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

Abstract

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.

Clinical Evaluation of a Novel Insulin Immunosensor

“…23 On the contrary, the sensing protocol can be improved by reducing the sample-to-answer time, which will bring this sensing approach even closer to real-time monitoring. 11,12 In this regard, insulin quantization with the immunosensor based on single-point calibrations [25][26][27][28][29] or calibration-free methodologies 30,31 should be explored in future experiments.…”

Simultaneous quantitation of kavalactones in kava dry extracts: comparison of multi‐standards and single standard validation approaches

“…In order to compare the influence of the number of analytical standards used for the kavalactone assay, the results obtained for the extract samples using the validated method for six kavalactone standards were compared with those obtained when only KAV was used as a standard. This single standard validation approach is presented in the WHO kava extract monograph 12 and is often used when the standard compounds required for sample validation and analysis are not all available 34 …”

“…This single standard validation approach is presented in the WHO kava extract monograph 12 and is often used when the standard compounds required for sample validation and analysis are not all available. 34 3 | RESULTS AND DISCUSSION…”

Strategies and Challenges in Method Development and Validation for the Absolute Quantification of Endogenous Biomarker Metabolites Using Liquid Chromatography‐tandem Mass Spectrometry

“…Despite the potential of ID-ISs, their application for absolute quantification of endogenous metabolites is increasing at a slow pace (Stanislaus et al, 2012;Khamis et al, 2017;Awad et al, 2019). Possible reasons include the additional derivatization work load needed during method development and validation, in comparison to nonderivatization (direct) methods, and the vigilance needed to avoid potential metabolite loss during the extra steps of sample handling (Khamis et al, 2017;Khamis et al, 2018c). On the contrary, the analytical improvements introduced by ID-ISs can outweigh the associated workload.…”