Chromatography Forum

Hello all,
In my work I develop and validate HPLC metods for examining drugs in biological matrix.
My question is for the calibration curve. According to the requirements of FDA for bioanalytical method validation the calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample with internal standard), and 5-8 non zero samples covering the expected range.
What is the meaning of the blank and zero sample? How have they been included in the curve? I work with Agillent liquid chromatograph series 1100 and I construct the calibration curve without including the zero point (blank, zero sample). Actually I think that I cannot include a zero amount in the curve. What do you think?
Thank you.

Blank and zero samples should not be included in the curve.
These samples are just as control of cross - contamination during sample preparation, carry -over in the instrument and absence of the analytes in the blank plasma.

Thank you.
But how shall I proceed in the case if (accidentally) there is a contamination in the blank sample in some of the analitycal series?
May I substract blank run from the calibration and/or matrix samples (by the software)? Or I should repeat the whole analytical run/series?
Is the meaning of the zero samples to calculate what percent of examined peak is from a contamination?

If there's an interference, it will show when you calculate your zero sample (internal standard only) as an unknown.
Depending on the results and the level of compendial compliance you are bound by, you may either scrap the run (or the method )or set a multiplier so that you don't report any results that are not at least (for example) 3x the zero sample.