0.067 M Phosphate buffer , pH 7.0—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. Adjust dropwise, if necessary, with dibasic sodium phosphate solution to a pH of 7.0.

Substrate solution— Dissolve 23.7 mg of N-acetyl-L-tyrosine ethyl ester, suitable for use in determining chymotrypsin, in about 50 mL of 0.067 M Phosphate buffer, pH 7.0 with warming. When cool, dilute with additional pH 7.0 buffer to 100 mL. (Substrate solution may be stored in the frozen state and used after thawing; it is important, however, to freeze immediately after preparation.)

Crystallized Trypsin solution— Dissolve a sufficient quantity of Crystallized Trypsin, accurately weighed, in 0.0010 N hydrochloric acid to obtain a solution containing 650 USP Trypsin Units per mL.

Procedure— Conduct the test 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 to ensure that the temperature does not change by more than 0.5. Pipet 200 µL of 0.0010 N hydrochloric acid and 3.0 mL of the Substrate solution into a 1-cm cell. Place this cell in the spectrophotometer, and adjust the instrument so that the absorbance reads 0.200 at 237 nm. Pipet 200 µL of Crystallized Trypsin solution into another 1-cm cell, add 3.0 mL of the Substrate solution, and place the cell in the spectrophotometer. [NOTE—This order of addition is to be followed.] At the time the Substrate solution is added, start a stopwatch, and 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 of less importance than the constancy of the rate of change of absorbance. If the rate of change does not remain constant for at least 3 minutes, repeat the run, and if necessary, use a lower concentration. The duplicate run at the same dilution should match the first run 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 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 test. Calculate the number of USP Chymotrypsin Units per mg of Crystallized Trypsin 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 Crystallized Trypsin in the volume of solution used in determining the absorbance. Not more than 50 USP Chymotrypsin Units per 2500 USP Trypsin Units is found, indicating the presence of not more than approximately 5% of chymotrypsin.

0.067 M Phosphate buffer , pH 7.6—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 13 mL of the monobasic potassium phosphate solution with 87 mL of the anhydrous dibasic sodium phosphate solution.

Substrate solution— Dissolve 85.7 mg of N-benzoyl-L-arginine ethyl ester hydrochloride, suitable for use in assaying Crystallized Trypsin (see NOTE), in water to make 100 mL. Dilute 10 mL of this solution with 0.067 M Phosphate buffer, pH 7.6 to 100 mL. Determine the absorbance of this solution, in a 1-cm cell, at 253 nm, in a suitable spectrophotometer equipped with thermospacers to maintain a temperature of 25 ± 0.1, using water as the blank. By the addition of 0.067 M Phosphate buffer, pH 7.6, or of the Substrate solution before dilution, adjust the absorbance so that it measures not less than 0.575 and not more than 0.585. Use this Substrate solution within 2 hours.

Crystallized Trypsin solution— Dissolve a sufficient quantity of Crystallized Trypsin, accurately weighed, in 0.0010 N hydrochloric acid to obtain a solution containing about 50 to 60 USP Trypsin Units per mL.

Procedure— Pipet 200 µL of 0.0010 N hydrochloric acid and 3.0 mL of the Substrate solution into a 1-cm cell. Place this cell in a spectrophotometer, and adjust the instrument so that the absorbance reads 0.050 at 253 nm. Pipet 200 µL of Crystallized Trypsin solution, containing 10 to 12 USP Trypsin Units, into another 1-cm cell, add 3.0 mL of Substrate solution, and place the cell in the spectrophotometer. At the time the Substrate solution is added, start a stopwatch, and read the absorbance at 30-second intervals for 5 minutes. Repeat the procedure on the same dilution at least once. Plot a curve of absorbance against time, and use only those values that form a straight line to determine the activity of the Crystallized Trypsin. If the rate of change does not remain constant for at least 3 minutes, repeat the run, and if necessary, use a lower concentration. One USP Trypsin Unit is the activity causing a change in absorbance of 0.003 per minute under the conditions specified in this Assay. Calculate the number of USP Trypsin Units per mg taken by the formula: in which A1 is the absorbance straight-line final reading, A2 is the absorbance straight-line initial reading, T is the elapsed time, in minutes, between the initial and final readings, and W is the weight, in mg, of Crystallized Trypsin in the volume of solution used in determining the absorbances.