Method for evaluating degradation pathways for pancreatin active pharmaceutical agents

1. A method of determining degradation of one or more proteins in a pancreatin API sample comprising the steps of:

a) providing independently a first and second pancreatin API sample in solution, wherein the second pancreatin API sample is a pancreatin API control sample;

b) providing independently a first and second reference sample in solution, wherein the first ad second reference samples comprise a reference enzyme in the absence of other enzymes, and wherein the second reference sample is a reference control sample;

c) subjecting the first pancreatin API sample and the first reference sample to a stress condition;

d) performing reverse-phase high performance liquid chromatography (RP-HPLC) on the subjected pancreatin API sample, the pancreatin API control sample, the subjected reference sample, and the reference control sample individually to afford first, second, third, and fourth results, respectively;

e) comparing the first results with the second results to determine a pancreatin API degradation of the one or more proteins in the subjected pancreatin API sample, wherein the pancreatic API degradation is expressed as % loss degradation;

f) comparing the third results with the fourth results to determine a reference sample degradation of the reference enzyme in the subjected reference sample, wherein the reference sample degradation is expressed as % loss degradation; and

g) comparing the pancreatin API degradation to the reference sample degradation to indicate the capability of the method to reveal the changes in the pancreatin API due to the stress condition,

wherein the stress condition is selected from the group consisting of acidic, basic, oxidative ad thermal, each stress condition making the subjected sample susceptible to degradation of the one or more proteins therein.

2. The method according to claim 1 further comprising conducting an enzymatic activity test for an enzyme on the first and second pancreatin API samples and the first and second reference samples to determine a difference in enzymatic activity, if any, for the enzyme, wherein the enzyme is selected from the group consisting of trypsin, elastase, lipase, protease and amylase.

3. The method according to claim 1 wherein the RP-HPLC has a stationary phase which is a C 4 derivatized column.

4. The method according to claim 3 wherein the RP-HPLC has a mobile phase which is a linear-segmented gradient of acetonitrile containing 0.1% trifluoroacetic acid (TFA).

5. The method according to claim 3 wherein the RP-HPLC has a mobile phase which is a 0.1% trifluoroacetic acid solution in water.

6. The method according to claim 3 wherein the oxidative stress condition comprises treating the first pancreatin API sample with hydrogen peroxide.

7. The method according to claim 3 wherein the acidic stress condition comprises treating the first pancreatin API sample and the first reference control sample with an aqueous solution of hydrochloric acid.

8. The method according to claim 3 wherein the basic stress condition comprises treating the first pancreatin API sample and the first reference control sample with an aqueous solution of sodium hydroxide.

9. The method according to claim 1 , wherein first reference enzyme is a protease.

10. The method of claim 9 , wherein the protease is selected from the group consisting of trypsin, chymotrypsin, elastase, lipase, carboxypeptidase-A (CPA), and carboxypeptidase-B (CPB).

11. The method according to claim 3 , wherein the RP-HPLC has a detection wavelength of 280 nm.