A simple surrogate approach for the quantitation of C4 (7α-hydroxy-4-cholesten-3-one) in human serum via LC-MS/MS and its application in a clinical study

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B Pub Date : 2025-05-14 DOI:10.1016/j.jchromb.2025.124651

Feng Yin , Mani Deepika Vakkalanka , Walter Wiley , M. Shane Woolf , Yousef Basir , Kumar Shah , Aaron M. Wheeler , Moucun Yuan , William R. Mylott Jr. , Mike Baratta

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引用次数: 0

Abstract

We present a validated LC-MS/MS assay for the quantitation of 7α-hydroxy-4-cholesten-3-one (C4), a key intermediate in the bile acid synthesis pathway from cholesterol, in human serum. A surrogate matrix approach was employed to overcome the challenges posed by the endogenous C4 levels in the biological matrix. Human serum samples were spiked with stable isotope labeled internal standard (SIL-IS), processed using supported liquid extraction (SLE), and analyzed by LC-MS/MS. Parallelism was successfully demonstrated between human serum (authentic matrix) and 5 % bovine serum albumin in phosphate buffered saline containing 0.1 % tween 20 (5 % BSA in PBST) (surrogate matrix). The assay's linear analytical range was established from 0.200 to 200 ng/mL. This validated LC-MS/MS method exhibited excellent accuracy and precision. The overall accuracy was between 97.9 % and 101 % with %CV less than 4.0 % for C4 in human serum. C4 was found to be stable in human serum for up to 24.7 h at room temperature, up to 34 days when stored at −25 °C or − 80 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human serum samples to support a clinical study.

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LC-MS/MS定量人血清中C4 （7α-羟基-4-胆甾醇-3-酮）的简易替代方法及其在临床研究中的应用

我们提出了一种有效的LC-MS/MS测定方法，用于测定人血清中7α-羟基-4-胆甾醇-3-酮（C4）的含量，C4是胆固醇合成胆汁酸途径的关键中间体。采用替代基质方法来克服生物基质中内源性C4水平带来的挑战。人血清样品采用稳定同位素标记内标（SIL-IS）加标，支持液萃取（SLE）处理，LC-MS/MS分析。成功地证明了人血清（真实基质）与含0.1% tween 20 （5% BSA在PBST中）（替代基质）的5%牛血清白蛋白在磷酸盐缓冲盐水中的平行性。该方法的线性分析范围为0.200 ~ 200 ng/mL。经验证的LC-MS/MS方法具有良好的准确度和精密度。人血清中C4的总体准确度在97.9% ~ 101%之间，%CV小于4.0%。发现C4在室温下在人血清中的稳定性可达24.7小时，在- 25°C或- 80°C保存时可达34天，经过5次冷冻/解冻循环。该分析已成功应用于人类血清样本，以支持临床研究。

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来源期刊

Journal of Chromatography B 医学-分析化学

CiteScore

5.60

自引率

3.30%

发文量

306

审稿时长

44 days

期刊介绍： The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.