Chromatography Forum

Hi to all,

Can anybody please help me for the extraction procedure for a drug containing amine and carboxilic groups as side chains. Extraction from Human plasma.

i have tried with oasis MAX anion exchange cartridges(SPE) eluting with 2% formic acid in methanol. I am getting the drug but i want different method either it is liquid/liquid or SPE with C18 or HLB cartridges.

Instead of MAX, have you tested OASIS-WAX? You don't explain if your problem is low recovery, but if it is, perhaps using weak anionic interaction you can elute your product easily with basic MeOH

You did not explain, why you want to switch the method - assuming that it works...

On a purely hydrophobic surface such as Oasis HLB, most zwitterions are less retained, when they are in the doubly charged state compared to the singly charged state. You could either load and wash at alkaline or at acidic pH, and elute at neutral pH. However, this requires a bit of homework to figure out, where your compound is retained and where it elutes. You can find procedures for this in the Oasis brochure on the Waters website.

If you are looking, you will also find a procedure, where a zwitterion has been cleaned up by retaining it on an MCX cartridge and then on an MAX cartridge (or vice versa), resulting in really superior sample cleanup.

thanks crlar and uwe,

Basically my problem is i am not getting the optimum recovery. Oasis wax cartridges are not available with us.

i have tried with basic methanol i.e., 2% ammonia in methanol with HLB as well as with MCX by changing the cleanup step with acidic and basic buffers, and methnol. i did several trials but my recovery of the drug is only 5-10% and i am getting interference with the blank plasma, eventhough i changed the plasma.

I think cleanup is not good enough. For zwitter ions can i go for liquid/liquid?

We analysed a compound with a prim. amine and a carboxillic group by mixing the plasma with 0.02 M KH2PO4 (1:3) and applied this to a 100 mg C18 SPE cartridge. After a wash with water, the analyte was eluted wit a mixture of organic solvent and water. A wash with a mixture of organic and water was not possible.....

regards Bert

Bert,

can you tell me the organic solvent? is it possible to mix organic solvent with water? either it is Methanol or ACN

For the elution of our compound, we used 15% iso-propanol in water. A mixture of water and acetonitrile or methanol could also be good for your analyte, but you have to do some homework, as Uwe said.

regards,

Bert

I did lot of home work regarding this product, with different cartridges, different buffers and different elution mixtures, but only thing is i am getting 10-15% recovery.

does it work with liquid/liquid extraction? can any body help me.

regards

rams

I do not understand what you have tried. I do not understand, why the Oasis MAX is not available to you. My first bet for a first-class sample cleanup for a zwitterion would be a combination of cation and anion exchange.

However, since you want to work with the HLB cartridges, I would like to give you a protocol that will tell you exactly what is going on and will permit you to select the best conditions. I know that you have done some homework, but it is not clear to me what you have done.

Here is the thought process: most zwitterionic compounds have a fair difference between the solvent elution strength needed to extract the positively, negatively charged or zwitterionic form of the molecule. Usually, the zwitterion is less retained. Whethter the positively charged form is more retained than the negatively charged form is not predictable.

Prepare three standard solutions of your analyte, one at low pH e.g. in formic acid, one at high pH, e.g in ammonia, and one at neutral pH, i.e. in phosphate buffer.

Load 10 cartridges each with these three sample preparations. Collect the effluent from the loading and analyze at least one effluent at each pH to see if there is any breakthrough of your analyte. If you hane no breakthrough at least under acidic or basic conditions, you have found a way to load the analyte on the HLB cartridge.

Now extract the 10 cartridges loaded under acidic conditions with the formic acid solution to which 10%, 20%, 30%, etc. methanol is added.

Extract the 10 cartridges loaded under neutral conditions with the neutral phosphate buffer solution to which 10%, 20%, 30%, etc. methanol is added.

Extract the 10 cartridges loaded under basic conditions with the basic ammonia solution to which 10%, 20%, 30%, etc. methanol is added.

Analyze these samples!

From this, you will now get the complete retention profile of your analyte as a function of pH and methanol concentration.

Assume that the analyte starts to wash out at 40% methanol under acidic conditions, 50% methanol under basic conditions, and 20% methanol under neutral conditions. Your best extraction protocol would be to load your analyte under basic conditions (with ammonia added), then wash with 45% methanol in ammonia, and then reextract with 50% (or slightly higher) methanol at neutral pH (phosphate). This will give you the cleanest sample solution.

If you want to avoid phosphate, you could also (for this example) wash with up to 45% methanol with ammonia, followed by a reextraction with maybe 60% methanol or higher at low pH. Since you need to vary the organic concentration more, you will get more interferences from this simpler protocol.

The most important part about this is that you need to create the complete information about the elution of your compound in order to optimize the extraction protocol. Unfortuantely, this is work. If you could get the MAX cartridges, you could potentially save yourself some of this work by adopting a MCX-MAX combined extraction protocol.

I like Uwe's method, it seems like a comprehensive way to address this situation.

I'd like to suggest an alternative, derivatization. Having done a lot of work with amino acids (zwitterions) there are a variety of methods to choose from. You can use something like FMOC-Cl, which will react with amine groups, and label it with a big, hydrophobic, fluorene group. The amine group becomes a carbamate (neutral) and the compound is now more hydrophobic and only anionic. You can now use anion exchange and/or C18 SPE with a much simpler protocol. Also, if available, desired, you can use fluorescence detection (Ex=260nm, Em=310nm)

To be fair, there are drawbacks to derivatization. First, you must look at reaction efficiency. You must perform the reaction at a pH where the amine is deprotonated, and if the pKa of the amine is high you could be at pH 10. At this high pH, there is a significant amount of OH- around, and this will compete to react with FMOC-Cl (usually this is not a problem for amino acids, can use pH 7.8 ). Also, if you are extracting from plasma, there will certainly be other things in solution that will be derivatized. Second thing to consider is time. The reaction itself seems to take up to an hour, which may or may not be acceptable. It's fine for me, add reagent to solution, go to lunch, come back, and extract.... Third is the potential for MS detection. Usually a derivatized amine will ionize worse than a free amine in ESI (on the other hand, the fluorescence detection is very sensitive, and selective for fluorescent molecules). One last thing, there will always be an extra peak from the product of the reaction of FMOC-Cl and water (FMOC-OH). If the derivatized compound is hydrophobic, it will likely be well separated from this.

A generic procedure. Prepare a reagent solution of FMOC-Cl in an aprotic solvent (acetonitrile, acetone). Prepare an aqueous buffer solution (borate, phosphate, no amine based buffers!) at a pH where the amine of your analyte is deprotonated. Take 500 uL of buffer, 500 uL of acetonitrile, and x uL of plasma (or whatever solution you will derivitize) (I add acetonitrile to the mix because FMOC-Cl is not very soluble in water and when you add the reagent to a solution that is 99% water, it will precipitate and react mainly with water). Mix this solution, and add x uL of the FMOC-Cl solution in acetonitrile (the reaction is 1:1 FMOC to amine group, work out the ratio so there is an excess of reagent).

Other reagents are DANSYL-Cl, and AQC (which it good because it reacts with amines much more quickly than water, SN2 vs. SN1 reaction or something like that)

Thanks uwe and Mitchell for your advises,

I againe tried with MAX cartridges, this time i got 60% recovery and calibration curve is also good. Once again thanks for your advises.

cheers
rams