title: Investigation of absolute and relative response for three different liquid chromatography/tandem mass spectrometry systems; the impact of ionization and detection saturation.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Global DMPK, Regulatory Bioanalysis, AstraZeneca R&D, Mölndal, Sweden. lars.b.nilsson@astrazeneca.com
authors.forename: Lars B
authors.initials: LB
authors.lastname: Nilsson
authors.forename: Patrik
authors.initials: P
authors.lastname: Skansen
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D007554:Isotopes; D057230:Limit of Detection; D012015:Reference Standards; D012044:Regression Analysis; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D007554:Isotopes