A: Thin-Layer Chromatographic Identification Test 201—

B: The retention time of the major peak in the chromatogram of the Test solution corresponds to that in the chromatogram of the Resolution solution, as obtained in the test for Limit of N-pyroglutamyl-aprotinin and related compounds.

Test solution— Dilute a concentrated solution of Aprotinin with water, or weigh out Aprotinin and dissolve in water, to obtain a solution containing about 4 to 7 USP Aprotinin Units per mL.

Standard solution— Dilute USP Aprotinin RS with water to obtain a solution having a concentration similar to that of the Test solution.

Capillary zone electrophoresis buffer— Dissolve 8.21g of monobasic potassium phosphate in 400 mL of water, adjust with phosphoric acid to a pH of 3.0, and dilute with water to 500 mL.

Capillary zone electrophoresis system (see Capillary Electrophoresis under Biotechnology-Derived Articles—Test 1047)— The capillary electropherograph is equipped with a 214-nm detector and a 45- to 60-cm uncoated fused silica capillary with an internal diameter of 75 µm with the temperature controlled at 25. Apply a field strength of 0.2 kV/cm for 30 minutes, using Capillary zone electrophoresis buffer as the electrolyte in both buffer reservoirs. Electropherograph the Standard solution, and record the peak responses as directed for Procedure: the relative migration times are about 0.98 for des-Ala-des-Gly-aprotinin, 0.99 for des-Ala-aprotinin, and 1 for aprotinin. The resolution, RS, between the des-Ala-des-Gly-aprotinin and des-Ala-aprotinin peaks is not less than 0.8, and the resolution between the des-Ala-aprotinin and aprotinin peaks is not less than 0.5. The migration time for the aprotinin peak is between 19 and 25 minutes. The tailing factor, T, of the aprotinin peak is not more than 3 (see Chromatography 621 for calculation). The baseline is stable and shows little drift. Rinse the capillary for at least 1 minute with at least 10 total capillary volumes of 0.1 N sodium hydroxide, followed by at least 10 total capillary volumes of water, and by at least 20 capillary volumes of Capillary zone electrophoresis buffer between injections.

Procedure— Transfer a volume of the Test solution, approximately 15 nL, into the anodic end of the capillary (apply differential pressure of 3.5 kPa for 3 seconds either by vacuum or pressure), record an electropherogram, and measure the peak areas. Calculate the percentage contents of des-Ala-des-Gly-aprotinin and des-Ala-aprotinin by the formula: in which ri is the peak response corresponding to des-Ala-des-Gly-aprotinin or des-Ala-aprotinin; and rs is the sum of the responses of des-Ala-des-Gly-aprotinin, des-Ala-aprotinin, and aprotinin peaks: not more than 8.0% des-Ala-des-Gly-aprotinin and not more than 7.5% des-Ala-aprotinin is found.

Solution A— Prepare a filtered and degassed solution containing 3.52 g of monobasic potassium phosphate and 7.26 g of dibasic sodium phosphate dissolved in 1000 mL of water.

Solution B— Prepare a filtered and degassed solution containing 3.52 g of monobasic potassium phosphate, 7.26 g of dibasic sodium phosphate, and 66.07 g of ammonium sulfate dissolved in 1000 mL of water.

Resolution solution— Prepare a solution of USP Aprotinin System Suitability RS containing about 5 USP Aprotinin Units per mL in Solution A.

Test solution— Prepare a solution of Aprotinin having a concentration of about 5 USP Aprotinin Units per mL. Dilute with Solution A.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 7.5-mm × 7.5-cm column that contains packing L52 and is maintained at a constant temperature of 40. The flow rate is 1 mL per minute. The chromatograph is programmed as follows. Chromatograph the Resolution solution as directed for Procedure: the retention time for aprotinin is between 17 and 20 minutes; the relative retention times are about 0.9 for N-pyroglutamyl-aprotinin, and 1.0 for aprotinin; the resolution, R, between N-pyroglutamyl-aprotinin and aprotinin is not less than 1.0; and the tailing factor for the aprotinin peak is not greater than 2.0.

Procedure— Inject 40 µL of the Test solution into the chromatograph, record the chromatogram, and measure the peak areas. Calculate the percentage of each impurity peak in the chromatogram by the formula: in which ri is the response of each impurity peak; and rs is the sum of the responses of all peaks in the chromatogram of the Test solution: not more than 1.0% N-pyroglutamyl-aprotinin is found; not more than 0.5% of any other impurity is found; and the sum of all unknown impurities is not more than 1.0%.

Mobile phase— Prepare a filtered and degassed mixture of water, glacial acetic acid, and acetonitrile (6:2:2).

Resolution solution— Prepare an aprotinin solution that contains about 5 USP Aprotinin Units per mL with about 2% aprotinin oligomers. [note—This solution can be obtained by heating lyophilized aprotinin at 112 for about 2 hours and dissolving the solid at the specified concentration in water.]

Test solution— Prepare a solution of Aprotinin in water that contains about 5 USP Aprotinin Units per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a series of three 7.8-mm × 30-cm columns containing packing L33. The flow rate is about 1.0 mL per minute. Chromatograph the Resolution solution as directed for Procedure: the retention time for aprotinin is between 24.5 and 25.5 minutes; the relative retention times are about 0.9 for the dimer and 1.0 for aprotinin; the resolution, R, between the dimer peak and the aprotinin peak is not less than 1.3; and the tailing factor for the aprotinin peak is not greater than 2.5.

Procedure— Inject about 100 µL of the Test solution into the chromatograph, record the chromatogram, and measure the peak areas. Calculate the percentage of each oligomer peak in the chromatogram by the formula: in which ri is the response of each peak having a retention time less than that of aprotinin monomer; and rs is the sum of the responses of all peaks: the sum of all oligomers is not more than 1.0%.

0.0015 M Borate buffer— Transfer about 0.93 g of boric acid into a 1000-mL volumetric flask, dissolve in 900 mL of water, adjust with 5 N sodium hydroxide to a pH of 8.0, dilute with water to volume, and mix. Transfer 100 mL of this solution into a 1000-mL volumetric flask, dilute with water to volume, and mix.

Assay preparation— Prepare a solution of Aprotinin with 0.0015 M Borate buffer to obtain a solution having about 1.67 USP Aprotinin Units per mL (about 0.6 mg per mL).

Trypsin solution— Prepare a solution of USP Trypsin Crystallized RS containing about 4300 USP Trypsin Units per mL, using 0.001 N hydrochloric acid as the solvent. Use a freshly prepared solution, and keep in ice water.

Trypsin and aprotinin solution— To 4.0 mL of the Trypsin solution, add 1.0 mL of the Assay preparation. Dilute immediately with 0.0015 M Borate buffer to 40.0 mL. Allow to stand at room temperature for 10 minutes, and then keep in ice water. [note—Use within 6 hours of preparation.]

Dilute trypsin solution— Dilute 0.5 mL of the Trypsin solution with 0.0015 M Borate buffer to 10.0 mL. Allow to stand at room temperature for 10 minutes, and then keep in ice water.

Substrate solution— Dissolve 69 mg of N-benzoyl-l-arginine ethyl ester hydrochloride in 10 mL of water. [note—Use within 2 hours.]

Procedure— Mix 9.0 mL of 0.0015 M Borate buffer and 1.0 mL of Substrate solution in a jacketed glass vessel with a capacity of about 30 mL and that contains a stirring device. The lid of the reaction vessel should contain five holes to accommodate the electrodes, the tip of a buret, a tube for the admission of nitrogen, and the introduction of reactants. An automated or manual titration apparatus may be used. Adjust to a pH of 8.0 by the addition of 0.1 N sodium hydroxide VS. Maintain an atmosphere of nitrogen within the vessel, and stir continuously. When the temperature has reached equilibrium at 25 ± 0.1, add 1.0 mL of Trypsin and aprotinin solution, and start a timer. Maintain at a pH of 8.0 by the addition of 0.1 N sodium hydroxide VS, and note the volume added every 30 seconds. Continue the reaction for 6 minutes. Determine the volume of 0.1 N sodium hydroxide added per second, in mL (n1). Carry out a similar titration using 1.0 mL of the Dilute trypsin solution. Determine the volume of 0.1 N sodium hydroxide added per second, in mL (n2). For the lyophilized powder, calculate the aprotinin activity in USP Aprotinin Units per mg using the formula: in which m is the quantity, in mg, of Aprotinin used to prepare 1 mL of the Assay preparation. For the concentrated solution, calculate the USP Aprotinin Units per mL using the following formula: in which D is the dilution factor of the concentrated solution used to prepare the Assay preparation.