Chromatography Forum

Hi, I am analysing fatty acids in human serum and quantifying amounts from internal standard C17 which I add to all my samples at 1mg/ml. I know from previous experience that quantities of the saturated palmitic acid in human blood samples are around 0.3-0.4mg/ml and I've always got that in my previous analyses, although in plasma.
However after setting up a new std curve and new method recently, I carefully extracted and analysed some samples and everything looks ok, bar a few fatty acids which are being quantified in much higher amounts than could possibly be present in serum (about 1.2 mg/ml).
I'm wondering if I've left something out in the quantification stage of method setup that could result in the overestimation of a compound relative to its internal std, e.g. could my Q ions be out of range/ should I be operating in SIM vs SCAN mode? Does anyone have any suggestions? I'm not an expert by any means and would appreciate any help.

Alison

Hi Alison

Contamination from fingerprints can easily cause this problem. Try running a blank with water or saline/buffer instead of the sample and check for background levels of target acids.

Peter

A,

Go back and re-prep your Internal STD (ISTD) C-17 at three dilution levels (1 mg_0.5mg_0.25mg) and then add them in BOTH blood and plasma test samples.

Conduct your extraction procedure. Plot the "recovered concentration" vs. "expected conc." for Both matrices and at all three ISTD levels that were added to your samples.

Do the plots have reasonably similar R-square values?
Or, is one plot tending to bias the response in a certain direction?
Are they similarly linear or is one biased up or down from the 'regression line"?

This experiment will at least help you to at least resolve the issue of sample prep variability and/or possible matrix effects on the recovery of the analyte or interference in the recovery of the ISTD.

Please report back to us.