Pralidoxime Chloride USP

[sdeboer]

Hi. I've been having trouble achieving consistent plate count of >4000 when using the USP Assay for Pralidoxime Chloride (2-PAM). I am using the Spherisorb ODS1 column specified by USP, and I've tried both the 4x250mm and 4.6x250mm. Anyone have success with this method? Thanks.

[tom jupille]

Can you get reasonable plate counts on that instrument with other methods?

• If not, you may be looking at extra-column volume (too much tubing?, a poorly assembled fitting?, too big an injection? ...? )
• If low plate count is unique to this method, are the columns new or have they been used for a long while?
• Does the peak shape look OK? Any excessive tailing, shoulder, . . .?

[sdeboer]

Hi. Yes, I have run other methods successfully. There is another peak that elutes before the Pralidoxime Chloride with plate count about 10,000. The Pralidoxime Chloride is fronting a lot - tailing is about 0.6. We think the compound is sticking to the silica, so I increased the TEAC modifier, but I can only increase it so much with a USP method. I have tried an older and a new column, and I've had the most success after flushing the column with mobile phase overnight.

[tom jupille]

Okay, you have covered the most likely possibilities. If the peak is fronting badly, increasing the temperature can help (the USP allows 10 degrees as an adjustment). Another possibility is to try an "equivalent" column from another source. The USP has two databases comparing column selectivity at:
http://apps.usp.org/app/USPNF/columns.html
The second one (the "PQRI") database is much larger than the first one.

[sdeboer]

I am having issues with base degradation in a purity method development. I have been adding 1N NaOH to my drug product and the base seems to split the peak, potentially into an isomer. I have successfully degraded the drug product with acid, heat, light and peroxide, and have peak purity with the method for those samples. Do I have to start over with the method since I am unable to separate the base deg? Thanks!

[sdeboer]

Thanks for the help. We were able to achieve the plate count by decreasing the injection volume.

I now have another issue with this. I modified this method for a purity method (this one was not sensitive enough), and the base degraded sample is not separated. The NaOH seems to significantly degrade the 2-PAM, it seems to an isomer, from papers I've read. It appears almost like a split peak, a large shoulder on the main peak. I have peak purity and good separation by all other degradation avenues. Any ideas?

[tom jupille]

Do I have to start over with the method since I am unable to separate the base deg?

I'm sorry to say this, but unless you can explain what's going on based on the compound's structure -- and can justify that it doesn't matter, the answer is "yes", you need a new method.

Since you're getting at least a partial of separation with the existing method, you may be able to get by with more plates (maybe smaller particle size), but that would mean cutting your injection volume down even further to avoid the initial problem you had.

[sdeboer]

Thank you, Tom. I appreciate the advice!