I recently encounted a problem regarding liquid liquid extraction (LLE). The compounds that I am dealing with are very polar and acidic. They can only be retained in mobile phase which contains 1% of ACN. When I was using LLE to deal with the plasma samples, very low recovery was observed. Different solvents, such as Ethyl acetate, MTBE have been tested, none of them gave a satisfactory result. Any comments, suggestions or even literatures? Thanks.

What pH did you try in your aqueous phase? You want your compounds to be non-ionic to go into the organic phase. So you may need to make the water very acidic.

I did a series of experiments varying the pH by adding formic acid. The recovery increases as the amount of formic acid increases. However, I noticed, at lower pH, the parent compound, which is less polar, started tailing. In the mean time, it seems that many other unwanted staff has also been extracted (noticable increasing amount of residue after evaporation). So I am hesitating to go to very low pH. Is lowering the pH my only option? Do I need more polar extraction solvent? Thanks again.

I'm not sure how adjusting the pH of your aqueous before LLE extraction could affect your chromatography (you mentioned tailing as an effect of lowering your aqueous pH). Salt, acid, and base aren't going to extract into organic (MTBE, Ethyl Acetate, etc.) so your starting aqueous shouldn't affect your chromatography.

If you want to extract an acidic compound by LLE you will have to lower the pH. If you keep increasing the polarity of the extraction solvent you're just going to end up with an extraction solvent that is miscible with water. To extract two compounds with very different pKas you can extract the same sample twice. Perform the LLE at pH ~7 for the neutral compound, separate the organic and aqueous layers, keeping both. At this point the organic layer will contain your analyte with the higher pKa and the aqueous layer will contain the low pKa analyte. Then acidify the aqueous layer and perform the LLE again. You can then combine the two organic portions and you should have both analytes.

This is a little cumbersome though. SPE might fit your needs better. Waters provides some great SPE methods for situations such as yours. Example extraction using Waters Oasis MAX Cartridges:

Load sample
Wash with pH 7 NaOAc
Elute neutral compounds with Methanol
Elute acidic compounds with 2% Formic Acid in Methanol

I highly recommend the Waters Oasis Application Notebook. I reference mine all the time. You can request a free copy at the following site:
www.waters.com

Here is a possibility: formic acid will extract into the organic phase with your analyte. This can cause the tailing, if this overloads the buffer of your mobile phase. If you would use another acid, for example phosphoric acid, it would increase the extraction even better than the formic acid did, but it will not go into the organic phase, and therefore the peak tailing will not occur.
Of course, I also agree with the last post: SPE is often less cumbersome than LLE.

Hi Angela,
In your suggested SPE procedure, any justification of using pH 7 NaOAc as washing solution? What is the concentration? Is it OK if I just use DI water? Thanks.

I still like the LLE approach. If formic acid is extracted, it can cause carry over of other stuff. If you do not want to use an inorganic acid as Uwe has suggested, you can use citric acid to control the pH and go to a lower pH without additional interferences.

The best solvents for extracting a very polar acidic compound are ethyl acetate, ethyl ether, or chloroform. MTBE is particularly worthless.

By all indications your compound is quite acidic. You might estimate a pKa by titration. If it is quite acidic, low pKa, use phosphoric or stronger mineral acid. I don't see the preference for organic acids for acidifying extractions. Multiple extractins may be necessary for quantitative extraction.

I am trying to find a method to detect acyclovir and ganciclovir in plasma samples for bioequivalence studies.
I finnaly found a mobile phase (phosphate buffer 10mM pH 5: acetonitrile:methanol (95:4:1) and chromatographic column (LiChrspher select B 250mm, 5um) to separate them, using DAD detector.

The problem now is the extraction procedure.
According to the literature, those compounds are so polar that is very difficult to use organic solvent. Most of the papers use deproteinization with perchloric acid and direct injection in HPLC system.
As I do not have perchloric acid, I tested 20% thrichloracetic acid (TCA), acetonitrile, methanol (MeOH).I also tested a salting out procedure adding NaCl in the sample and then I extracted it with diethyleter (DEE) , dichloromethane and hexane:DEE. No results at all.

Do you have any suggestion ????

I developed two methods for acyclovir and ganciclovir. Both have strong fluorescence at low pHs. (LOQ 10 and 30 ng/mL respectively). Try Oasis for SPE. It worked with me. Sorry, no further info. I work for a CRO!

What was the method on Oasis?

what is the spe method for ganciclovir