pH6.8Phosphate buffer— On the day of use,dissolve 13.6g of monobasic potassium phosphate in water to make 500mLof solution.Dissolve 14.2g of anhydrous dibasic sodium phosphate in water to make 500mLof solution.Mix 51mLof the monobasic potassium phosphate solution with 49mLof the dibasic sodium phosphate solution.If necessary,adjust by the dropwise addition of the appropriate solution to a pHof 6.8.

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

Standard preparation— Weigh accurately about 20mg of USP Pancreatin Amylase and Protease RSinto a suitable mortar.Add about 30mLof pH6.8Phosphate buffer,and triturate for 5to 10minutes.Transfer the mixture with the aid of pH6.8Phosphate bufferto a 50-mLvolumetric flask,dilute with pH6.8phosphate bufferto 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 4times the amylase activity of the USP Pancreatin Amylase and Protease RS,weigh accurately about 10mg 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 mLcorresponding approximately to that of the Standard preparation.]Add about 3mLof pH6.8Phosphate buffer,and triturate for 5to 10minutes.Transfer the mixture with the aid of pH6.8Phosphate bufferto a 100-mLvolumetric flask,dilute with pH6.8Phosphate bufferto volume,and mix.

Procedure— Prepare four stoppered,250-mLconical flasks,and mark them S,U,BS,and BU.Pipet into each flask 25mLof Substrate solution,10mLof pH6.8Phosphate buffer,and 1mLof sodium chloride solution (11.7in 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 BUand BSadd 2mLof 1Nhydrochloric acid,mix,and return the flasks to the water bath.To flasks Uand BUadd 1.0-mLportions of Assay preparation,and to flasks Sand BSadd 1.0mLof Standard preparation.Mix each,and return the flasks to the water bath.After 10minutes,accurately timed from the addition of the enzyme,add 2-mLportions of 1Nhydrochloric acid to flasks Sand U,and mix.To each flask,with continuous stirring,add 10.0mLof 0.1Niodine VS,and immediately add 45mLof 0.1Nsodium hydroxide.Place the flasks in the dark at a temperature between 15and 25for 15minutes.To each flask add 4mLof 2Nsulfuric acid,and titrate with 0.1Nsodium thiosulfate VSto the disappearance of the blue color.Calculate the amylase activity,in USP Units per mg,of the Pancrelipase taken by the formula: in which CSis the amylase activity of the Standard preparation,in USP Units per mL,WUis the amount,in mg,of Pancrelipase taken,and VU,VS,VBU,and VBSare the volumes,in mL,of 0.1Nsodium thiosulfate consumed in the titration of the solutions in flasks U,S,BU,and BS,respectively.

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

Olive oil substrate— Combine 165mLof Acacia solution,20mLof olive oil,and 15g 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 15minutes,intermittently cooling in an ice bath to prevent the temperature from exceeding 30.

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

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

Standard test dilution— Suspend about 200mg of USP Pancreatin Lipase RS,accurately weighed,in about 3mLof cold water in a mortar,triturate for 10minutes,and add cold water to a volume necessary to produce a concentration of 8to 16USP 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 5to 10mLof the cold suspension,and allow the temperature to rise to 20before pipeting the exact volume.

Assay test dilution— Suspend about 200mg of Pancrelipase,accurately weighed,in about 3mLof cold water in a mortar,triturate for 10minutes,and add cold water to a volume necessary to produce a concentration of 8to 16USP 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 5to 10mLof the cold suspension,and allow the temperature to rise to 20before pipeting the exact volume.

Procedure— Mix 10.0mLof Olive oil substrate,8.0mLof Buffer solution,2.0mLof Bile salts solution,and 9.0mLof water in a jacketed glass vessel of about 50-mLcapacity,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 0.1Nsodium hydroxide VS,from a microburet inserted through an opening in the cover,to adjust the pHto 9.20potentiometrically using a calomel-glass electrode system.Add 1.0mLof Assay test dilution,and then continue adding the 0.1Nsodium hydroxide VSfor 5minutes to maintain the pHat 9.0.Determine the volume of 0.1Nsodium hydroxide VSadded after each minute.

Calculation of potency— Plot the volume of 0.1Nsodium hydroxide VStitrated 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 1.25g of finely powdered casein in a 100-mLconical flask containing 5mLof water,shake to form a suspension,add 10mLof 0.1Nsodium hydroxide,shake for 1minute,add 50mLof water,and shake for about 1hour to dissolve the casein.If necessary,adjust to a pHof about 8,using 1Nsodium hydroxide or 1Nhydrochloric acid.Transfer the solution quantitatively to a 100-mLvolumetric flask,dilute with water to volume,and mix.Use this substrate on the day it is prepared.

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

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

Filter paper— Determine the suitability of the filter paper by filtering a 5-mLportion of Trichloroacetic acid solutionthrough the paper and measuring the absorbance of the filtrate at 280nm,using an unfiltered portion of the same Trichloroacetic acid solutionas 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 solutionuntil the absorbance of the filtrate,determined as above,is not more than 0.04.

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

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

Procedure— Label test tubes in duplicate S1,S2,and S3for the standard series,and Ufor the sample.Pipet into tubes S12.0mL,into S2and U1.5mL,and into S31.0mLof Buffer solution.Pipet into tubes S11.0mL,into S21.5mL,and into S32.0mLof the Standard test dilution.Pipet into tubes U1.5mLof the Assay test dilution.Pipet into one tube each of S1,S2,S3,and U5.0mLof Trichloroacetic acid solution,and mix.Designate these tubes as S1B,S2B,S3B,and UB,respectively.Prepare a blank by mixing 3mLof Buffer solutionand 5mLof Trichloroacetic acid solutionin a separate test tube marked B.Place all the tubes in a 40water bath,insert a glass stirring rod into each tube,and allow for temperature equilibration.At zero time,add to each tube,at timed intervals,2.0mLof the Casein substrate,preheated to the bath temperature,and mix.Sixty minutes,accurately timed,after the addition of the Casein substratestop the reaction in tubes S1,S2,S3,and Uby adding 5.0mLof Trichloroacetic acid solutionat the corresponding time intervals,stir,and remove all the tubes from the bath.Allow to stand at room temperature for 10minutes for complete protein precipitation,and filter.The filtrates must be free from haze.Determine the absorbances of the filtrates,in 1-cm cells,at 280nm,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 S3by 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 dilutionused.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.