pH 6.8 Phosphate buffer— On the day of use, dissolve 13.6 g of monobasic potassium phosphate in water to make 500 mL of solution. Dissolve 14.2 g of anhydrous dibasic sodium phosphate in water to make 500 mL of solution. Mix 51 mL of the monobasic potassium phosphate solution with 49 mL of the dibasic sodium phosphate solution. If necessary, adjust by the dropwise addition of the appropriate solution to a pH of 6.8.

Substrate solution— On the day of use, stir a portion of purified soluble starch equivalent to 2.0 g of dried substance with 10 mL of water, and add this mixture to 160 mL of boiling water. Rinse the beaker with 10 mL of water, add it to the hot solution, and heat to boiling, with continuous mixing. Cool to room temperature, and add water to make 200 mL.

Standard preparation— Weigh accurately about 20 mg of USP Pancreatin Amylase and Protease RS into a suitable mortar. Add about 30 mL of pH 6.8 Phosphate buffer, and triturate for 5 to 10 minutes. Transfer the mixture with the aid of pH 6.8 Phosphate buffer to a 50-mL volumetric flask, dilute with pH 6.8 phosphate buffer to volume, and mix. Calculate the activity, in USP Units of amylase activity per mL, of the resulting solution from the declared potency on the label of the Reference Standard.

Assay preparation— For Pancrelipase having about 4 times the amylase activity of the USP Pancreatin Amylase and Protease RS, weigh accurately about 10 mg of Pancrelipase into a suitable mortar. [NOTE—For Pancrelipase having a different amylase activity, weigh accurately the amount necessary to obtain an Assay preparation having amylase activity per mL corresponding approximately to that of the Standard preparation.] Add about 3 mL of pH 6.8 Phosphate buffer, and triturate for 5 to 10 minutes. Transfer the mixture with the aid of pH 6.8 Phosphate buffer to a 100-mL volumetric flask, dilute with pH 6.8 Phosphate buffer to volume, and mix.

Procedure— Prepare four stoppered, 250-mL conical flasks, and mark them S, U, BS, and BU. Pipet into each flask 25 mL of Substrate solution, 10 mL of pH 6.8 Phosphate buffer, and 1 mL of sodium chloride solution (11.7 in 1000), insert the stoppers, and mix. Place the flasks in a water bath maintained at 25 ± 0.1, and allow them to equilibrate. To flasks BU and BS add 2 mL of 1 N hydrochloric acid, mix, and return the flasks to the water bath. To flasks U and BU add 1.0-mL portions of Assay preparation, and to flasks S and BS add 1.0 mL of Standard preparation. Mix each, and return the flasks to the water bath. After 10 minutes, accurately timed from the addition of the enzyme, add 2-mL portions of 1 N hydrochloric acid to flasks S and U, and mix. To each flask, with continuous stirring, add 10.0 mL of 0.1 N iodine VS, and immediately add 45 mL of 0.1 N sodium hydroxide. Place the flasks in the dark at a temperature between 15 and 25 for 15 minutes. To each flask add 4 mL of 2 N sulfuric acid, and titrate with 0.1 N sodium thiosulfate VS to the disappearance of the blue color. Calculate the amylase activity, in USP Units per mg, of the Pancrelipase taken by the formula: in which CS is the amylase activity of the Standard preparation, in USP Units per mL, WU is the amount, in mg, of Pancrelipase taken, and VU, VS, VBU, and VBS are the volumes, in mL, of 0.1 N sodium thiosulfate consumed in the titration of the solutions in flasks U, S, BU, and BS, respectively.

Acacia solution— Centrifuge a solution of acacia (1 in 10) until clear. Use only the clear solution.

Olive oil substrate— Combine 165 mL of Acacia solution, 20 mL of olive oil, and 15 g of crushed ice in the cup of an electric blender. Cool the mixture in an ice bath to 5, and homogenize at high speed for 15 minutes, intermittently cooling in an ice bath to prevent the temperature from exceeding 30.

Buffer solution— Dissolve 60 mg of tris(hydroxymethyl)aminomethane and 234 mg of sodium chloride in water to make 100 mL.

Bile salts solution— Prepare a solution to contain 80.0 mg of USP Bile Salts RS in each mL.

Standard test dilution— Suspend about 200 mg of USP Pancreatin Lipase RS, accurately weighed, in about 3 mL of cold water in a mortar, triturate for 10 minutes, and add cold water to a volume necessary to produce a concentration of 8 to 16 USP Units of lipase activity per mL, based upon the declared potency on the label of the Reference Standard. Maintain the suspension at 4, and mix before using. For each determination withdraw 5 to 10 mL of the cold suspension, and allow the temperature to rise to 20 before pipeting the exact volume.

Assay test dilution— Suspend about 200 mg of Pancrelipase, accurately weighed, in about 3 mL of cold water in a mortar, triturate for 10 minutes, and add cold water to a volume necessary to produce a concentration of 8 to 16 USP Units of lipase activity per mL, based upon the estimated potency of the test material. Maintain the suspension at 4, and mix before using. For each determination withdraw 5 to 10 mL of the cold suspension, and allow the temperature to rise to 20 before pipeting the exact volume.

Procedure— Mix 10.0 mL of Olive oil substrate, 8.0 mL of Buffer solution, 2.0 mL of Bile salts solution, and 9.0 mL of water in a jacketed glass vessel of about 50-mL capacity, the outer chamber of which is connected to a thermostatically controlled water bath. Cover the mixture, and stir continuously with a mechanical stirring device. With the mixture maintained at a temperature of 37 ± 0.1, add 0.1 N sodium hydroxide VS, from a microburet inserted through an opening in the cover, to adjust the pH to 9.20 potentiometrically using a calomel-glass electrode system. Add 1.0 mL of Assay test dilution, and then continue adding the 0.1 N sodium hydroxide VS for 5 minutes to maintain the pH at 9.0. Determine the volume of 0.1 N sodium hydroxide VS added after each minute.

Calculation of potency— Plot the volume of 0.1 N sodium hydroxide VS titrated against time. Using only the points which fall on the straight-line segment of the curve, calculate the mean acidity released per minute by the test specimen and the Standard. Taking into consideration the dilution factors, calculate the lipase activity, in USP Units, of the Pancrelipase taken by comparison to the activity of the Reference Standard, using the lipase activity stated on the label of USP Pancreatin Lipase RS.

Casein substrate— Place 1.25 g of finely powdered casein in a 100-mL conical flask containing 5 mL of water, shake to form a suspension, add 10 mL of 0.1 N sodium hydroxide, shake for 1 minute, add 50 mL of water, and shake for about 1 hour to dissolve the casein. If necessary, adjust to a pH of about 8, using 1 N sodium hydroxide or 1 N hydrochloric acid. Transfer the solution quantitatively to a 100-mL volumetric flask, dilute with water to volume, and mix. Use this substrate on the day it is prepared.

Buffer solution— Dissolve 6.8 g of monobasic potassium phosphate and 1.8 g of sodium hydroxide in 950 mL of water in a 1000-mL volumetric flask, adjust to a pH of 7.5 ± 0.2, using 0.2 N sodium hydroxide, dilute with water to volume, and mix. Store this solution in a refrigerator.

Trichloroacetic acid solution— Dissolve 50 g of trichloroacetic acid in 1000 mL of water. This solution may be stored at room temperature.

Filter paper— Determine the suitability of the filter paper by filtering a 5-mL portion of Trichloroacetic acid solution through the paper and measuring the absorbance of the filtrate at 280 nm, using an unfiltered portion of the same Trichloroacetic acid solution as the blank: the absorbance is not more than 0.04. If the absorbance is more than 0.04, wash the filter paper repeatedly with Trichloroacetic acid solution until the absorbance of the filtrate, determined as above, is not more than 0.04.

Standard test dilution— Add about 100 mg of USP Pancreatin Amylase and Protease RS, accurately weighed, to 100.0 mL of Buffer solution, and mix by shaking intermittently at room temperature for about 25 minutes. Dilute quantitatively with Buffer solution to produce a concentration of about 2.5 USP Units of protease activity per mL, based upon the declared potency on the label of the Reference Standard.

Assay test dilution— Weigh accurately about 100 mg of Pancrelipase into a suitable mortar. Add about 3 mL of Buffer solution, and triturate for 5 to 10 minutes. Transfer the mixture with the aid of Buffer solution to a 100-mL volumetric flask, dilute with Buffer solution to volume, and mix. Dilute quantitatively with Buffer solution to obtain a dilution that corresponds in activity to the Standard test dilution.

Procedure— Label test tubes in duplicate S1, S2, and S3 for the standard series, and U for the sample. Pipet into tubes S1 2.0 mL, into S2 and U 1.5 mL, and into S3 1.0 mL of Buffer solution. Pipet into tubes S1 1.0 mL, into S2 1.5 mL, and into S3 2.0 mL of the Standard test dilution. Pipet into tubes U 1.5 mL of the Assay test dilution. Pipet into one tube each of S1, S2, S3, and U 5.0 mL of Trichloroacetic acid solution, and mix. Designate these tubes as S1B, S2B, S3B, and UB, respectively. Prepare a blank by mixing 3 mL of Buffer solution and 5 mL of Trichloroacetic acid solution in a separate test tube marked B. Place all the tubes in a 40 water bath, insert a glass stirring rod into each tube, and allow for temperature equilibration. At zero time, add to each tube, at timed intervals, 2.0 mL of the Casein substrate, preheated to the bath temperature, and mix. Sixty minutes, accurately timed, after the addition of the Casein substrate stop the reaction in tubes S1, S2, S3, and U by adding 5.0 mL of Trichloroacetic acid solution at the corresponding time intervals, stir, and remove all the tubes from the bath. Allow to stand at room temperature for 10 minutes for complete protein precipitation, and filter. The filtrates must be free from haze. Determine the absorbances of the filtrates, in 1-cm cells, at 280 nm, with a suitable spectrophotometer, using the filtrate from the blank (tube B) to set the instrument.

Calculation of potency— Correct the absorbance values for the filtrates from tubes S1, S2, and S3 by subtracting the absorbance values for the filtrates from tubes S1B, S2B, and S3B, respectively, and plot the corrected absorbance values against the corresponding volumes of the Standard test dilution used. From the curve, using the corrected absorbance value (U UB) for the Pancrelipase taken, and taking into consideration the dilution factors, calculate the protease activity, in USP Units, of the Pancrelipase taken by comparison with that of the Standard, using the protease activity stated on the label of USP Pancreatin Amylase and Protease RS.