I tried to measure my drug sunitinib 0.1mg/kg (0.07mM)samples in mice plasma and used an internal stranded verapamil 50nM and made a calibration standreds from 1nM to 3000nM but the problem is in some samples I GOT NO peaks and also the intercept was negative so please can you help me in addressing the problem
Dear Ella Hasan,
Do you get no peak in standards and QCs or in unknown samples? Could it be that the concentrationa are actually below Limit of detection?
If you are concerned with response in real sample extracts compared to standards you can test over-spike / standard addition (add known concentration in selected real samples). This will reveal issues with e.g. ion Suppression, formulation interference.
It is not unusual for intercept to be negative when using all non-Zero standards, as is common practice in bioanalytics.It is not necessarily an issue, as Long as standrds and QCs are reproducible and robust, and you do not extrapolate concentrations out of your Standard curve.
Are you taking special care with light protection of your samples, working solutions...? Sunitinib is very light sensitive, could this be part of the problems you observed? For some compounds stability may be fine in plasma but more sensitive in extracts..
Best of luck
Patrice
Dear Ella
Absence of peaks may be due to column blockage or autosampler needle blockage.check for any carryover or extend your chromatographic run.try to change to any c18 100mm,5micron column.Use 1mm ammonium acetate with acetonitrile as mobile phase ideally it should elute faster in the column using high organic mobile phase.check for your ion source for proper spray.
Good luck
Kannadhasan
Very useful information for my work! I will gladly follow further discussion!
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