Chymotrypsin

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Chymotrypsin is a proteolytic enzyme crystallized from an extract of the pancreas gland of the ox, Bos taurus L. (Fam. Bovidae). It contains NLT 1000 USP Chymotrypsin Units/mg, calculated on the dried basis, and NLT 90.0% and NMT 110.0% of the labeled potency, as determined by the Assay.

Add the following:

2 IDENTIFICATION

A. It meets the requirements in the Assay.

B.

Solution A: 0.1% Phosphoric acid in water

Solution B: 0.1% Phosphoric acid in acetonitrile

Mobile phase: See Table 1.

Table 1

Diluent: 1 mM hydrochloric acid

Standard solution: 1 mg/mL of USP Chymotry psin RS in Diluent Sample solution: 1 mg/mL of Chymotrypsin in Diluent

2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 280 nm

Column: 4.6-mm × 25-cm; 5-µm packing L26, pore size 300 Å Temperatures

Column: 60°

Autosampler: 5° Flow rate: 1.0 mL/min

Injection volume: 10 µL

2.2 System suitability

Sample: Standard solution

[NOTE—The retention time for the major peak of chymotrypsin is 16.65–20.35 min.]

Suitability requirements

Relative standard deviation: NMT 2.0% for the chymotrypsin peak, from triplicate injections

2.3 Analysis

Samples: Standard solution and Sample solution

Acceptance criteria: The retention time of the major peak of the Sample solution corresponds to that of the major peak of the Standard solution. (USP 1-Dec-2021)

3 ASSAY

PROCEDURE

Monobasic potassium phosphate solution: 9.08 mg/mL of monobasic potassium phosphate in water Dibasic sodium phosphate solution: 9.46 mg/mL of anhy drous dibasic sodium phosphate in water

Phosphate buffer: Mix 38.9 mL of Monobasic potassium phosphate solution and 61.1 mL of Dibasic sodium phosphate solution. If necessary, adjust by the dropwise addition of 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 assaying Chymotrypsin, in 50 mL of Phosphate buffer, with warming. When the solution is cool, dilute with additional Phosphate 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.]

Sample solution: Dissolve a quantity of Chymotrypsin in 0.0012 N hydrochloric acid to yield a solution containing 12–16 USP Chymotrypsin Units/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-s interval.

Blank solution: Mix 0.2 mL of 0.0012 N hydrochloric acid and 3 mL of water. Analysis

Samples: Substrate solution, Sample solution, and Blank solution

[NOTE—Determine the suitability of the substrate and check the adjustment of the spectrophotometer by performing the Analysis using USP Chymotry psin RS in place of the Sample solution.]

Conduct the Assay in a suitable spectrophotometer equipped to maintain a temperature of 25 ± 1.0° in the cell compartment. Determine the temperature in the reaction cell before and after the absorbance measurement to ensure that the temperature does not change by more than 1.0°. Pipet 3.0 mL of Blank solution into a 1-cm cell. Place the cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.00 at 237 nm. Pipet 0.2 mL of Sample 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-s intervals for NLT 5 min. 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 NLT 3 min, repeat the test and, if necessary, use a lower concentration. The duplicate determination of the Sample solution matches the first determination, of the same dilution, in rate of absorbance change.

Determine the average absorbance change per min, using only the values within the 3-min 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/min under the conditions specified in the Assay.

Calculate the number of USP Chymotrypsin Units/mg in the portion of Chymotrypsin taken:

Result = (A₂− A₁)/(T × W × F)

A₂ = absorbance straight-line initial reading

A₁ = absorbance straight-line final reading

T = time elapsed between the initial and final readings (min)

W = weight of Chymotrypsin in the volume of solution used in determining the absorbance (mg)

F = Chymotrypsin activity conversion factor, 0.0075/min

Acceptance criteria: NLT 1000 USP Chymotrypsin Units/mg on the dried basis; 90.0%–110.0% of the labeled potency

4 IMPURITIES

4.1 RESIDUE ON IGNITION 〈281〉

NMT 2.5%

Change to read:

4.2 LIMIT OF TRYPSIN

Tris buffer: 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 hy drochloride, suitable for use in assaying trypsin, to a 25- mL volumetric flask. Add 5 mL of Tris buffer, and swirl until the substrate dissolves. Add 0.25 mL of methyl red–methylene blue TS, and dilute with water to volume.

Sample solution: 10 mg/mL of Chymotrypsin in water Analysis

[NOTE—Determine the suitability of the substrate by performing the Analysis using the appropriate amount of USP Try psin Bovine RS (USP 1- Dec-2021) in place of the Sample solution.]

By means of a micropipet, transfer 50 µL of Sample solution to a depression on a white spot plate. Add 0.2 mL of Substrate solution. Acceptance criteria: No purple color develops within 3 min (NMT 1% of trypsin).

5 SPECIFIC TESTS

Change to read:

5.1 MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR SPECIFIED MICROORGANISMS 〈62〉

Total aerobic microbial count should be NMT 102 cfu/g. The total yeasts and molds count should be NMT 101 cfu/g. (USP 1-Dec-2021) It meets the requirements of the tests for absence of Pseudomonas aeruginosa, Salmonella species, Escherichia coli, (USP 1-Dec-2021) and Staphylococcus aureus.

5.2 LOSS ON DRYING 〈731〉

Analysis: Dry under vacuum at 60° for 4 h. Acceptance criteria: NMT 5.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, and avoid exposure to excessive heat. Change to read:

USP REFERENCE STANDARDS 〈11〉

USP Chymotrypsin RS

USP Trypsin Bovine RS (USP 1-Dec-2021)