Cifuentes Girard Maria Fernanda, Ruskic David, Böhm Günter, Picenoni Renzo, Hopfgartner Gérard
Life Sciences Mass Spectrometry, Department of Inorganic and Analytical Chemistry, University of Geneva, 24 Quai Ernest Ansermet, CH-1211, Geneva 4, Switzerland.
CTC Analytics, AG, Zwingen, Switzerland.
Anal Chim Acta. 2020 Aug 29;1127:198-206. doi: 10.1016/j.aca.2020.06.030. Epub 2020 Jul 8.
For metabolite profiling chemical derivatization has been used to improve MS sensitivity and LC retention. However, for multi-analytes quantification, the number of commercially available isotopically labelled internal standards is limited. Besides, there is no single workflow which can provide large-scale metabolomics coverage in particular for polar metabolites. To overcome these limitations and to improve reproducibility a fully automated dual derivatization approach was developed. Differential Isotope Labeling (DIL) was adopted by derivatizing carbonyl, amino and phenol metabolites with two isotopic forms. Urine samples were derivatized with C-dansyl chloride (DnsCl) and C-dansylhydrazine (DnsHz). Suitable quantification standards were generated by derivatized 40 standards including amino acids, sex hormones and other highly polar metabolites with labelled C-dansyl chloride and C-dansylhydrazine. The derivatization of the standards and the urine sample was performed using a PAL RTC autosampler in-line to column-switching LC-HRMS analysis with data independent acquisition (SWATH-MS). The parallel reactions were completed in 15 min inside of two agitators at different conditions overlapping with the LC-MS analysis time which was of 25 min. The column switching setup is critical to remove the excess of reagents which can negatively affect the ionization efficiency and deteriorate the chromatographic performance. The combination of dual DIL with SWATH-MS acquisition enables post-identification of unknown metabolites and quantitation at precursor (MS1) and specific tag fragment (MS2) levels. The inter- and intra-batch accuracy and precision of the method fall in the range ±15% using single point calibration, and at MS1 or MS2 level providing full flexibility. The method was successfully applied to the analysis of human urine samples.
对于代谢物谱分析，化学衍生化已被用于提高质谱灵敏度和液相色谱保留率。然而，对于多分析物定量，市售的同位素标记内标数量有限。此外，没有单一的工作流程能够提供大规模的代谢组学覆盖范围，特别是对于极性代谢物。为了克服这些限制并提高重现性，开发了一种全自动双衍生化方法。采用差异同位素标记（DIL），用两种同位素形式对羰基、氨基和酚类代谢物进行衍生化。尿液样本用C-丹磺酰氯（DnsCl）和C-丹磺酰肼（DnsHz）进行衍生化。通过用标记的C-丹磺酰氯和C-丹磺酰肼对40种标准品（包括氨基酸、性激素和其他高极性代谢物）进行衍生化，生成了合适的定量标准品。标准品和尿液样本的衍生化使用PAL RTC自动进样器在线进行柱切换液相色谱-高分辨质谱分析，并采用数据非依赖采集（SWATH-MS）。平行反应在两个不同条件的搅拌器内15分钟内完成，与25分钟的液相色谱-质谱分析时间重叠。柱切换设置对于去除可能对电离效率产生负面影响并恶化色谱性能的过量试剂至关重要。双DIL与SWATH-MS采集的结合能够对未知代谢物进行鉴定后，在前体（MS1）和特定标签片段（MS2）水平进行定量。使用单点校准，该方法的批间和批内准确度和精密度在±15%范围内，并且在MS1或MS2水平提供了充分的灵活性。该方法已成功应用于人类尿液样本的分析。
