College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada.
Brandenburg University of Technology Cottbus-Senftenberg, Großenhainer Strasse 57, 01968, Senftenberg, Germany.
Anal Bioanal Chem. 2018 Sep;410(23):5899-5913. doi: 10.1007/s00216-018-1205-5. Epub 2018 Jul 13.
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.
靶向代谢组学需要通过串联质谱 (MS/MS) 分析对候选生物标志物进行准确而精确的定量。差示同位素标记 (DIL) 通过用两种相同试剂的同位素形式对代谢物进行衍生化，从而改善代谢组学中的质谱 (MS) 分析。尽管具有优势，但当需要完全验证的定量方法时，DIL-液相色谱 (LC)-MS/MS 会导致工作量的大量增加。为了减少工作量，我们假设可以使用单点校准或相对定量作为替代方法。这两种方法都将在资源和时间上节省大量成本。为了验证我们的假设，选择了六种尿代谢物作为模型化合物。使用经过充分验证的多点丹磺酰氯-DIL-LC-MS/MS 方法分析尿液样本。使用单点校准和相对定量模式重新处理样品。结果表明，单点校准或相对定量的性能对于一些代谢物，不如多点校准。单点校准无法对大多数参与者样本中的乙醇胺进行定量，其定量下限失败。此外，在丝氨酸和乙醇胺定量过程中，其精密度在一个参与者中不可接受。另一方面，相对定量会产生最不准确的数据。事实上，在相对定量得到的丝氨酸数据中，没有一个数据与多点校准得到的数据可比。最后，虽然单点校准对大多数模型化合物的表现整体上可接受，但我们不能将这些发现推断到同一分析运行中的其他代谢物。分析人员应评估单点校准为预期定量方法的每个代谢物的准确性和精密度。
