Multiple-tracer approaches for investigating glucose metabolism in humans usually involve the administration of stable and radioactive glucose tracers and the subsequent determination of tracer enrichments in sampled blood. When using conventional, low-resolution mass spectrometry (LRMS), the number of spectral interferences rises rapidly with the number of stable tracers employed. Thus, in LRMS, both computational effort and statistical uncertainties associated with the correction for spectral interferences limit the number of stable tracers that can be simultaneously employed (usually two). Here we show that these limitations can be overcome by applying high-resolution mass spectrometry (HRMS). The HRMS method presented is based on the use of an Orbitrap mass spectrometer operated at a mass resolution of 100 000 to allow electrospray-generated ions of the deprotonated glucose molecules to be monitored at their exact masses. The tracer enrichment determination in blood plasma is demonstrated for several triple combinations of ¹³C- and ²H-labeled glucose tracers (e.g., [1-²H₁]-, [6,6-²H₂]-, [1,6-¹³C₂]glucose). For each combination it is shown that ions arising from ²H-labeled tracers are completely differentiated from those arising from ¹³C-labeled tracers, thereby allowing the enrichment of a tracer to be simply calculated from the observed ion intensities using a standard curve with curve parameters unaffected by the presence of other tracers. For each tracer, the HRMS method exhibits low limits of detection and good repeatability in the tested 0.1–15.0% enrichment range. Additionally, due to short sample preparation and analysis times, the method is well-suited for high-throughput determination of multiple glucose tracer enrichments in plasma samples.

中文翻译：

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使用高分辨率质谱法测定¹³ C和² H标签的葡萄糖示踪剂的同位素富集：在双重和三重追踪研究中的应用

用于研究人体内葡萄糖代谢的多示踪剂方法通常涉及稳定和放射性葡萄糖示踪剂的施用以及随后在采样血液中确定示踪剂富集的方法。当使用常规的低分辨率质谱（LRMS）时，频谱干扰的数量会随着所采用的稳定示踪剂的数量而迅速增加。因此，在LRMS中，与频谱干扰校正相关的计算工作和统计不确定性都限制了可以同时使用的稳定示踪剂的数量（通常为两个）。在这里，我们表明可以通过应用高分辨率质谱（HRMS）来克服这些限制。提出的HRMS方法基于使用质量分辨率为100000的Orbitrap质谱仪，以电喷雾生成的去质子化葡萄糖分子的离子以其精确质量进行监控。血浆中示踪剂富集的测定证明了几种的三重组合。¹³ C和² H标记的葡萄糖示踪剂（例如[ ^1-2 H ₁ ]-，[6,6- ² H ₂ ]-，[1,6- ¹³ C ₂ ]葡萄糖）。对于每种组合，均显示出将^2个H标记示踪剂产生的离子与^13个示踪剂产生的离子完全区分开使用C标记的示踪剂，从而可以使用标准曲线简单地从观察到的离子强度中计算出示踪剂的富集，而曲线参数不受其他示踪剂的存在的影响。对于每种示踪剂，HRMS方法在测试的0.1–15.0％富集范围内均显示出较低的检测限和良好的重复性。此外，由于样品制备和分析时间短，该方法非常适合于高通量测定血浆样品中多种葡萄糖示踪剂的富集。