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I am developing LC-MS method for a novel compound and we are running through all kinds of problems that seem to give us almost zero recovery
I-Recovery from ACN-extracted plasma spiked post-extraction:
..Recovery is time dependent and it goes down from 90% within 15 min to <1% over 24 hrs
..It is not a matrix effect, the flow is split between MS and PDA, UV data shows the disappearance of the peak, just like MS data, and there are no new peaks showing up in UV
..Adduct/Mass shift in matrix: We did LC-neutral loss, precursor ion, and enhanced Q1 to look for adducts or degradants and couldn't find any
..Degradation by matrix (unlikely enzymatic because this is all post-extraction after ACN crash): pretreated samples with boiling or trypsin, slight improvement in recover to about 20%??!! With trypsin
..LLE and salting out (methods that results into phase separation) resulted in a 100% post-extraction recovery
..All protein precipitation, LLE, and SPE methods resulted in 0% recovery
..All direct-injection methods (no evaporation and reconstitution) by injecting the supernatant of ACN precipitation resulted in zero recovery
..The mentioned LLE and salting out methods resulted in 0% pre-extraction recovery because the compound is very polar.
We are suspecting a mass shift by adduct formation and/or enzymatic/chemical degradation, which survives ACN crash, but we are not able to prove anything so far.
Any thoughts please?!
-- Nagsen Gautam
Dept. of Pharmaceutical Sciences
University of Nebraska Medical Center,
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