0.067M Phosphate buffer ,pH7.0—Dissolve 4.54g of monobasic potassium phosphate in water to make 500mLof solution.Dissolve 4.73g of anhydrous dibasic sodium phosphate in water to make 500mLof solution.Mix 38.9mLof the monobasic potassium phosphate solution with 61.1mLof dibasic sodium phosphate solution.Adjust dropwise,if necessary,with dibasic sodium phosphate solution to a pHof 7.0.

Substrate solution— Dissolve 23.7mg of N-acetyl-L-tyrosine ethyl ester,suitable for use in determining chymotrypsin,in about 50mLof 0.067M Phosphate buffer,pH7.0with warming.When cool,dilute with additional pH7.0buffer to 100mL.(Substrate solutionmay 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 0.0010Nhydrochloric acid to obtain a solution containing 650USP Trypsin Units per mL.

Procedure— Conduct the test in a suitable spectrophotometer equipped to maintain a temperature of 25±0.1in 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 200µLof 0.0010Nhydrochloric acid and 3.0mLof the Substrate solutioninto a 1-cm cell.Place this cell in the spectrophotometer,and adjust the instrument so that the absorbance reads 0.200at 237nm.Pipet 200µLof Crystallized Trypsin solutioninto another 1-cm cell,add 3.0mLof the Substrate solution,and place the cell in the spectrophotometer.[NOTE—This order of addition is to be followed.]At the time the Substrate solutionis added,start a stopwatch,and read the absorbance at 30-second intervals for not less than 5minutes.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 3minutes,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 0.0075per 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 A2is the absorbance straight-line initial reading,A1is the absorbance straight-line final reading,Tis the elapsed time,in minutes,between the initial and final readings,and Wis the weight,in mg,of Crystallized Trypsin in the volume of solution used in determining the absorbance.Not more than 50USP Chymotrypsin Units per 2500USP Trypsin Units is found,indicating the presence of not more than approximately 5%of chymotrypsin.

0.067M Phosphate buffer ,pH7.6—Dissolve 4.54g of monobasic potassium phosphate in water to make 500mLof solution.Dissolve 4.73g of anhydrous dibasic sodium phosphate in water to make 500mLof solution.Mix 13mLof the monobasic potassium phosphate solution with 87mLof the anhydrous dibasic sodium phosphate solution.

Substrate solution— Dissolve 85.7mg of N-benzoyl-L-arginine ethyl ester hydrochloride,suitable for use in assaying Crystallized Trypsin (see NOTE),in water to make 100mL.Dilute 10mLof this solution with 0.067M Phosphate buffer,pH7.6to 100mL.Determine the absorbance of this solution,in a 1-cm cell,at 253nm,in a suitable spectrophotometer equipped with thermospacers to maintain a temperature of 25±0.1,using water as the blank.By the addition of 0.067M Phosphate buffer,pH7.6,or of the Substrate solutionbefore dilution,adjust the absorbance so that it measures not less than 0.575and not more than 0.585.Use this Substrate solutionwithin 2hours.

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

Procedure— Pipet 200µLof 0.0010Nhydrochloric acid and 3.0mLof the Substrate solutioninto a 1-cm cell.Place this cell in a spectrophotometer,and adjust the instrument so that the absorbance reads 0.050at 253nm.Pipet 200µLof Crystallized Trypsin solution,containing 10to 12USP Trypsin Units,into another 1-cm cell,add 3.0mLof Substrate solution,and place the cell in the spectrophotometer.At the time the Substrate solutionis added,start a stopwatch,and read the absorbance at 30-second intervals for 5minutes.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 3minutes,repeat the run,and if necessary,use a lower concentration.One USP Trypsin Unit is the activity causing a change in absorbance of 0.003per minute under the conditions specified in this Assay.Calculate the number of USP Trypsin Units per mg taken by the formula: in which A1is the absorbance straight-line final reading,A2is the absorbance straight-line initial reading,Tis the elapsed time,in minutes,between the initial and final readings,and Wis the weight,in mg,of Crystallized Trypsin in the volume of solution used in determining the absorbances.