A: The retention time of the vasopressin peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

B: Mass spectral analysis—

Mobile phase— Dissolve 6.6 g of dibasic ammonium phosphate in about 950 mL of water, and adjust with concentrated phosphoric acid to a pH of 3.0. Dilute with water to 1 L, and mix. To 870 mL of this solution add 130 mL of acetonitrile, and mix. Filter under vacuum through a 0.45-µm nylon membrane. [note—The retention time of the vasopressin peak is very sensitive to small changes in acetonitrile concentration in the Mobile phase.]

Diluent— Dissolve 5.0 g of chlorobutanol in 5.0 mL of glacial acetic acid, add 5.0 g of alcohol, 1.1 g of sodium acetate, and 1000 mL of water, and mix.

Standard preparation— Dissolve the entire contents of a vial of USP Vasopressin RS in a known volume of Diluent. [note—The solution may be diluted as necessary to a working concentration range for the assay.]

Assay preparation— Transfer about 10 mg of Vasopressin, accurately weighed, to a 100-mL volumetric flask. Dissolve in 0.25% glacial acetic acid, and dilute with the same solvent to volume. Mix, and pipet 5.0 mL of this solution into a 100-mL volumetric flask, and dilute with 0.25% glacial acetic acid to volume.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a variable wavelength detector set at 220 mm and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is 1.0 mL per minute. The column is allowed to equilibrate for 1 hour before making the first injection. Determine the suitability of the system (see System Suitability under Chromatography 621) as follows. Inject 20 µL of the Standard preparation into the equilibrated liquid chromatograph, allow about 60 minutes for complete elution, and record the chromatogram as directed for Procedure: the retention time of the vasopressin peak is between 6 and 9 minutes, and is completely resolved from adjacent peaks; the resolution, R, between vasopressin and the nearest adjacent peak is not less than 1.5; and the relative standard deviation for replicate injections is not more than 2.0% for vasopressin.

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 responses for the major peaks. Calculate the potency of Vasopressin, in USP Vasopressin Units per mg, by the formula: in which V is the volume of sample solution in which the sample was dissolved; and W is the amount, in mg, of vasopressin dissolved in the sample solution; C is the concentration in USP Vasopressin Units per mL in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.