Dear all,Back to the Top
I am new to LCMS quantification analysis, as I am trying to quantify
time) the drugs using SRM, I face the follwing problem,
1. I got good results on Drug and Internal Standard in Mobile phase
2. In the case of Plasma samples (in-vitro) I got only 10-20% recovery
HPLC results shows around 80-90% recovery...
I got a very good linearity for the plain samples upto 5 ng.
But In plasms samples, I face problem, Generally MS is sensitive than
Is the plasma interfere the ionsation of my drug and internal standard?
have changed the method by increasing the flow rate, changing the
increasing the run time and changing the mobile phase... Still our
is around 40%...
Is this due to the suppression of Ionization by matrix components or
other interference? Is any body came across a similar problem?
Your valuable suggestions are welcome.
Thanks & Regards,
It depends what solvent you are using for your extraction. Solvents likeBack to the Top
Methanol and DMSO usually suppress ionization. Some salts that are
release from the plasma during the extraction can also do it. Other
thing that you can try is divert the valve to waste in the first minute.
Dr.B.Sivakumar -Back to the Top
You may be experiencing ion suppression due to the presence of other
interfering substances in the sample extract. You can conveniently
assess the extent of ion suppression by the following procedure:
1. infuse the drug into the MS at a constant rate
2. monitor the ion current for the drug
3. inject an extract of a negative control plasma sample
4. determine the extent of ion suppression of the drug at its retention
An excellent example of the use of this technique during method
validation to assess ion suppression effects was reported by Luo et al.
in "Quantification and confirmation of flunixin in equine plasma by
liquid chromatography - quadrupole time-of-flight tandem mass
spectrometry" in Journal of Chemistry B 801: 173-184 (2004).
Richard Sams, Ph.D.
Dear Dr. SivakumarBack to the Top
For clarification of your problem we need to know the Retention Time of
both drug and IS. We observed the suppression in degree of ionisation
of drug when the RT of drug and IS are same.
Try to change the final reconstitution solvent for increasing the
Final approach can be to change the ionisation source because in some
cases APCI gives better result than ESI.
If problem persist than a detailed discussion of the method may be
Feel free to contact in case of any doubt.
Sr. Research Associate
Dear Dr.SivakumarBack to the Top
Hope you have taken care of Capacity Factor for main peak as to be at
least 2-3, so that endogenous components ofbiological matrixelute
The most appropriate way of doing recovery studies using LC-MS is as
1. Spike the known concentration of drug in to drug free matrix and
extract with the your established procedure and quantitate the peak
2. process drug free matrix with your extraction procedure and spike
the known concentration of drug into that just before reconstitution
and quantitate the peak response.
3. Compare response of 1 againest 2 to estimate the exact recoveries.
Because the matrix effects and suppression of ionization by matrix may
create havoc when you compare the responses of extracted samples
againest neat solutions.
Thanking you and Regards
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