Urea C13 »Urea C13contains not less than 99.0percent and not more than 100.5percent of 13CH4N2O. Packaging and storage— Preserve in well-closed containers at room temperature. USP Reference standards á11ñ USP Urea C13RS. Limit of biuret— Standard solution— Dissolve an accurately weighed quantity of biuret in water,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 0.033mg per mL. Test solution— Transfer 100.0mg of Urea C13to a test tube,and dissolve in 3mLof water. Procedure— To the Test solutionand to 3mLof the Standard solutionadd 3mLof sodium hydroxide solution (10in 100)and about 3drops of copper sulfate solution (0.5in 100),mix,and allow to stand for 5minutes.Any reddish violet color in the Test solutionis not more intense than that obtained from the Standard solution:not more than 0.1%of biuret is found. Isotopic purity— Test solution— Prepare a solution in methanol containing about 12mg of Urea C13per mL. Chromatographic system (see Chromatography á621ñandMass Spectrometry á736ñ)— The gas chromatograph is connected to a mass spectrometer,and is equipped with a 0.25-mm ×15-m capillary column coated with a 0.1-µm film of phase G47.The injection port is maintained at a temperature of 250,the detector is maintained at a temperature of 200,and the transfer line to the mass spectrometer is maintained at a temperature of 265.Helium is used as the carrier gas.The mass spectrometer is operated in a single-ion response mode.The electron energy is 70eV. Procedure— Inject about 1µLof the Test solutioninto the gas chromatograph,record the total ion chromatogram,and combine all of the mass spectra scans across the entire major peak.Record the peak intensities at mass-to-charge ratios of 60,61,62,and 63.Calculate the percentage of carbon that is C13in the portion of Urea C13taken by the formula: 100[(I61+I63)/(I60+I61+I63)], in which I60,I61,and I63are the relative peak intensities at mass-to-charge ratios of 60,61,and 63,respectively:not less than 99%is found.Calculate the percentage of oxygen that is O18in the portion of Urea C13taken by the formula: 100[(I62+I63)/(I60+I61+I62+I63)], in which I62is the relative peak intensity at a mass-to-charge ratio of 62,and the other terms are as defined above:not more than 15%is found. Other requirements— It meets the requirements for Identificationtests Aand B,Melting range,Residue on ignition,Alcohol-insoluble matter,Chloride,Sulfate,and Heavy metalsunder Urea. Assay— Mobile phase— Prepare a filtered and degassed mixture of acetonitrile,methanol,and water (89:10:1).Make adjustments if necessary (seeSystem Suitabilityunder Chromatography á621ñ). Biuret stock solution— Transfer about 15mg of biuret,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.Transfer 1.0mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix. System suitability preparation— Transfer about 25mg of urea,accurately weighed,to a 10-mLvolumetric flask.Pipet 1.0mLof Biuret stock solutioninto the flask,dilute with Mobile phaseto volume,and mix. Standard preparation— Dissolve an accurately weighed quantity of USP Urea C13RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 2mg per mL. Assay preparation— Transfer about 100mg of Urea C13,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 200-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L8.The flow rate is about 0.8mLper minute.Chromatograph the System suitability preparationand the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between urea and biuret is not less than 1.5;and the relative standard deviation for replicate injections is not more than 1%. Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of 13CH4N2Oin the portion of Urea C13taken by the formula: (MU/MS)50C(rU/rS), in which MUand MSare the molecular weights of Urea C13and USP Urea C13RS,respectively;Cis the concentration,in mg per mL,of USP Urea C13RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively. Auxiliary Information— Staff Liaison:Andrzej Wilk,Ph.D.,Senior Scientific Associate Expert Committee:(RMI)Radiopharmaceuticals and Medical Imaging Agents USP28–NF23Page 354 Phone Number:1-301-816-8305