the retention time for gonadorelin is between 24 and 30 minutes; the column efficiency is not less than 5000 theoretical plates; and the tailing factor is not more than 2.0. If necessary, adjust the flow rate (to between 0.8 and 2 mL per minute) or, alternatively, change (by not more than 3%) the percentages of Solution A and Solution B at 3 minutes and at 20 minutes.

Procedure— Separately inject equal volumes (about 100 µL) of Solution A, each of the Standard solutions, and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Correct the peak responses, using the chromatogram of Solution A as a blank. Plot the corrected responses of the Standard solutions, and determine the regression line, using the least-squares method. The coefficient of variation from the regression line is not more than 3.0%. From the graph so obtained, determine the concentration of each impurity in the Test solution: not more than 3.0% of any individual impurity is found, and not more than 5.0% of total impurities is found.

Buffer solution— Dissolve 6.8 g of monobasic potassium phosphate in water, and dilute with water to 1000 mL. Adjust with 1 N potassium hydroxide to a pH of 6.5.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (82:18). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparations— Quantitatively dissolve accurately weighed quantities of USP Gonadorelin Hydrochloride RS in Mobile phase to obtain solutions having known concentrations of about 0.08 mg per mL, 0.10 mg per mL, and 0.12 mg per mL. [note—These Standard preparations may be stored in a refrigerator for 2 months. Remove suitable portions and warm to room temperature before use.]

Assay preparation— Dissolve an accurately weighed quantity of Gonadorelin Hydrochloride in Mobile phase to obtain a solution containing 0.10 mg per mL.

Identification solution— Mix equal volumes of the Assay preparation and the Standard preparation containing 0.10 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. [note—Condition the column with Mobile phase until a stable baseline is obtained.] Chromatograph about 20 µL of the Identification solution: the ratio, RR, of the retention times of the gonadorelin peaks obtained from the Assay preparation and the Standard preparation is 1.00 ± 0.05. Chromatograph the Standard preparation containing 0.08 mg per mL, and record the peak responses as directed for Procedure: the retention time for gonadorelin is between 8 and 11 minutes; the column efficiency is not less than 900 theoretical plates; the tailing factor is not more than 2.5; and the relative standard deviation for not fewer than three replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of each of the Standard preparations and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. [note—If more than five specimens are analyzed, reinject the Standard preparations before injecting further specimens of the Assay preparation.] Plot the responses of the gonadorelin peaks versus concentration, in mg per mL, of gonadorelin in each of the Standard preparations, and determine the regression line, using the least-squares method. The coefficient of variation from the regression line is not more than 3.0%. From the graph so obtained, determine the concentration, C, of gonadorelin in the Assay preparation. Calculate the percentage of C55H75N17O13·2HCl in the portion of Gonadorelin Hydrochloride taken by the formula: