Bioequivalence and Bioavailability Forum

Hi Angus & Lyn,

repetitive extraction/back-extraction is indeed an excellent procedure to get very “clean” extracts. Unfortunately it requires the analyte to be stable in all pHs employed. Omeprazole has a half-life of a couple of seconds (!) in strong acidic pH. Remaining options:

• LLE at ~neutral pH with ethyl acetate. Dirty extracts, since EtAc after centrifugation contains ~5% H₂0. Quite often greasy residues which are difficult to dissolve in mobile phase.
  Et₂O would be another option allowing back-extraction to basic buffer.
  
• LLE in strongly basic conditions with unpolar solvents like CH₂Cl₂ and/or hexane. Does not remove basic contaminants – which are more prevalent than acidic ones by far…
  
• SPE: Not so nice as well. Only mild washing with low percentages of organic modifier possible.
  
• Protein precipation: Most commonly applied method for omeprazole. High dilution may help in limiting carrying over contaminants to the LC.

@Lyn: Do you use a pre-column? Did you consider online-SPE or (probably even better) column-switching (i.e., the first column diol / CN or even RP in HILC-style and heart-cutting only the analyte-fraction to the chiral column)? Both should protect the main column from most of the nasty stuff.