Test solution: 10mg per mL,in water.

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

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

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

Chromatographic system (see Chromatography á621ñ)— 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 30mLper minute.Chromatograph the Standard solutioncontaining 1.2mg 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 0.5and 3minutes;and the relative standard deviation for not less than three replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes (1to 3µL)of each of the Standard solutions,Test solution,and Anhydrous methanolinto the chromatograph,and measure the responses for the first (water)and second (methanol)major peaks,correcting the peak areas obtained from the Test solutionand the Standard solutionsagainst the Anhydrous methanolblank.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 Wis the weight,in mg,of Gonadorelin Hydrochloride in the Test solution:not more than 7.0%is found.

Mobile phase— To 500mLof water in a 1-Lvolumetric flask,add 1mLof 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 Suitabilityunder Chromatography á621ñ).

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

Test solution— Transfer about 50mg of Gonadorelin Hydrochloride,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— 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 0.5mLper minute.[NOTE—Do not allow the flow rate to exceed 0.6mLper minute.Condition the column for about 60minutes until a stable baseline is obtained.]Chromatograph the Acetate standard solutioncontaining 100µg of sodium acetate trihydrate per mL,and record the peak responses as directed for Procedure:the retention time of the acetate peak is between 10and 16minutes;the column efficiency is not less than 2000theoretical 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 100µL)of each Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.[NOTE—If more than five specimens are analyzed,reinject the Standard solutionsbefore injecting further specimens of the Test solution.]Plot the responses of the Standard solutionsversus 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 59.03and 136.08are the molecular weights of acetate and sodium acetate trihydrate,respectively;and Wis the weight,in mg,of Gonadorelin Hydrochloride in the Test solution:not more than 1.0%is found.

Solution A— Dissolve 13.6g of monobasic potassium phosphate in water,dilute with water to 2000mL,and mix.Filter and degas.Adjust with 1Npotassium hydroxide to a pHof 6.5.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.

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

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

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm UVdetector and a 4.6-mm ×15-cm column containing 5-µm packing L1,and is programmed to provide variable mixtures of Solution Aand Solution B,beginning with 100%of Solution A,changing after 3minutes to a mixture of 82%Solution Aand 18%Solution B,maintained at that composition for the next 17minutes,then changed linearly over the next 10minutes so that it consists of a mixture of 30%Solution Aand 70%Solution Bat 30minutes,and maintained at that composition for the next 5minutes,then changed linearly over the next 3minutes so that the composition at 38minutes is again 100%Solution A.Pump Solution Athrough the column at a flow rate of about 1mLper minute for about 30minutes or until a stable baseline is obtained,then inject 100µLof Solution A,and run the gradient elution program to completion to condition the column.Again inject 100µLof Solution A,and run the gradient elution program to completion.Chromatograph the Standard solutioncontaining 40µg per mL,and record the peak responses as directed for Procedure:the retention time for gonadorelin is between 24and 30minutes;the column efficiency is not less than 5000theoretical plates;and the tailing factor is not more than 2.0.If necessary,adjust the flow rate (to between 0.8and 2mLper minute)or,alternatively,change (by not more than 3%)the percentages of Solution Aand Solution Bat 3minutes and at 20minutes.

Procedure— Separately inject equal volumes (about 100µL)of Solution A,each of the Standard solutions,and the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.Correct the peak responses,using the chromatogram of Solution Aas 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 6.8g of monobasic potassium phosphate in water,and dilute with water to 1000mL.Adjust with 1Npotassium hydroxide to a pHof 6.5.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand acetonitrile (82:18).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

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

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

Identification solution— Mix equal volumes of the Assay preparationand the Standard preparationcontaining 0.10mg per mL.

Chromatographic system (see Chromatography á621ñ)— 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 1.5mLper minute.[NOTE—Condition the column with Mobile phaseuntil a stable baseline is obtained.]Chromatograph about 20µLof the Identification solution:the ratio,RR,of the retention times of the gonadorelin peaks obtained from the Assay preparationand the Standard preparationis 1.00±0.05.Chromatograph the Standard preparationcontaining 0.08mg per mL,and record the peak responses as directed for Procedure:the retention time for gonadorelin is between 8and 11minutes;the column efficiency is not less than 900theoretical 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 20µL)of each of the Standard preparationsand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.[NOTE—If more than five specimens are analyzed,reinject the Standard preparationsbefore 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: