Bioanalysis of physiological (endogenous) compounds

[Christian Eckhoff]

For GLP assays you typically need analyte-free matrix, to prepare calibration and QC samples. I am interested to hear how colleagues are approaching GLP assays of physiological compounds where matrix invariably contains the analyte.

There seem to be two schools of thinking:

1. Strip matrix of the analyte, usually with charcoal. I found that this can be difficult to accomplish when using highly sensitive LC-MS methods. Some people also say that while it's okay for preparing calibration samples, you should never prepare QC samples with stripped matrix, particularly QC samples that you use for stability assessments. Unfortunately, the same people are not offering advice then on how to prepare QC samples for physiological analytes.

2. Use the standard addition method for calibration. This is not all that easy because my data system doesn't understands how to do it leaving me with a non-validated (Excel) data system to calculate sample concentrations. I also have samples from small animals so I can not afford to analyze each sample 4 or 5 times because I only have 200 µL to begin with. Can you forgo sample stability assessments with standard addition method?


So, if you have been in this situation yourself I'd love to hear from you, particularly from a GLP perspective. Many thanks!

[Keaka]

The other option is to find a surrogate matrix (i.e. the biological matrix diluted significantly, say 100x, in phosphate buffer).

You would have to validate in such a way to demonstrate calibration of your analyte in surrogate matrix applies to actual matrix samples as well.