Investigation of absolute and relative response for three different liquid chromatography/tandem mass spectrometry...

The investigations in this article were triggered by two observations in the laboratory; for some liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems it was possible to obtain linear calibration curves for extreme concentration ranges and for some systems seemingly linear calibration curves gave good accuracy at low concentrations only when using a quadratic regression function.

The absolute and relative responses were tested for three different LC/MS/MS systems by injecting solutions of a model compound and a stable isotope labeled internal standard. The analyte concentration range for the solutions was 0.00391 to 500 μM (128,000×), giving overload of the chromatographic column at the highest concentrations. The stable isotope labeled internal standard concentration was 0.667 μM in all samples.

The absolute response per concentration unit decreased rapidly as higher concentrations were injected. The relative response, the ratio for the analyte peak area to the internal standard peak area, per concentration unit was calculated. For system 1, the ionization process was found to limit the response and the relative response per concentration unit was constant. For systems 2 and 3, the ion detection process was the limiting factor resulting in decreasing relative response at increasing concentrations.

For systems behaving like system 1, simple linear regression can be used for any concentration range while, for systems behaving like systems 2 and 3, non-linear regression is recommended for all concentration ranges. Another consequence is that the ionization capacity limited systems will be insensitive to matrix ion suppression when an ideal internal standard is used while the detection capacity limited systems are at risk of giving erroneous results at high concentrations if the matrix ion suppression varies for different samples in a run.