Gehrke Sarah, Reisz Julie A, Nemkov Travis, Hansen Kirk C, D'Alessandro Angelo
Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, 12801 East 17th Ave, Aurora, CO, 80045, USA.
Rapid Commun Mass Spectrom. 2017 Sep 15;31(17):1445-1452. doi: 10.1002/rcm.7916.
In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited by a lack of rapid extraction methods.
Here we characterized the efficacy of fast liquid-liquid extractions for characterization of hydrophilic compounds through ultra-high-pressure liquid chromatography/mass spectrometry. Internal stable-isotope-labeled standards were used to quantitatively characterize markers of energy and oxidative metabolism in human whole blood, plasma and red blood cells - three common matrices of clinical relevance.
For all the tested matrices, vortexing time (4-60 min) did not significantly affect extraction yields for the tested hydrophilic metabolites. Coefficients of variations <<20% for all tested compounds, except for the redox-sensitive metabolite cystine (accumulating over time). Internal standards and second extractions confirmed recoveries >80% for all tested metabolites, except for basic amino acids and polyamines, which showed reproducible yields ranging from 50 to 75%. Global profiling and absolute quantitation of 24 metabolites revealed similarities between the plasma and red blood cell metabolomes.
Rapid extraction (~4 min) of hydrophilic compounds is a viable and potentially automatable strategy to perform quantitative analysis of whole blood, plasma and red blood cells for research or clinical applications.
在过去五年中，高通量代谢组学显著推动了科学研究，并有望在临床代谢组学和个性化医疗领域取得重大进展。虽然流动注射质谱和三分钟代谢组学方法领域的创新使研究人员现在每天能够处理数百至数千个样本，但时间敏感的临床应用，尤其是在急诊科，因缺乏快速提取方法而受到限制。
在此，我们通过超高压液相色谱/质谱法表征了快速液液萃取用于亲水性化合物表征的效果。使用内部稳定同位素标记标准物对人全血、血浆和红细胞（三种具有临床相关性的常见基质）中的能量和氧化代谢标志物进行定量表征。
对于所有测试的基质，涡旋时间（4 - 60分钟）对测试的亲水性代谢物的提取产率没有显著影响。除了对氧化还原敏感的代谢物胱氨酸（随时间积累）外，所有测试化合物的变异系数均<<20%。内标和二次萃取证实，除碱性氨基酸和多胺外，所有测试代谢物的回收率均>80%，碱性氨基酸和多胺的产率在50%至75%之间可重复。对24种代谢物的全局分析和绝对定量揭示了血浆和红细胞代谢组之间的相似性。
快速提取（约4分钟）亲水性化合物是一种可行且可能可自动化的策略，可用于对全血、血浆和红细胞进行研究或临床应用的定量分析。
