Analytical and Bioanalytical Chemistry

, Volume 410, Issue 23, pp 5899–5913 | Cite as

Comparison of accuracy and precision between multipoint calibration, single point calibration, and relative quantification for targeted metabolomic analysis

  • Mona M. Khamis
  • Nancy Klemm
  • Darryl J. Adamko
  • Anas El-AneedEmail author
Research Paper


Targeted metabolomics requires accurate and precise quantification of candidate biomarkers, often through tandem mass spectrometric (MS/MS) analysis. Differential isotope labeling (DIL) improves mass spectrometric (MS) analysis in metabolomics by derivatizing metabolites with two isotopic forms of the same reagent. Despite its advantages, DIL-liquid chromatographic (LC)-MS/MS can result in substantial increase in workload when fully validated quantitative methods are required. To decrease the workload, we hypothesized that single point calibration or relative quantification could be used as alternative methods. Either approach will result in significant saving in resources and time. To test our hypothesis, six urinary metabolites were selected as model compounds. Urine samples were analyzed using a fully validated multipoint dansyl chloride-DIL-LC-MS/MS method. Samples were reprocessed using single point calibration and relative quantification modes. Our results demonstrated that the performance of single point calibration or relative quantification was inferior, for some metabolites, to multipoint calibration. The lower limit of quantification failed in the quantification of ethanolamine in most of participant samples using single point calibration. In addition, its precision was not acceptable in one participant during serine and ethanolamine quantification. On the other hand, relative quantification resulted in the least accurate data. In fact, none of the data generated from relative quantification for serine was comparable to that obtained from multipoint calibration. Finally, while single point calibration showed an overall acceptable performance for the majority of the model compounds, we cannot extrapolate the findings to other metabolites within the same analytical run. Analysts are advised to assess accuracy and precision for each metabolite in which single point calibration is the intended quantification mean.


Metabolomics Single point calibration Validation Quantification Differential isotope labeling, dansyl chloride 



The authors acknowledge the valuable comments and discussions with Dr. Randy Purves, University of Saskatchewan, concerning the project.

Funding information

Ms. Khamis thanks the College of Pharmacy and Nutrition for scholarship funding. Funding for the project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant and the QTRAP 6500 instrument was purchased through a Western Diversification Grant from the Western Economic Diversification Canada.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Mona M. Khamis
    • 1
  • Nancy Klemm
    • 2
  • Darryl J. Adamko
    • 3
  • Anas El-Aneed
    • 1
    Email author
  1. 1.College of Pharmacy and NutritionUniversity of SaskatchewanSaskatoonCanada
  2. 2.Brandenburg University of Technology Cottbus-SenftenbergSenftenbergGermany
  3. 3.Department of Pediatrics, College of MedicineUniversity of SaskatchewanSaskatoonCanada

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