Back to the Top
I am trying to develop a new method of analyzing a drug found in plasma for eventual PK studies. However, with subsequent injections, the reverse phase column degenerates. i.e. retention time of standard changes and psi increases. Sample prep involves acetonitrile ppt followed by an SPE extraction phase. The final prep is resuspended into mobile phase (IPA-H2O-acetonitrile) and injected. The compound is somewhat acid sensitive. After 10 samples, I cannot regenerate the column with acid washes, Acetonitrile, dichloromethane, IPA, etc. Any suggestions on either cleaning the column between sample runs, or cleaning the sample better first?
Back to the Top
Sample processing is not a problem it seems.Same procedure of sample processing is frequently being used in many industries without any issue of column degeneration.
You are using two processing techniques, ACN precipitation and SPE,which will give you clean sample.
Add some steps:
Filter your mobile phase through 0.22u filter prior to analysis.
In precipitation technique increase the dilution, Increase centrifugal speed (rpm,12000) and run for longer time to reduce your junk load.
Carefully withdraw supernatant keeping ice pack below the sample (Lower temperature coagulates protein and increases clarity of supernatant)
Use gradient method to remove plasma phospholipids (long runner)
Use guard column if required.
Hope this will help
Want to post a follow-up message on this topic?
If this link does not work with your browser send a follow-up message to PharmPK@boomer.org with "Drug analysis in plasma - hplc cleaning" as the subject
Support PharmPK by using the
Copyright 1995-2011 David W. A. Bourne (email@example.com)