Problems with USP 467

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Dr. Analytical
Dr. Analytical's picture
Problems with USP 467

Originally posted by ziggy and moved to here.
Hi, I'm currently validating USP <467>, water soluble articles. The main problem with this method is standard solution preparation for class 2A. I cannot get homogenous standard solution regarding non polar compounds (cyclohexane and methylcyclohexane). The moment I add USP Class 2A standard stock solution to water I get emulsion, which is not stable. Shaking worsens the situation as dropplets of the solvents mentioned earlier, merge into larger ones. Typical RSD for six injections of standard solution are about 3% for water soluble solvent and up to 50% for cyclohexane and methylcyclohexane. I also tried mixing solution on magnetic stirer in ultrasonic bath and achieved moderate improvement...
Does anyone managed to get good result with this procedure?

Dr. Analytical
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I moved your topic to a separate post so that it will be visible to more people.
You are experiencing the same problems that we found with this method: the water soluble compounds like methanol and acetonitrile work well, but the non-water soluble compounds (cyclohexane, methylcyclohexane) produce poor results. I think the main problem is that the cyclohexane concentration is too high, and it is not soluble in water.  In some solution, you can see droplets on the side of the flask.  If you let the solution stand, sometimes a separate layer of solvent forms at the top.  To summarize, the soluion, as described in the method, is not stable.
Here are some things you can try:

  • Substitute 10% DMSO in water for all of your solutions.  The stock solutions are prepared in DMSO, so this will help solubility.  We only see the cloudy Class 2A solution sometimes, not every time.
  • The solution should be agitated immediately before removing the 1 mL for the standard.  We found that shaking did not help as much as simple inversion: invert twice, remove cap, remove 1 mL.  If the solution must stand for more than a minute, you must repeat this step before you remove any more solution.

Try these experiments and write back with your results.

ziggy's picture
I agree with your proposal

I agree with your proposal (adding 10% DMSO to all solutions) but my customer doesn't allow me to change anything in the procedure :(

Dr. Analytical
Dr. Analytical's picture
It will be difficult to

It will be difficult to succeed with this method if you can not make modifications.  I believe USP does allow modifications if they are documented and validated.
Another idea would be to rapidly stir the solution (stirring bar/stir plate) and quickly remove a 1 mL solution.
Also, the caps on the headspace vials must be very tight.  Check that they are sealed completely.

GG2369's picture
Greetings, I hope you can

Greetings, I hope you can help me with a problem.
I've been running USP467 for almost a year now with good success overall.  The only problem, and it drives me absolutely nuts, is passing a low RSD requirement on hexane in the 2b usp standard.   I get varying responses in area from vial to vial, even though I was carefull in all the preparations.
I am using headspace analysis.  Prep of sample and standard is done as written in water insoluble articles (DMSO)  and instrument parameters and column are from Procedure A.      
Do you have any suggestions for me on achieving consistent areas from vial to vial?  

Dr. Analytical
Dr. Analytical's picture
We have found similar

We have found similar problems with the 2A standard using the water soluble methods.  Do you see acceptable results for the water soluble components in the same mix?
I do not have the method with me right now, but can you tell us how the standards are prepared?  If they are prepared in water, than you may have a similar problem to what we see.
Hexane is "insoluble" in water.  It has some solubility, but will form a separate phase above its solubility limit.  As you get near the solubility limit, I believe the hexane tends to "float" toward the top of the solution (it is less dense than water).  Although you may not have a separate phase, I think the solution near the surface is enriched with hexane and other water insolubles.  So, if this is in a volumetric flask, when you remove a 1 mL sample, you are sampling an enriched solution, and you get a higher number.  You may observe that the second and third (and fourth) draws from the same flask show decreasing hexane, because you removed most of it with the first draw.
This is a simplified picture of what I think is happening, but there are many variables.  How far into the solution do you put the tip of the pipet to withdraw the sample?  How long does the solution stand before/between sampling?  What kind of agitation do you do, and how often?
I recommend trying to invert the flask a few times before each draw.  Another option would be to use a stir bar/stir plate to stir the solution during the draws.  Of course, this may increase loss of volatile analytes, and there is always a chance of extraction from solution onto the Teflon surface of the stir bar.
Let me know if these ideas make any sense?

Climberszen's picture
The company that I work at

The company that I work at just completed and released <467> Standards traceable to the USP standards.  During the qualification process we learned the smallest issues can become huge issues.  One of the key issues is that some of the analytes are not very soluble in the DMSO.

Serbia's picture
How to prepare Solvent

How to prepare Solvent solution in USP 467 (residual solvents)? How much solvents should I weit?
I do not understand why this solution should have a concentration of the solvents 1/20 of the limits stated in Table 2. ?
You can take methanol for example.