Chymotrypsin solution— Dissolve 100mg in 10.0mLof water.

pH8.1Tris (hydroxymethyl)aminomethane buffer,0.08M—Dissolve 294mg of calcium chloride in 40mLof 0.20Mtris(hydroxymethyl)aminomethane,adjust with 1Nhydrochloric acid to a pHof 8.1,and dilute with water to 100mL.

Substrate solution— Transfer 98.5mg of p-toluenesulfonyl-L-arginine methyl ester hydrochloride,suitable for use in assaying trypsin,to a 25-mLvolumetric flask.Add 5mLof pH8.1Tris(hydroxymethyl)aminomethane buffer,0.08M,and swirl until the substrate dissolves.Add 0.25mLof methyl red–methylene blue TS,and dilute with water to volume.

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

pH7.0phosphate buffer ,fifteenth-molar—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.If necessary,adjust to a pHof 7.0by the dropwise addition of dibasic sodium phosphate solution.

Substrate solution— Dissolve 23.7mg of N-acetyl-L-tyrosine ethyl ester,suitable for use in assaying Chymotrypsin,in about 50mLof pH7.0phosphate buffer,fifteenth-molar,with warming.When the solution is cool,dilute with additional pH7.0buffer to 100mL.[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 0.0012Nhydrochloric acid to yield a solution containing between 12and 16USP Chymotrypsin Units per mL.The dilution is correct if,during the conduct of the assay,there is a change in absorbance of between 0.008and 0.012in 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 RSin place of the assay specimen.]Conduct the assay 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 in order to ensure that the temperature does not change by more than 0.5.Pipet 0.2mLof 0.0012Nhydrochloric acid and 3.0mLof Substrate solutioninto a 1-cm cell.Place this cell in the spectrophotometer,and adjust the instrument so that the absorbance will read 0.200at 237nm.Pipet 0.2mLof Chymotrypsin solutioninto another 1-cm cell,add 3mLof 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 5minutes.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 3minutes,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 0.0075per minute under the conditions specified in this assay.Calculate the number of USP Chymotrypsin Units per mg 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 Chymotrypsin in the volume of solution used in determining the absorbance.