Bioequivalence and Bioavailability Forum

Dear Kumar,

❝ Helmutz/ElMaestro/Olhbe:

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Back to you questions:

• it is seen quite often in LC-MS/MS that the response from the detector, and possibly the retention times, evolve gradually over time when switching from water or solvent-based samples to plasma samples. I'm not sure back pressure is the only reason for it. It also involves conditioning of the ion source, gradual accumulation of phospholipids on the column etc. As this gradual evolution may affect the analyte and the IS differently, resulting in an evolution of the peak area ratio, the idea of injecting stabilisation samples before the run is to get to a stable status of the system before you start injecting your CC/QC/subject samples. As this effect is analyte- and method-dependent, you may or may not need to inject stabilisation samples, and the number of samples required to obtain a stable response may vary.
  
• there is indeed a risk of "mis-use or improper use" of these samples. See here for an example... and its consequences.
  
• strong recommendation: in order to avoid problems, don't use CC, QC or subject samples from other / the same run as equilibration samples. You could get accused of cherry picking (injecting them till you are satisfied that the calibration curve and QCs will pass). Use either pooled samples, or samples especially prepared for that purpose, at a concentration different from the CC/QC. I don't think there is a need to inject samples at all CC levels of concentration. One medium level, or possibly just one low and one high, should be enough.
  
• look at the draft revised FDA guidance, lines 928-934.