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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 on this?
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This problem is really tricky, I haven't met any question like this one before.
I think there's must be something in ACN-treated samples that led to the transformation of this chemical, and probably this compound would have a high protein-binding rate. Protein-precipitated supernatant have a more complex chemical profile than LLE supernatants, and that may led to the time-dependent degradation of this compound.
Is there any structure in this compound that could be easily degraded? I think you should use a SCAN mode and compare the blank plasma sample with the spiked samples, see if you could find something unusual.
As the sample disappears so quickly in ACN-treated samples, I think the half life in blood should be rather short. Zero-recovery from LLE, SPE and protein precipitation makes it almost impossible to analyze using LC-MS. Try using microdialysis and check out if there's any luck.
-- Yours, Xinting Wang
Key Laboratory of Drug Metabolism & Pharmacokinetics
China Pharmaceutical University
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