» Corticotropin Injection is a sterile solution, in a suitable diluent, of the material containing the polypeptide hormone having the property of increasing the rate of secretion of adrenal corticosteroids, which is obtained from the anterior lobe of the pituitary of mammals used for food by man. Its potency is not less than 80.0 percent and not more than 125.0 percent of the potency stated on the label in USP Corticotropin Units. It may contain a suitable antimicrobial agent.
Packaging and storage Preserve in single-dose or multiple-dose containers, preferably of Type I glass. Store in a cold place.
Labeling If the labeling of Injection recommends intravenous administration, include specific information on dosage.
USP Reference standards 11
USP Ascorbic Acid RS.
USP Corticotropin RS.
USP Endotoxin RS.
USP Vasopressin RS.
Bacterial endotoxins 85 It contains not more than 3.1 USP Endotoxin Units per USP Corticotropin Unit.
Phosphate buffer 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.
Mobile phase Prepare a filtered and degassed mixture of Phosphate buffer and acetonitrile (87:13). [noteThe retention time of the vasopressin peak is very sensitive to small changes in the acetonitrile concentration.]
Standard solution Dissolve the entire contents of a vial of USP Vasopressin RS in a known volume of Phosphate buffer, and dilute with Phosphate buffer to obtain a final solution containing 0.1 USP Vasopressin Unit per mL.
Test solution Dissolve the entire contents of a vial of the Injection in a known volume of Phosphate buffer, and dilute with Phosphate buffer to obtain a final solution containing 2.0 USP Corticotropin Units per mL.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a variable wavelength detector set at 220 nm and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the vasopressin activity in USP Vasopressin Unit per USP Corticotropin Unit by the formula:
C(rU / rS)/2in which C is the concentration, in USP Vasopressin Units per mL, of the Standard solution; and rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively. The vaopressin activity is not more than 0.05 USP Vasopressin Unit per USP Corticotropin Unit.
pH 791: between 3.0 and 7.0.
Particulate matter 788: meets the requirements for small-volume injections.
Other requirements It meets the requirements under Injections 1.
Standard preparation Pipet 2.5 mL of gelatin TS into an opened container of USP Corticotropin RS, and mix, to obtain a solution having a concentration of 2.0 USP Corticotropin Units per mL. Using gelatin TS as a diluent, prepare three diluted standard solutions such that the respective concentrations of corticotropin constitute a geometric series such as 1:2:4 or 1:3:9 and such that the quantity of corticotropin in each 0.5 mL lies within the range of 10 to 300 milli-units.
Assay preparation In the same manner, using the same diluent, dilute the Injection to give three test solutions corresponding to those of the standard.
The animals Select healthy rats, of the same but either sex, that have been raised on a diet fully adequate with respect to vitamin and mineral content. Anesthetize the rats with ether, and remove the hypophysis from each by application of gentle suction through a fine-tipped tube. Between 16 and 48 hours after the operation, select those rats weighing between 80 and 180 g, but restrict the selection so that no rat is more than 30% heavier than the lightest, and the number of rats is an exact multiple of 6. Separate the selected rats into 6 groups, equal in size, of not less than 6 rats each, and assign at random one of the three diluted standard solutions or of the three test solutions to each group.
Procedure Inject all rats of each group subcutaneously with the assigned test doses. Three hours after the injection, anesthetize the rats, and remove both adrenal glands from each rat, free them from adhering tissue, and promptly weigh each pair on a suitable balance to the nearest 0.2 mg. Place the weighed glands from each rat in suitable vessels each containing 8.0 mL of metaphosphoric acid solution (1 in 40), and comminute the glands as by grinding with a small quantity of washed sand. Cover each vessel, and proceed similarly until all glands have been extracted.
Ascorbic acid determination Filter the metaphosphoric acid extracts, and pipet 4 mL of each filtrate into suitable vessels each containing 4.0 mL of indophenol-acetate TS. Mix by shaking, and read the absorbance at 520 nm, with a suitable spectrophotometer. From the observed absorbance and the standard curve prepared as directed in the next paragraph, calculate the amount of ascorbic acid in terms of mg of ascorbic acid in each 100 g of adrenal gland tissue.
Prepare a standard concentration-absorbance curve, using three standard solutions containing in each mL, respectively, 6.0, 8.0, and 10.0 µg of USP Ascorbic Acid RS in metaphosphoric acid solution (1 in 40). Pipet into each of 3 suitable vessels, preferably spectrophotometer cells, 4 mL of indophenol-acetate TS. Add 4.0 mL of one of the three standard ascorbic acid solutions to one of the cells, mix, and promptly read the absorbance in the same instrument and under the same conditions as for the adrenal gland extracts. Repeat the process for the other two standard ascorbic acid solutions, plot the concentration-absorbance values, and draw the straight line best fitting the 3 plotted points.
Calculation Tabulate the observed concentration of ascorbic acid in the adrenal glands of each rat, designated by the symbol y, for each dosage group of f rats. If the data from one or more rats are missing, adjust to groups of equal size by suitable means (see Replacement of Missing Values 111). Total the values of y in each group, and designate each total as T, subscripts 1 to 3 for the three successive dosage levels and subscripts S and U for the Standard and the Injection, respectively. Test both the agreement in slope of the dosage-response lines for the Standard and for the Injection, and the lack of curvature, as directed for a 3-dose balanced assay (see Tests of Assay Validity 111). If the combined discrepancy as measured by F3 exceeds its tabular value in Table 9 (see Combination of Independent Assays 111), regard these data as preliminary and repeat the assay.
Determine the logarithm of potency of the Injection by the formula:
M = (4iTa / 3Tb) + log R,
in which Ta = S(TU TS); Tb = S(T3 T1); i is the interval between successive log doses of both the Standard preparation and the Assay preparation; and R = vS / vU is the ratio of the high dose of the Standard in USP Units (vS) to the high dose of the Injection in mL (vU).
Compute the log confidence interval L (see Confidence Intervals for Individual Assays 111).
Replication Repeat the entire determination at least once. Test the agreement among the two or more independent determinations, and compute the weight for each (see Combination of Independent Assays 111). Calculate the weighted mean log-potency M and its confidence interval, Lc (see Confidence Intervals for Individual Assays 111). The potency, P*, is satisfactory if P* = antilog M is not less than 80% and not more than 125% of the labeled potency and if the confidence interval does not exceed 0.40.
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USP32NF27 Page 2025Pharmacopeial Forum: Volume No. 28(6) Page 1783
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.