Phosphate buffer— Dissolve 6.6g of dibasic ammonium phosphate in about 950mLof water,and adjust with concentrated phosphoric acid to a pHof 3.0.Dilute with water to 1L,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate bufferand acetonitrile (87:13).[NOTE—The 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 RSin a known volume of Phosphate buffer,and dilute with Phosphate bufferto obtain a final solution containing 0.1USP 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 2.0USP Corticotropin Units per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a variable wavelength detector set at 220nm and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1.5mLper 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 20µL)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: in which Cis the concentration,in USP Vasopressin Units per mL,of the Standard solution;and rUand rSare the peak responses obtained from the Test solution and the Standard solution,respectively.The vaopressin activity is not more than 0.05USP Vasopressin Unit per USP Corticotropin Unit.

Standard preparation— Pipet 2.5mLof gelatin TSinto an opened container of USP Corticotropin RS,and mix,to obtain a solution having a concentration of 2.0USP Corticotropin Units per mL.Using gelatin TSas a diluent,prepare three diluted standard solutions such that the respective concentrations of corticotropin constitute a geometric series such as 1:2:4or 1:3:9and such that the quantity of corticotropin in each 0.5mLlies within the range of 10to 300milli-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 16and 48hours after the operation,select those rats weighing between 80and 180g,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 6groups,equal in size,of not less than 6rats 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 0.2mg.Place the weighed glands from each rat in suitable vessels each containing 8.0mLof metaphosphoric acid solution (1in 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 4mLof each filtrate into suitable vessels each containing 4.0mLof indophenol-acetate TS.Mix by shaking,and read the absorbance at 520nm,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 100g of adrenal gland tissue.

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 frats.If the data from one or more rats are missing,adjust to groups of equal size by suitable means (see Replacement of Missing Values á111ñ).Total the values of yin each group,and designate each total as T,subscripts 1to 3for the three successive dosage levels and subscripts Sand Ufor 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 á111ñ).If the combined discrepancy as measured by F3exceeds its tabular value in Table 9(see Combination of Independent Assays á111ñ),regard these data as preliminary and repeat the assay.

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 á111ñ).Calculate the weighted mean log-potency bar(M)and its confidence interval,Lc(see Confidence Intervals for Individual Assays á111ñ).The potency,P*,is satisfactory if P*=antilog bar(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.