A:Infrared Absorption á197Kñ.

B: The retention times of the component H2B1apeak and the component H2B1bpeak in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Test solution: 5mg per mL,in methanol.

Internal standard solution— Dilute 0.5mLof isopropyl alcohol with water to 100mL,and mix.

Standard solution 1— Transfer 2.0mLof dehydrated alcohol to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution 2— Transfer 1.0mLof formamide to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution 3— Transfer 5.0mLof Standard solution 1and 5.0mLof Standard solution 2to a 50-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having concentrations of formamide and alcohol of 0.001and 0.002mLper mL,respectively.Transfer 2.0mLof this solution to a 15-mLcentrifuge tube,add 2.0mLof m-xylene,insert the stopper,mix,and centrifuge.Remove the upper m-xylene layer,and extract it with 2.0mLof water.Discard the upper layer,combine the two retained lower aqueous layers,add 1.0mLof Internal standard solution,and mix.Each mLof this solution contains about 0.0008mLof alcohol and 0.0004mLof formamide.

Standard solution 4— Transfer 10.0mLof Standard solution 1and 10.0mLof Standard solution 2to a 50-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having concentrations of alcohol and formamide of 0.004and 0.002mLper mL,respectively.Transfer 2.0mLof this solution to a 15-mLcentrifuge tube,add 2.0mLof m-xylene,insert the stopper,mix,and centrifuge.Remove the upper m-xylene layer,and extract it with 2.0mLof water.Discard the upper layer,combine the two retained lower aqueous layers,add 1.0mLof Internal standard solution,and mix.Each mLof this solution contains about 0.0016mLof alcohol and 0.0008mLof formamide.

Test solution— Transfer 120mg of Ivermectin,accurately weighed,to a 15-mLcentrifuge tube,and dissolve in 2.0mLof m-xylene,heating in a water bath at 45±5,if necessary.Add 2.0mLof water,mix,and centrifuge.Transfer the m-xylene layer to a 15-mLcentrifuge tube,and extract with 2.0mLof water.Discard the upper m-xylene layer,combine the two retained lower aqueous layers,add 1.0mLof Internal standard solution,and mix.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm ×30-m fused-silica analytical column coated with a 3-µm G43stationary phase.The carrier gas is helium,with a 1:5split ratio and a linear velocity of about 35cm per second.The chromatograph is programmed as follows.The column temperature is maintained at about 40for 5minutes after injection,then increased at a rate of 20per minute to 180and maintained at 180for 2minutes.The injection port temperature is maintained at about 140,and the detector temperature is maintained at about 250.

Procedure— Separately inject equal volumes (about 2µL)of Standard solution 3,Standard solution 4,and the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses for alcohol,formamide,and isopropyl alcohol.Plot the ratios of the peak responses for alcohol and isopropyl alcohol and for formamide and isopropyl alcohol versus concentrations,in mLper mL,of alcohol and formamide,respectively,obtained from Standard solution 3and Standard solution 4.From the graphs so obtained,and using the ratios of the peak responses for alcohol and isopropyl alcohol and for formamide and isopropyl alcohol obtained from the chromatogram of the Test solution,determine the concentrations,C,of alcohol and formamide in the Test solution.[NOTE—In the event that the peak responses of the Test solutionare significantly outside the ranges of peak responses obtained from Standard solution 3and Standard solution 4,prepare additional Standard solutions,and chromatograph them to obtain peak responses bracketing those obtained with the Test solution.]Calculate the percentages of alcohol and formamide in the portion of Ivermectin taken by the formula: in which Cis the concentration of alcohol or formamide,as appropriate,in mLper mL,in the Test solution;Dis the density of alcohol (0.79)or formamide (1.13);and Wis the weight,in mg,of Ivermectin taken:not more than 5.0%of alcohol and 3.0%of formamide are found.

Mobile phaseand Chromatographic system— Proceed as directed in the Assay.

Standard stock solution— Proceed as directed for Standard preparationin the Assay.

Standard solution 1— Transfer 1.0mLof the Standard stock solutionto a 100-mLvolumetric flask,dilute with methanol to volume,and mix.

Standard solution 2— Transfer 5.0mLof Standard solution 1to a 100-mLvolumetric flask,dilute with methanol to volume,and mix.

Test solution— Use the Assay preparation.

Procedure— Separately inject equal volumes (about 20µL)of Standard solution 1,Standard solution 2,and the Test solutioninto the chromatograph,record the chromatogram of the Test solutionfor a period of time equivalent to twice the retention time of the main peak in the chromatogram obtained from Standard solution 1,and measure the peak areas.Calculate the percentage of each impurity by the formula: in which riis the peak area for each individual impurity in the Test solutionchromatogram;rsis the sum of all peaks in the Test solutionchromatogram;and rbis the total area of all peaks in a blank chromatogram:not more than 2.5%is found for the sum of all peaks with a relative retention time of about 1.3to 1.4(corresponding to H4B1aisomers and D2,3H2B1a);not more than 1%is found for the peak with a relative retention time of about 0.7(corresponding to 8a-oxo-H2B1a);not more than 0.7%is found for the peak with a relative retention time of about 0.5(corresponding to Avermectin B1a);not more than 0.5%is found for any other individual impurity peak;not more than 1%is found for the sum of all other individual peaks;and not more than 4%is found for the sum of the areas of all the peaks,apart from the two main peaks (H2B1aand H2B1b).Disregard any peak with an area less than that of the two main peaks (H2B1aand H2B1b)in the chromatogram of Standard solution 2(0.05%).

Mobile phase— Prepare a mixture of acetonitrile,methanol,and water (53:35:12),pass through a filter having a 1-µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).Increasing the proportion of water increases the elution times and allows better separation of impurities.

Standard preparation— Dissolve an accurately weighed quantity of USP Ivermectin RSin methanol to obtain a solution having a known concentration of about 0.8mg per mL.

Assay preparation— Transfer about 80mg of Ivermectin,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.Sonicate,if necessary.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-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 Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.8for component H2B1band 1.0for component H2B1a;the resolution,R,between component H2B1band component H2B1ais not less than 3.0;the column efficiency determined from the component H2B1apeak is not less than 2000theoretical plates;the tailing factor for the component H2B1apeak is not more than 2.5;and the relative standard deviation for replicate injections is not more than 1.0%determined from the component H2B1apeak.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses for component H2B1aand component H2B1b.Calculate the quantity,in mg,of component H2B1a(C48H74O14)and component H2B1b(C47H72O14)in the portion of Ivermectin taken by the formula: in which Cis the concentration,in mg per mL,of component H2B1aor component H2B1bin the Standard preparation;and rUand rSare the peak responses for component H2B1aor component H2B1bobtained from the Assay preparationand the Standard preparation,respectively.