Sample precipitation during HPLC analysis.

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

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Hi all,

Currently i am verifying USP38 S2, LIMIT OF METHACRYLIC ACID AND ETHYL ACRYLATE for Type C copolymer by using HPLC system.

The sample preparation are as follows:

Sodium perchlorate solution: 35 mg/mL of sodium perchlorate. This solution has a concentration of 0.25 M.

Mobile phase: Add phosphoric acid dropwise to water to obtain a solution with a pH of 2.0. Prepare a mixture of this acidified water and methanol (80:20), and degas.

Sample solution: Transfer about 3 g of Methacrylic Acid and Ethyl Acrylate Copolymer to a 50-mL volumetric flask, dilute with methanol to volume, and mix. Add 10.0 33 mL of this solution dropwise, while continuously stirring, to a beaker that contains 5.0 mL of Sodium perchlorate solution. Remove the precipitated polymer to obtain a clear supernatant by centrifugation (e.g., NLT 5000 ×g for NLT 5 min). Use the clear supernatant.


However, after sample injection, the HPLC system shut down due to the system overpressure. It is suspected that the copolymer precipitated in the injection loop due to the mobile phase ratio (Methanol: acidic water, 20:80), which resulted in poor solubility of Type C copolymer.

It was observed that the solution is clear but dense and no precipitated polymer formed after mixing, which is different to that described in monograph.

As it is a monogrph method, again, nothing i can change but follow the procedures unless justify or validate the modified proceudres. In my opinion, most of the copolymer should be removed after centrifugation. But in fact, the copolymer dissolved in that solvent system (Methanol:Sodium perchlorate solution, 10:5), therefore, copolymer could not be removed even after centrifugation. I was wondering that if someone could provide me any information regarding sample solution preparation to avoid the copolymer precipitation? Thank you very much.
Here I just want to tell a good news. USP will make an errata for this analytical procedures. It was confirmed inapplicable for this excipient.
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