School of Pharmacy, College of Medicine , National Taiwan University , Taipei 10050 , Taiwan.
The Metabolomics Core Laboratory, Centers of Genomic and Precision Medicine , National Taiwan University , Taipei 10055 , Taiwan.
Anal Chem. 2019 Aug 20;91(16):10702-10712. doi: 10.1021/acs.analchem.9b02050. Epub 2019 Aug 8.
Dried blood spots (DBSs) have gained increasing attention recently with their growing importance in precision medicine. DBS-based metabolomics analysis provides a powerful tool for investigating new biomarkers. Until now, very few studies have discussed measures for improving analytical accuracy with the consideration of the special characteristics of DBSs. The present study proposed a postcolumn infused-internal standard (PCI-IS) assisted strategy to improve data quality for DBS-based metabolomics studies. An efficient sample preparation protocol with 80% acetonitrile as the extraction solvent was first established to improve the metabolite recovery. The PCI-IS assisted liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was used to simultaneously estimate the blood volume and correct the signal change caused by ion source contamination and the matrix effect to evaluate the spot volume effect and hematocrit (Hct) variation effect on target metabolites. Phenylalanine- was selected as the single PCI-IS to correct the matrix effect. For calibration of errors caused by the blood volume difference, 75% of the test metabolites showed good correlation ( ≥ 0.9) between the spot volume and the signal intensity after PCI-IS correction compared to less than 50% metabolites with good correlation before calibration. The spot volume was further calibrated by the same PCI-IS. Investigation of the Hct variation effect on target metabolites revealed that it affected the concentrations of metabolites in the DBS samples depending on their abundance in the red blood cell (RBC) or plasma; it is essential to preinvestigate the distribution of metabolites in blood to minimize the comparison bias in metabolomics studies. Finally, the PCI-IS assisted method was applied to study acetaminophen-induced liver toxicity. The results indicated that the proposed PCI-IS strategy could effectively remove analytical errors and improve the data quality, which would make the DBS-based metabolomics more feasible in real-world applications.
干血斑 (DBS) 因其在精准医学中的重要性日益增加而备受关注。基于 DBS 的代谢组学分析为研究新的生物标志物提供了有力的工具。到目前为止，很少有研究讨论过在考虑 DBS 特殊性质的情况下提高分析准确性的措施。本研究提出了一种柱后注入内标 (PCI-IS) 辅助策略，以提高基于 DBS 的代谢组学研究的数据质量。首先建立了一种高效的样品制备方案，以 80%乙腈作为提取溶剂，以提高代谢物的回收率。采用 PCI-IS 辅助的液相色谱-电喷雾电离质谱 (LC-ESI-MS) 法同时估算血斑体积，并校正离子源污染和基质效应引起的信号变化，以评估目标代谢物的斑点体积效应和血细胞比容 (Hct) 变化效应。选择苯丙氨酸作为单一 PCI-IS 来校正基质效应。对于由血斑体积差异引起的校准误差，与校准前 50%左右的代谢物具有良好相关性相比，75%的测试代谢物在 PCI-IS 校正后与斑点体积和信号强度之间具有良好的相关性 (≥0.9)。进一步用相同的 PCI-IS 对斑点体积进行校准。研究目标代谢物的 Hct 变化效应表明，它取决于代谢物在红细胞 (RBC) 或血浆中的丰度，影响 DBS 样品中代谢物的浓度；在代谢组学研究中，有必要预先研究代谢物在血液中的分布，以最小化比较偏倚。最后，将 PCI-IS 辅助方法应用于研究对乙酰氨基酚诱导的肝毒性。结果表明，所提出的 PCI-IS 策略可以有效消除分析误差，提高数据质量，这将使基于 DBS 的代谢组学在实际应用中更可行。
