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.
About 18 hours prior to the test, select a male rat weighing between 275 g and 325 g. Inject, subcutaneously, 1 mL per kg of body weight of a solution prepared by dissolving 50 mg of phenoxybenzamine hydrochloride in 0.1 mL of alcohol, acidifying with 1 drop of hydrochloric acid, and diluting with saline TS
to 5 mL. On the day of the test, anesthetize the rat, using an anesthetic substance that favors the maintenance of a uniform blood pressure. Secure the animal, and cannulate the trachea for artificial respiration. Arrange to obtain a continuous record of the blood pressure from the carotid artery. Arrange for intravenous injections by means of a suitable cannula approximately 1 mm in external diameter inserted into a femoral or jugular vein. Keep the animal warm during its preparation and during the test. Determine by trial the dose of the Standard preparation
which, when injected intravenously at regular intervals of 12 to 15 minutes, will produce consistent blood pressure elevations of between 20 and 70 mm of mercury. Select the 2 doses that would be in a ratio of approximately 2 to 3, and prepare 2 doses of the Test preparation
that correspond to the doses of the selected Standard preparations
. Inject the rat with each dose in replicate of the Standard preparation
and the Test preparation
in a random fashion at regular intervals of 12 to 15 minutes, and record the blood pressures. The requirements of the test are met if the increase in blood pressure between the low dose and the high dose of the Standard preparation
is comparable to that of the Test preparation
Oxytocic activity (for product labeled of animal origin)
Proceed as directed in the Assay
, except that a suitable dilution of the USP Oxytocin RS
will contain approximately 1.2 USP Oxytocin Units per mL of Standard preparation
. The oxytocic activity of the Test preparation
is not more than 1.2 USP Oxytocin Units per mL.
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. [NOTEThe retention time of the vasopressin peak is very sensitive to small changes in acetonitrile concentration in the Mobile phase.]
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.
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.]
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.
(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.
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:
20C(rU / rS)(V / W),
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
are the peak responses obtained from the Assay preparation
and the Standard preparation