Chromatography Forum

I'm currently working on a SPE method for the determination of amoxicillin.

We used the Waters Oasis 2x4 strategy and found following method:
Oasis MCX cartridge,
condition/equilibrate with 1 ml MeOH, followed by 1ml water.
Then 1 ml sample load. Then there is a wash with 2% formic acid. And a elution step with 100% MeOH. After this, amoxicillin was eluted with 5% ammonia in MeOH.

Our samples were clean, no matrix effects at all, but: after 30 minutes, our amoxicillin was gone. This was due to the high pH of the ammonia solution. We then switched to 1% ammonia in MeOH, and before eluting the samples, there was enough 2% formic acid in the tubes to lower the pH from 11 to 4.5.

Unfortunately this was'nt enough and we still get recoveries of 66% instead of 100%.

We also tried HLB cartridges (reversed phase), but the amoxicillin elutes in the wash step.

Anyone who has some ideas?


Thanks

Bart

Can I ask which kind of sample you want to analyse?

If I understand you correctly, you lowered the concentration of the elution solution so that you end up with a low pH in the collection vials after mixing inside the collection vials with some of the residual low-pH solvent in the cartridge.

If this is correct, the lower pH regime during elution is the cause of the lack of complete elution. My suggestion is to go back to the elution with a high concentration of ammonia (maybe you do not need 5%, but you need more than the current 1%) combined with adding some acid into the collection vials before elution to keep the pH low.

I lowered the ammonia concentration, but the 66% recovery was due to the high pH, and I found 34% of degradation.

The pH of the solution was more than 2 pH units above the pKa of the amoxicillin, so this isn't the reason for the low recovery.

OK. Then the acid in the collection vial should solve the problem of the degradation.

OK. Then the acid in the collection vial should solve the problem of the degradation.

It doesn't, the short contact of amoxicillin with pH 11 is enough to breakdown the amoxicillin for about 34%. (Besides the fact that there was 2% formic acid added to tubes before eluting to lower the pH to 4.5)

Maybe I should switch to other SPE chemistries?

Bart

An alternative is to do HILIC SPE and as for sample work up.

I've attached the reference to a rather recent bioanalytic method for the determination of amoxicillin in plasma where the authors have used hydrophilic interaction solid-phase extraction followed by RPLC-UV for quantitation.

Development of a Solid Phase Extraction-Liquid Chromatographic Method for the Determination of Amoxicillin in Plasma
N. Lindegårdh, T. Singtoroj, A. Annerberg, N. Day
Therapeutic Drug Monitoring, 27 (2005) 503-508

You may try using Oasis WCX instead. It possesses weak cation exchange property which you can maneuver the charge of SPE sorbent by adjusting the pH of washing / eluting solvent. Generic method makes use of loading samples at neutral pH buffer, washing with same buffer and methanol to remove interferents, and eluting the analyte with acidic methanol. If amoxillicin is stable in low pH, I guess you may get a clean extract with good analyte recovery.

Hubert

A lot of betalactams undergo ready methanolysis. Try swapping acetonitrile for the MeOH in your method.