how to increase the response of a method for an analyte

[parveenqa]

i want to increase thae response of my method (ion pair RPLC) for my analyte but i m having limited plasma volume(200uL) and extraction is through protein pptn as my drug is highly basic(pKa 12. and soluble in aqueous media.

[Uwe Neue]

Protein precipitation is a dilution exercise, and you need to evaporate to dryness and reconstitute.

Alternatively, a better approach (cleaner sample, concentration) is SPE. Methods are available on the Waters website under Oasis. Roughly, I would use the Oasis WCX weak cation exchanger, bind the analyte under neutral conditions, wash with an aqueous soluition, then wash again with methanol, then elute with methanol and acid. Procedures in the Oasis handbook give you more details.

[danko]

Hi Parveenqa,

How large is the diameter of the column you’re using?

Best Regards

[AdrianF]

What detection method are you using? If the substance is fluorescent you can get much greater sensitivity. Alternatively you may be able to derivatize to give a fluorescent product.