Quantitation of endogenous analytes in biofluid without a true blank matrix
Quantitation of endogenous analytes in biofluid without a true blank matrix
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DOI：
10.1021/ac034505u
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发表时间：
2003-11-01
影响因子：
7.4
通讯作者：
Cohen, LH
中科院分区：
文献类型：
--
作者：
Li, WL;Cohen, LH
提出了一种方法，描述了一种可靠且实用的程序，用于定量空白（未处理）生物基质中以相对高背景水平存在的分析物。使用“替代分析物”方法，在含有非常低（< 10 ng/mL）和高（>2000 ng/mL）背景水平的所需分析物的各种生物基质中定量内源性分析物。这种定量“替代分析物”方法在开发HPLC/MS方法期间应用。酮异己酸（W），其被鉴定为支链氨基酸转移酶抑制剂活性的潜在生物标志物。使用氘标记的KIC（d（3））作为替代分析物，而不是内标物，以生成校准曲线，可以基于KIC对KIC-d（3）的回归方程和响应因子反算生物流体中EX的浓度。特别是，这种方法可以在控制生物流体如血浆中制备标准品，这极大地促进了方法开发的过程。对于经验证的方法，观察到KIC-d（3）的线性范围为10-5000 ng/mL。KIC-d（3）的日内和日间实验准确度（以误差百分比计算）小于或等于10%。该方法简单、快速、可靠，可用于定量血浆、脑匀浆、脑脊液和其他来自发现和药理学研究的生物样品中的KIC。
A method is presented that describes a reliable and practical procedure for quantitation of an analyte present at relatively high background levels in blank (untreated) biological matrixes. Using a "surrogate analyte" approach, an endogenous analyte was quantitated in a variety of biological matrixes containing both very low (< 10 ng/mL) and high (>2000 ng/mL) background levels of the desired analyte. This quantitative "surrogate analyte" approach was applied during the development of an HPLC/MS method for a.-ketoisocaproic acid (W), which was identified as a potential biomarker for branched chain amino acid transferase inhibitor activity. Using deuterium-labeled KIC (d(3)) as a surrogate analyte, not an internal standard, to generate the calibration curve, the concentration of EX in biofluid could be back-calculated based on the regression equation and response factor of KIC to KIC-d(3). In particular, this approach made it possible to prepare standards in control biofluid such as plasma, which greatly facilitated the process of method development. For the validated method, a linear range of 10-5000 ng/mL for KIC-d(3) was observed. Intraday and interday experimental accuracy, calculated as percent error, were in the range of less than or equal to 10% for KIC-d(3). This method is simple, rapid, and reliable for the quantitation of KIC in plasma, brain homogenate, cerebrospinal fluid, and other biological samples from discovery and pharmacological studies.