Specificity - How to Improve it

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I currently have a related substances USP Monograph isocratic method with failing specificity when trying to validate. Thoughts are this could be an old method that might need some work.

What would be first port of call. Placebo peaks and Impurities of interest are all eluting together near the solvent front. Is trying a gradient elution the way forward?
Mouleyre wrote:
Is trying a gradient elution the way forward?

Yes, sure, if the gradient elution is possible (it is barely possible with ion-pair LC).
Possibly, but if this were my problem I would start by verifying that the retention of the API matches that expected from the monograph. If it doesn't, I would check my column and mobile phase before trying something like a gradient.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
I found it was not uncommon to find USP Monograph procedures that were antiquated and inadequate. Or downright confusing.
tom jupille wrote:
Possibly, but if this were my problem I would start by verifying that the retention of the API matches that expected from the monograph. If it doesn't, I would check my column and mobile phase before trying something like a gradient.


The problem here though is that the Monograph doesn't quote the retention time only the relative retention times. I'm interested to hear how you would usually verify the retention time.
Check if the monograph references a published article; if it does, check that. If you are using the exact same column then it's possible that your column is bad. If you are using an "equivalent" column, bear in mind that the USP "L" categories are, in fact, only a very rough guide to equivalence. The USP itself recognizes that and has made available a column equivalency database (actually, two databases) that allows a much closer match to the original column: https://apps.usp.org/app/USPNF/columns.html

All of that said, I can't see any harm in making a quick try with a wide-range gradient (as has been pointed out, if this is an ion-pair method, a gradient would be problematic) to see what's going on.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Mouleyre wrote:
The problem here though is that the Monograph doesn't quote the retention time only the relative retention times.

Please specify the monograph under discussion.
tom jupille wrote:
Check if the monograph references a published article; if it does, check that. If you are using the exact same column then it's possible that your column is bad. If you are using an "equivalent" column, bear in mind that the USP "L" categories are, in fact, only a very rough guide to equivalence. The USP itself recognizes that and has made available a column equivalency database (actually, two databases) that allows a much closer match to the original column: https://apps.usp.org/app/USPNF/columns.html

All of that said, I can't see any harm in making a quick try with a wide-range gradient (as has been pointed out, if this is an ion-pair method, a gradient would be problematic) to see what's going on.


It looks like it is ion-pair based on Mobile phase composition below:

Solution A:6.5 g/L of sodium 1-octanesulfonate in water. To each L add 2.9 mL of phosphoric acid, and adjust with 5 N sodium hydroxide solution to a pH of 3.0.

Mobile phase:Acetonitrile and Solution A (43:57)


Would that be correct that is ion-pair?
Mouleyre wrote:
Solution A:6.5 g/L of sodium 1-octanesulfonate in water. To each L add 2.9 mL of phosphoric acid, and adjust with 5 N sodium hydroxide solution to a pH of 3.0.

Mobile phase:Acetonitrile and Solution A (43:57)


Would that be correct that is ion-pair?

Yes, this is an ion-pair HPLC method. Also it is now clear to me that the USP monograph in question is Fluoxetine Tablets.

Did you try to use the column (Zorbax SB C8) listed at https://www.uspchromcolumns.com ?

What retention times and relative retention times of fluoxetine and its identified impurities (aminomethyl-1-phenylpropanol and fluoxetine related compound B) do you obtain? Are these impurities separated with Rs not less than 4.5 as required in the monograph? What retention times range corresponds to your phrase "Placebo peaks and Impurities of interest are all eluting together near the solvent front"?
vmu wrote:
Mouleyre wrote:
Solution A:6.5 g/L of sodium 1-octanesulfonate in water. To each L add 2.9 mL of phosphoric acid, and adjust with 5 N sodium hydroxide solution to a pH of 3.0.

Mobile phase:Acetonitrile and Solution A (43:57)


Would that be correct that is ion-pair?

Yes, this is an ion-pair HPLC method. Also it is now clear to me that the USP monograph in question is Fluoxetine Tablets.

Did you try to use the column (Zorbax SB C8) listed at https://www.uspchromcolumns.com ?

What retention times and relative retention times of fluoxetine and its identified impurities (aminomethyl-1-phenylpropanol and fluoxetine related compound B) do you obtain? Are these impurities separated with Rs not less than 4.5 as required in the monograph? What retention times range corresponds to your phrase "Placebo peaks and Impurities of interest are all eluting together near the solvent front"?


There is a new method coming up to replace this method as stated in PF 46(5) so will try feasibility studies on that.
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