How to resolve interference from Theophylline peak

[ghie malig]

Hi Everyone!

My colleague is modifying a method for determining Theophylline in human plasma. The problem is, she cannot remove or resolve the interference from Theophylline. She's getting a resolution of 1. She uses direct precipitation of the plasma with 20 % Perchloric acid and is back extracted using DCM/Hexane (1:1) to further remove the impurities. The column is Chromolith RP-18 150 x 4.6 and the MP is composed of 0.02 M Acetate buffer/MeOH (80:20 ) pH 5.5 with the flow rate of 1.0 mL/min. She already played with the pH of the mobile phase and its ratio but it seems that she cannot resolve the problem. Any suggestions? Your feedback is very much appreciated. Thank you so much!

Best regards,
Ghie

[amaryl]

Hi Everyone!

My colleague is modifying a method for determining Theophylline in human plasma. The problem is, she cannot remove or resolve the interference from Theophylline. She's getting a resolution of 1. She uses direct precipitation of the plasma with 20 % Perchloric acid and is back extracted using DCM/Hexane (1:1) to further remove the impurities. The column is Chromolith RP-18 150 x 4.6 and the MP is composed of 0.02 M Acetate buffer/MeOH (80:20 ) pH 5.5 with the flow rate of 1.0 mL/min. She already played with the pH of the mobile phase and its ratio but it seems that she cannot resolve the problem. Any suggestions? Your feedback is very much appreciated. Thank you so much!

Best regards,
Ghie

Hello Ghie,

Happy New year to you

Having worked with pH and ratio of mobile phase. Try playing out with flow rate provided the peak shape doesn't deteriorates. Say make it 0.98 ml/min...though I have never worked with this. Just a suggestion. I think a resolution of 1.5 will be obtained.

But I am wondering why didn't a change in ratio of mobile phase didn't work?

Regards,

Amaryl.

[juddc]

First, precisely what was done with the pH? Up? Down? What with?

Second, you might try switching from MeOH to acetonitrile or other solvent as your organic because that will alter your selectivity. Also, if your column has active silanols (I have no experience with the column you're using), triethylamine might help if your counter ion is not ammonium or if you switch to a phosphate or other buffer system, but be careful not to raise the pH of the MP beyond what your column can tolerate.

If nothing else, a longer column or a smaller particle size would improve things.

Another option might be to see what effect an ion-pairing agent might have on selectivity. Be VERY careful if you choose to try this route, however, as things can get messy quickly. I was able to do this to good effect once on a reversed phase assay for ascorbic acid in biological media. I didn't have much flexibility with the mobile phase from the standpoint of organic solvent selection because the MP was 100% aqueous, yet I had an issue with a co-eluter arising from the media. The IP agent (alkyl sulfonate) didn't affect the ascorbic acid, but DID move the co-eluter away quite nicely and the assay worked well. If you choose to try this, only do it with an isocratic method, dedicate your column, leave lots of time for equilibration, and be sure to rinse the IP out of your system after you're done with the analysis. As I said, this can work, but should be tried as a last resort.

You may be able to try something on the sample prep side, too. SPE may be a good route as theophylline can be ionized at high pH and there are SPE supports that can work at very high pH's (Waters' Oasis line comes to mind), so you have flexibility there. I might try SPE at a variety of pH's on this sample to see what happens. On a RP-SPE, theophylline would likely be retained at a pH below 8.00 and eluted at 10 or so (pKa = 8.77). This would require validation, however and could actually end up being a fair amount of work. I'd be inclined try to improve your separation first.

Hope this helps or at least gives some fodder for comment!

[SIELC_Tech]

Ghie,

Check the following method for Theophylline. It retained around 10 minutes on Primesep column. At this conditions plasma components will be positively charged and repel from Primesep stationary phase. Most of proteins will come in void:
http://www.sielc.com/compound_090.html

Your selectivity will change with the buffer nature; you can use ammonium formate or acetate for LC/MS detection

It is going to be something similar to our "Direct Plasma" analysis:
http://www.sielc.com/Technology_DirectP ... lysis.html

Contact me if you have questions.

regards,

Vlad

[juddc]

Nice looking application...watch the spelling of "Xanthine" though.

[SIELC_Tech]

Thanks Chris,

Getting rusty with my "old english"....LOL

Vlad

[Uwe Neue]

I am not sure that the problem is as simple as presented by SIELC tec. If the interference does not respond to pH changes, it may not respond to ion-exchange either. On the other hand, you did not say by how much the pH was varied, and the suggestion by juddc to change the pH DRASTICALLY might work just fine.

[ghie malig]

Hello!

Thanks for all your posts. They were indeed helpful. My colleague increased the pH from 5.5 to 7 and decrased it to 3. It is within the pH limit of the column. The limit of chromolith RP 18 column is from pH 2- 7.5. The interference and Theophylline were not affected with these changes in pH. She is hoping that at least the interference will move away from Theophylline. She also used ion-pair reagent Tetrabutyl ammonium sulfate and it did not retain her analytes. She used Sulfamethoxazole as internal standard and it was well resolved from Theophylline. Her only problem was the interference. She used Perchloric acid as precipitant. She also tried precipitation with Acetonitrile and back extract with DCM/Hexane. And it seems that she did not acquire a clean blank. The best she tried was perchloric to clean up the blank plasma except for that small interference which she cannot resolve from Theophylline peak. She wanted to attain a resolution of 2. We are looking at the possibility of using SPE unless we have other ways of resolving that really small interference peak. We haven't tried SPE cartridges in our lab because it is costly. We usually analyze more than 1000 samples per study. SPE cartridges in our country is expensive. But if don't have any other resort, will use SPE. Thanks! Hope to hear from all of you again. Have a good day!

-ghie malig-

[juddc]

Thanks for the description.

Tetrabutylammonium sulfate will work for negatively charged species, but it won't have an effect on positively charged ones...I might still be inclined to try an alkyl sulfonate at low MP pH to see what happens there as it will do just the opposite. It might also slightly alter the retention of the theophylline.

Alternately, you might want to screen some other columns to see if there is another RP column that could give the selectivity you need. Be careful of some of the reversed phase - amide columns, though, as they are often not quite as durable as other RP supports.

You might also want to try looking at an amino column. These tend to be quite robust - especially ones on polymer supports such as the Asahipak NH2 P50 and can give interesting selectivity, though you would need to redevelop your method from scratch and I'm not sure how well theophylline would be retained. It might be fun to try a run at pH 10 on a polymer based amino column to see what you get. It'll certainly be different from your RP assay...

Please keep us tuned in!

[SIELC_Tech]

Ghie,

In our application you can see how selectivity changes with the change of the acid. Any other buffer will give you different selectivity which might be enough to achieve desired separation.
Primesep B or Primesep D columns are basic in nature and have alkyl chain, these two functionalities help resolving critical pairs.
Do you know if interference comes from plasma component or from impurity/metabolites?
What is you retention time for specific mobile phases? Can you send me chromatograms to see?

Regards,

Vlad

[HW Mueller]

I have never done Theophylline so this is general: It often happens with such biological systems that if you managed to modify your technique to separate one interference, another takes its place, especially if you work near the detection limit. Thus it often helps to do a multistep (generally called multidimensional) chromatography. Including a SPE step is about the simplest of this type. If you (they..) can not afford to throw away these cartridges check whether they can be washed and reused, or better: Get empty precolumns (from Upchurch, as an example) dryfill them yourself, use them inline (via Rheodyne switchers, etc....), thus washing them continously under pressure.....

[Rafael Chust]

My first injection into an HPLC over 20 years ago was exactly teophylline from human plasma.

It was a very old Waters system with fixed wavelenght detector (254 nm) and µBondapak C18 column.

As far as I remember, we only precipitate the proteins with TFA, filtrate and inject in isocratic MeOH/H20 - no interferences was found. We used some IS and no interferences was found. Detection limits were great, linearity also...

In fact, I can not see were the problem might reside...

[ghie malig]

Thanks so much. Got so much help from you already. My colleague is now trying the effect of addition of acid in MP in the separation of xanthines as suggested by vlad. thanks again.

-ghie-

[SIELC_Tech]

Ghie,

Changing acid will not have the same effect on reverse phase column as on Primesep columns. Primesep has reverse phase and ion-exchange properties. Carbon chain has embedded polar ionizable group and that helps you to use it for your advantage.

What is concentration of xanthine in plasma? We have plasma in the lab and can try to mimic your separation.

Contact us if you want to use this opportunity.

Regards,

Vlad

[HW Mueller]

Rafael, do you still know the concentrations of Theophylline in your study? It may help ghie malig´s colleague to know whether she is doing something wrong, or whether there might be some special problems involved. Thus it would also be instructive to find out what ghie malig´s colleague´s concentations are.