Chymotrypsin solution— Dissolve 100 mg in 10.0 mL of water.

pH 8.1 Tris (hydroxymethyl)aminomethane buffer, 0.08 M—Dissolve 294 mg of calcium chloride in 40 mL of 0.20 M tris(hydroxymethyl)aminomethane, adjust with 1 N hydrochloric acid to a pH of 8.1, and dilute with water to 100 mL.

Substrate solution— Transfer 98.5 mg of p-toluenesulfonyl-l-arginine methyl ester hydrochloride, suitable for use in assaying trypsin, to a 25-mL volumetric flask. Add 5 mL of pH 8.1 Tris(hydroxymethyl)aminomethane buffer, 0.08 M, and swirl until the substrate dissolves. Add 0.25 mL of methyl red–methylene blue TS, and dilute with water to volume.

Procedure— [note—Determine the suitability of the substrate by performing the Procedure using the appropriate amount of USP Trypsin Crystallized RS in place of the test specimen.] By means of a micropipet, transfer 50 µL of Chymotrypsin solution to a depression on a white spot plate. Add 0.2 mL of Substrate solution: no purple color develops within 3 minutes (not more than 1% of trypsin).

pH 7.0 phosphate buffer , fifteenth-molar—Dissolve 4.54 g of monobasic potassium phosphate in water to make 500 mL of solution. Dissolve 4.73 g of anhydrous dibasic sodium phosphate in water to make 500 mL of solution. Mix 38.9 mL of the monobasic potassium phosphate solution with 61.1 mL of dibasic sodium phosphate solution. If necessary, adjust to a pH of 7.0 by the dropwise addition of dibasic sodium phosphate solution.

Substrate solution— Dissolve 23.7 mg of N-acetyl-l-tyrosine ethyl ester, suitable for use in assaying Chymotrypsin, in about 50 mL of pH 7.0 phosphate buffer, fifteenth-molar, with warming. When the solution is cool, dilute with additional pH 7.0 buffer to 100 mL. [note—Substrate solution may be stored in the frozen state and used after thawing, but it is important to freeze it immediately after preparation.]

Chymotrypsin solution— Dissolve a sufficient quantity of Chymotrypsin, accurately weighed, in 0.0012 N hydrochloric acid to yield a solution containing between 12 and 16 USP Chymotrypsin Units per mL. The dilution is correct if, during the conduct of the assay, there is a change in absorbance of between 0.008 and 0.012 in each 30-second interval.

Procedure— [note—Determine the suitability of the substrate and check the adjustment of the spectrophotometer by performing the Procedure using USP Chymotrypsin RS in place of the assay specimen.] Conduct the assay in a suitable spectrophotometer equipped to maintain a temperature of 25 ± 0.1 in the cell compartment. Determine the temperature in the reaction cell before and after the measurement of absorbance in order to ensure that the temperature does not change by more than 0.5. Pipet 0.2 mL of 0.0012 N hydrochloric acid and 3.0 mL of Substrate solution into a 1-cm cell. Place this cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.200 at 237 nm. Pipet 0.2 mL of Chymotrypsin solution into another 1-cm cell, add 3 mL of Substrate solution, and place the cell in the spectrophotometer. [note—Carefully follow this order of addition, and begin timing the reaction from the addition of the Substrate solution.] Read the absorbance at 30-second intervals for not less than 5 minutes. Repeat the procedure on the same dilution at least once. Absolute absorbance values are less important than a constant rate of absorbance change. If the rate of change fails to remain constant for not less than 3 minutes, repeat the test and, if necessary, use a lower concentration. The duplicate determination at the same dilution matches the first determination in rate of absorbance change. Determine the average absorbance change per minute, using only the values within the 3-minute portion of the curve where the rate of absorbance change is constant. Plot a curve of absorbance against time. One USP Chymotrypsin Unit is the activity causing a change in absorbance of 0.0075 per minute under the conditions specified in this assay. Calculate the number of USP Chymotrypsin Units per mg taken by the formula: in which A2 is the absorbance straight-line initial reading, A1 is the absorbance straight-line final reading, T is the elapsed time, in minutes, between the initial and final readings, and W is the weight, in mg, of Chymotrypsin in the volume of solution used in determining the absorbance.