Electrolyte solution— Use filtered and buffered saline electrolyte solution.1

Diluent— Prepare a solution that contains,in each liter of water,1.5g of sodium lauryl sulfate and 0.1g of thimerosal.Prior to use,pass the solution through a 0.2-µm nylon filter.[NOTE—The Diluentis to be used exclusively to prepare the Reference stock preparationdescribed below;it must not be used to prepare the Sample preparation.]

Reference stock solution— Transfer a quantity of NISTtraceable microspheres suspension containing about 0.5g of microspheres directly into a tared centrifuge tube equipped with a cap,and weigh.2Using the density and concentration of the microspheres obtained from the Certificate of Analysis,calculate the volume occupied by the microspheres and the number of microspheres in the portion taken.Calculate the target total volume,in mL,by dividing the number of microspheres in the portion taken by the target concentration of 2.0×108microspheres per mL.Calculate the target Diluentvolume by subtracting the volume occupied by the microspheres from the target total volume.Transfer the target volume of Diluentto the centrifuge tube containing the portion of microspheres taken,and mix the tube vigorously for 1hour.The prepared Reference stock solution,which contains a suspension of microspheres with a target mean particle diameter of 5.2µm and a concentration of 2.0×108microspheres per mL,is divided into smaller containers and stored at 5.

Reference solution— Equilibrate the Reference stock solutionto room temperature,and mix thoroughly.Immediately transfer 20µLof the Reference stock solutionto a beaker containing 200mLof Electrolyte solution,mix,and analyze immediately.

Blank solution— Use 200mLof Electrolyte solution.

Test solution— Allow the Perflutren Protein-Type A Microspheres for Injection to equilibrate to room temperature.Invert the vial,and gently rotate to resuspend the microspheres.[NOTE—After resuspension,the contents should appear as a homogeneous,opaque,milky-white suspension.]Immediately withdraw a 20-µLaliquot,transfer to a beaker containing 200mLof Electrolyte solution,mix,and analyze immediately.

Test apparatus— Use a multichannel particle analyzer that operates on the electrical zone-sensing principle.3The analyzer is fitted and calibrated with an aperture tube having a 50-µm orifice.The multichannel particle analyzer is equipped with software capable of data-smoothing,data extrapolation,distribution graphing,and data conversion.Analyze the Blank solution,the Reference solution,and the Test solutionas directed for Procedure:the total count in the Blank solutionis not more than 500;the mean particle diameter of microspheres in the Reference solutionis within 5%of the mean particle diameter of microspheres in the Reference stock solution;the concentration of the Reference solutionis within 10%of the concentration of the Reference stock solution;and the coincidence effect in the analysis of the Test solutionis not more than 5%.

Procedure— Rinse the orifice of the aperture tube with Electrolyte solutionbefore and after analyzing each preparation.Place the Blank solutionin the apparatus,and adjust the vacuum on the sample stand so that the counting begins about 12seconds after the analyzer is set to the counting position.Set the data acquisition to stop when one of the following conditions is met:preset length of time,preset volume,preset number of counts in any channel,or total counts.Collect the count versus the channel data for the Blank solution,and analyze using the data-smoothing,data extrapolation,distribution graphing,and data conversion features of the system software.In the same manner,analyze the Reference solutionand the Test solution.The Test solutiondata are normalized and expressed as the number of microspheres per mL:the concentration of microspheres is between 5.0×108and 8.0×108per mL.Calculate the percentage of microspheres less than 10µm in size in the portion of Perflutren Protein-Type A Microspheres for Injection taken by the formula: in which PAis the number of microspheres in the 1-to 10-µm size range,and PBis the number of microsphere particles in the 1-to 32-µm size range.Not less than 93%of microsphere particles is smaller than 10µm.

Reference solutions— Use 99%perflutren reference standard4(electronic grade perflutren gas of at least 99molar %purity)and 60%perflutren reference standard4(an electronic grade gas in air mixture containing 60molar %perflutren gas).

Blank solution— Use ambient air.

Test solution— Use gas from the container headspace.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a thermal conductivity detector and a 0.53-mm ×25-m fused-silica (porous layer open tubular)column coated with Al2O3/KCl (aluminum oxide deactivated with potassium chloride).5The carrier gas is helium with a flow rate adjusted to obtain a retention time of about 1.5to 1.8minutes for perflutren.The column temperature is maintained at about 65,the injection port at about 130,and the detector at about 180.Chromatograph the Reference solutionsas directed for Procedure:the resolution,R,between perflutren and air is not less than 2;and the relative standard deviation for replicate injections is not more than 5%.The measured value for the 99%perflutren reference standard is within 5%of the nominal value.

Procedure— Using a gas-tight syringe,separately inject 10µLof the Blank solution,the Reference solutions,and the Test solutioninto the chromatograph.Record the chromatograms,and measure the responses for the major peaks.The percentages of perflutren in the 99%perflutren reference standard and in the Test solutionare calculated by comparing the peak areas in each with the peak areas obtained from the 60%perflutren reference standard.The container headspace of Perflutren Protein-Type A Microspheres for Injection contains not less than 60%of perflutren gas.

Reference stock solutions— The 97%decafluorobutane reference standard is decafluorobutane gas of at least 97molar %purity.6The 5%decafluorobutane-5%perflutren reference standard is a mixture containing 5molar %decafluorobutane gas and 5molar %perflutren gas in air.7The 99%perflutren reference standard is electronic grade perflutren gas of at least 99molar %purity.4

Analysis vial— Transfer 100µLof 97%decafluorobutane reference standard gas and 100µLof glacial acetic acid to a 2-mLvial equipped with a septum cap.

Reference solution— Transfer 100µLof 99%perflutren reference standard and 0.75mLof 1%Albumin Human to an Analysis vial,and incubate by mixing for at least 3hours.

Test solution— Allow a vial of the Perflutren Protein-Type A Microspheres for Injection to equilibrate to room temperature.Invert the vial,and gently rotate to resuspend the microspheres.[NOTE—After resuspension,the contents of the vial should appear as a homogeneous,opaque,milky-white suspension.]Withdraw 0.75mLof Perflutren Protein-Type A Microspheres for Injection,and transfer to another Analysis vial.Incubate the Test solutionby mixing for at least 3hours.

Chromatographic system (see Chromatography á621ñ)— Prepare as directed for Container headspace content.The carrier gas is helium with a flow rate adjusted to obtain the following retention times:1.0to 1.1minutes for air,1.3to 1.5minutes for perflutren,and 1.5to 2.5minutes for decafluorobutane.The column temperature is maintained at about 85,and then after elution of the perflutren the temperature is increased at a rate of 50per minute to 120,and maintained at 120for 2minutes.The injection port temperature is maintained at about 130,and the detector at about 180.Chromatograph the 5%decafluorobutane-5%perflutren reference standard and the Reference solutionas directed for Procedure:the resolution,R,between air and perflutren is not less than 2;the resolution,R,between perflutren and decafluorobutane is not less than 5;the relative standard deviation determined from the perflutren peak response for the Reference solutionis not more than 5%;and the relative standard deviation determined from the response ratios for replicate injections of the 5%decafluorobutane-5%perflutren reference standard is not more than 5%.

Procedure— Inject 20µLof the headspace gas from the vials containing the Reference solutionand the Test solutioninto the gas chromatograph.Record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg per mL,of perflutren in the portion of Perflutren Protein-Type A Microspheres for Injection taken by the formula: in whichMis the number of µmols of decafluorobutane in an Analysis vialafter addition of the Perflutren Protein-Type A Microspheres for Injection;Vis the volume,in mL,of Perflutren Protein-Type A Microspheres for Injection added to the Analysis vial;and RSand RUare the peak area ratios of decafluorobutane to perflutren obtained from the Reference solutionand the Test solution,respectively.The quantity of perflutren in the Perflutren Protein-Type A Microspheres for Injection is between 0.11and 0.33mg per mL.

Diluted antifoam reagent— Transfer 100µLof antifoam reagent8to a suitable container,and dilute with water to 10mL.

Blank preparation— Transfer 500µLof Sodium Chloride Injection to a culture tube.Dilute the contents of the tube with water to 2mL,and add 10µLof Diluted antifoam reagent.

Standard preparations— Transfer 25-,50-,62.5-,75-,and 100-µLaliquots of protein standard solution9containing 8g per dLinto separate tubes.Dilute the contents of each tube with water to 2.00mL,and add 10µLof the Diluted antifoam reagentto each tube.During the Procedure,the addition of 3.0mLof biuret reagent TSto each of the tubes produces Standard preparationswith protein concentrations of 0.4,0.8,1.0,1.2,and 1.6mg per mL.

Assay preparation— Equilibrate each container of Perflutren Protein-Type A Microspheres for Injection to room temperature,and mix each for at least 5minutes to ensure a homogenous suspension.Vent the container,and transfer 500-µLaliquots into separate tubes.Dilute the contents of each tube with water to 2mL,and add 10µLof Diluted antifoam reagent.

Procedure— To each of the tubes containing the Blank preparation,Standard preparations,and Assay preparationadd 3.0mLof biuret reagent TS,mix,and allow to stand for 30minutes,accurately timed,for maximum color development.The Blank preparation,Standard preparations,and Assay preparationare treated identically.Using the Blank preparation,set the absorbance equal to zero.Determine the absorbance of each of the Standard preparationsand the Assay preparationin 1-cm cells with a suitable spectrophotometer at a wavelength of 540nm.Using linear regression,analyze the data obtained for each of the Standard preparations.Calculate the correlation coefficient,slope,and y-intercept values:the correlation coefficient is not less than 0.995.Calculate the quantity,in mg,of protein in each mLof the Perflutren Protein-Type A Microspheres for Injection by the formula: in which 10is the dilution factor;and AUis the absorbance of the Assay preparation:the calculated quantity of protein in the Perflutren Protein-Type A Microspheres for Injection is between 8mg per mLand 12mg per mL.