†Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
‡Clinical Biochemistry Service, Central University Hospital of Asturias, 33011 Oviedo, Spain.
Anal Chem. 2015 Apr 7;87(7):3755-63. doi: 10.1021/acs.analchem.5b00769. Epub 2015 Mar 19.
This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.
本工作描述了使用 (13)C 标记的有机化合物多次尖峰同位素稀释程序。介绍了一种双尖峰同位素稀释方法，该方法能够校正和定量分析人血清中两种化合物时发生的肌酸-肌酐相互转化。血清肌酐的测定可能会受到样品制备过程中肌酸和肌酐之间相互转化的影响，或者由于固相萃取 (SPE) 对这些化合物的化学分离效率不高而受到影响。该方法基于使用不同标记的 (13)C 类似物 ((13)C1-肌酐和 (13)C2-肌酸)，通过液相色谱-串联质谱 (LC-MS/MS) 测量肌酸和肌酐的同位素分布，并应用多元线性回归。分析了五个不同冻干的基于血清的对照品和两个认证的人血清参考物质 (ERM-DA252a 和 ERM-DA253a)，以评估所提出的双尖峰 LC-MS/MS 方法的准确性和精密度。该方法应用于研究 LC-MS/MS 和气相色谱-质谱 (GC-MS) 分析过程中的肌酸-肌酐相互转化，以及传统气相色谱-同位素稀释质谱 (GC-IDMS) 参考方法中 SPE 步骤的分离效率，该方法用于测定血清肌酐。通过 GC-MS 对真实血清样品的分析表明，SPE 对肌酸-肌酐的分离可能是一个非定量步骤，在含有高浓度肌酸的样品中，可能会导致肌酐高估高达 28%。此外，在 LC-MS/MS 样品制备过程中观察到肌酸向肌酐的可检测转化。所开发的双尖峰 LC-MS/MS 改进了当前用于人血清中肌酐的同位素稀释质谱 (IDMS) 测定的最新技术，因为它对样品制备步骤中可能产生的所有误差进行了校正。
