Urea C 13
» Urea C 13 contains not less than 99.0 percent and not more than 100.5 percent of 13CH4N2O.
Packaging and storage Preserve in well-closed containers at room temperature.
USP Reference standards 11
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 0.033 mg per mL.
Test solution Transfer 100.0 mg of Urea C 13 to a test tube, and dissolve in 3 mL of water.
Procedure To the Test solution and to 3 mL of the Standard solution add 3 mL of sodium hydroxide solution (10 in 100) and about 3 drops of copper sulfate solution (0.5 in 100), mix, and allow to stand for 5 minutes. Any reddish violet color in the Test solution is not more intense than that obtained from the Standard solution: not more than 0.1% of biuret is found.
Test solution Prepare a solution in methanol containing about 12 mg of Urea C 13 per mL.
Chromatographic system (see Chromatography 621 and Mass Spectrometry 736) 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 70 eV.
Procedure Inject about 1 µL of the Test solution into 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 C 13 in the portion of Urea C 13 taken by the formula:
100[(I61 + I63)/(I60 + I61 + I63)]in which I60, I61, and I63 are 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 O 18 in the portion of Urea C 13 taken by the formula:
100[(I62 + I63)/(I60 + I61 + I62 + I63)]in which I62 is 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 Identification tests A and B, Melting range, Residue on ignition, Alcohol-insoluble matter, Chloride, Sulfate, and Heavy metals under Urea.
Mobile phase Prepare a filtered and degassed mixture of acetonitrile, methanol, and water (89:10:1). Make adjustments if necessary (see System Suitability under Chromatography 621).
Biuret stock solution Transfer about 15 mg of biuret, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix. Transfer 1.0 mL of this solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.
System suitability preparation Transfer about 25 mg of urea, accurately weighed, to a 10-mL volumetric flask. Pipet 1.0 mL of Biuret stock solution into the flask, dilute with Mobile phase to volume, and mix.
Standard preparation Dissolve an accurately weighed quantity of USP Urea C 13 RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 2 mg per mL.
Assay preparation Transfer about 100 mg of Urea C 13, 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 200-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L8. The flow rate is about 0.8 mL per minute. Chromatograph the System suitability preparation and 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 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of 13CH4N2O in the portion of Urea C 13 taken by the formula:
(MU / MS)50C(rU / rS)in which M U and MS are the molecular weights of Urea C 13 and USP Urea C 13 RS, respectively; C is the concentration, in mg per mL, of USP Urea C 13 RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2495