The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. The addition of stable labels ensures the subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared with the current semi-quantitation method, which utilizes isotopic interfering radio isotopologs of metabolites alone as calibrants. The proof-of-concept is illustrated with (¹⁴C,¹³C₂)-acetaminophen where in vitro biotransformation produced (¹⁴C,¹³C₂)-sulfate and (¹⁴C,¹³C₂)-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry. Quantitative data obtained by this method fell within 82–86% of the values from conventional LC–MS/MS method.

中文翻译：

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双功能化同位素作为校准物，通过液相色谱结合辐射测定法和质谱法对血浆中的药物代谢物进行无标准定量

所报告的方法涉及一种新的工作流程，该工作流程无需使用具有放射性和稳定同位素的单一多同位素标记化合物来定量生物样品中的药物代谢物的真实参考标准。所得母体药物的放射性和稳定双功能化同位素 (RADSTIL) 用作体外的底物将代谢物生物转化为靶向 RADSTILs 作为校准物。包含无线电标记可以实现辐射测量和质谱检测。添加稳定的标记可确保随后对临床前和临床样品中未标记的代谢物进行无同位素干扰的定量。与当前的半定量方法相比，这提供了更准确的定量工作流程，后者仅利用代谢物的同位素干扰放射性同位素作为校准物。^{用 ( 14} C, ¹³ C ₂ )-对乙酰氨基酚说明概念验证，其中体外生物转化产生 ( ¹⁴ C, ¹³ C ₂ )-硫酸盐和 ( ¹⁴C, ¹³ C ₂ )-葡萄糖苷酸校准物。对乙酰氨基酚代谢物的绝对定量然后通过液相色谱结合辐射测定和质谱来实现。通过这种方法获得的定量数据在传统 LC-MS/MS 方法的 82-86% 范围内。