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C43H66N12O12S2 1007.19

Oxytocin [50-56-6].
» Oxytocin is a nonapeptide hormone having the property of causing the contraction of uterine smooth muscle and of the myoepithelial cells within the mammary gland. It is prepared by synthesis or obtained from the posterior lobe of the pituitary of healthy domestic animals used for food by man. Its oxytocic activity is not less than 400 USP Oxytocin Units per mg.
Packaging and storage— Preserve in tight containers, preferably of Type I glass, in a refrigerator.
Microbial enumeration tests 61 and Tests for specified microorganisms 62 The total bacterial count does not exceed 200 cfu per g. For products of animal origin, it meets also the requirements of the tests for absence of Salmonella species and Escherichia coli.
A: The retention time of the oxytocin peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation as obtained in the Assay.
B: Obtain the uterus from a 120- to 200-g rat in diestrus. Suspend one horn of the uterus in a bath at 32 containing, in each L of water, 9.0 g of sodium chloride, 0.42 g of potassium chloride, 0.16 g of calcium chloride, 0.50 g of sodium bicarbonate, 0.25 g of dextrose, and 0.0053 g of magnesium chloride. Oxygenate the bath solution with a mixture of 95% oxygen and 5% carbon dioxide. Record the contractions of the muscle on a recorder, using an isotonic and linear transducer. Add to the bath two appropriate dilutions of USP Oxytocin RS, and record the contraction of the muscle following each dilution. The appropriate dilutions are determined to give clearly distinctive submaximal contractions. Replace the solution in the bath with a fresh solution, and wait until the muscle is relaxed. Dissolve or dilute the preparation to be tested in a suitable diluent to obtain responses on the addition of two doses similar to the one used with the Standard solution. The magnitude of the contractions obtained with the Standard solution is comparable to the contractions obtained with the test solution.
Vasopressor activity (for product labeled of animal origin)— Proceed as directed in the Assay under Vasopressin, except to prepare a dilution of the Standard solution of USP Vasopressin RS containing 0.1 USP Vasopressin Unit per mL. The vasopressic activity of the test preparation is not more than 0.1 USP Vasopressin Unit per mL.
Ordinary impurities— The sum of the responses of impurities in the chromatogram of the Assay preparation obtained in the Assay is not more than 5% of the area of the oxytocin peak.
Mobile phase A— Prepare a buffer solution of 0.1 M monobasic sodium phosphate.
Mobile phase B— Prepare a filtered and degassed mixture of acetonitrile in water (1:1). Make adjustments if necessary (see System Suitability under Chromatography 621).
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 Oxytocin 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— Dissolve an accurately weighed quantity of Oxytocin in Diluent to obtain a solution containing about 10 USP Oxytocin Units per mL.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a variable wavelength detector set at 220 nm and a 12.0-cm × 4.6-mm column that contains 5-µm packing L1, and is programmed to provide variable mixtures of Mobile phase A and Mobile phase B. The column is maintained at room temperature, and the flow rate is about 1.5 mL per minute. The system is equilibrated with a mixture of 70% Mobile phase A and 30% Mobile phase B. After each injection of the Standard preparation and the Assay preparation, the composition of the mobile phase is changed linearly over the next 20 minutes so that it consists of a mixture of 50% Mobile phase A and 50% Mobile phase B. Chromatograph the Standard preparation, and record the chromatograms as directed for Procedure. Adjust the flow rate or the composition of the Mobile phase such that the retention time of oxytocin is approximately 10 minutes and between 15 and 17 minutes for chlorobutanol. The resolution, R, between oxytocin 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 oxytocin.
Procedure— Separately inject three equal volumes (about 100 µL) of the Assay preparation and the Standard preparation into the chromatograph, and record the chromatograms as described under Chromatographic system. Identify the peaks, and determine the area of the oxytocin peak. Calculate the potency of oxytocin in USP Oxytocin Units per mg by the formula:
C(rU / rS)(V / W)
in which C is the concentration, in USP Oxytocin Units per mL, of the Standard preparation; and rU and rS are the mean peak responses obtained from the Assay preparation and the Standard preparation, respectively; V is the volume of sample solution in which the sample was dissolved; and W is the amount, in mg, of oxytocin dissolved in the sample solution.
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Topic/Question Contact Expert Committee
Monograph Larry N. Callahan, Ph.D.
Senior Scientist
(BBPP05) Biologics and Biotechnology - Proteins and Polysaccharides
Reference Standards Lili Wang, Technical Services Scientist
61 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
(MSA05) Microbiology and Sterility Assurance
62 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
(MSA05) Microbiology and Sterility Assurance
USP32–NF27 Page 3185
Pharmacopeial Forum: Volume No. 34(3) Page 647
Chromatographic Column—
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.