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LC–MS/MS quantification of 7α-hydroxy-4-cholesten-3-one (C4) in rat and monkey plasma
Journal of Chromatography B ( IF 3 ) Pub Date : 2017-09-05 , DOI: 10.1016/j.jchromb.2017.09.006
Lijuan Kang , Thomas M. Connolly , Naidong Weng , Wenying Jian

7α-hydroxy-4-cholesten-3-one (C4) is an oxidative enzymatic product of cholesterol metabolism via cholesterol 7α-hydroxylase, an enzyme also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1). C4 is a stable intermediate in the rate limiting pathway of bile acid biosynthesis. Previous studies showed that plasma C4 levels correlated with CYP7A1 enzymatic activity and could serve as a biomarker for bile acid synthesis. Here we developed and qualified a simple and robust high-throughput method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) to quantify C4 in rat and monkey plasma. As C4 being an endogenous compound, this method used calibration standards in 50/50: acetonitrile/water (v/v). In order to mimic the incurred samples, quality control samples were prepared in the authentic plasma. Stable isotope labeled C4 (C4-d7) was used as the internal standard. The sample volume for analysis was 20 μL and the sample preparation method was protein precipitation with acetonitrile. The average endogenous C4 concentrations, from 10 different lots of rat and monkey plasma, were 53.0 ± 16.5 ng/mL and 6.8 ± 5.6 ng/mL, respectively. Based on these observed endogenous C4 levels, the calibration curve ranges were established at 1–200 ng/mL and 0.5–100 ng/mL for rat assay and monkey assay, respectively. The method was qualified with acceptable accuracy, precision, linearity, and specificity. Matrix effect, recovery, and plasma stability of bench-top, freeze-thaw, and long-term frozen storage were also evaluated. The method has been successfully applied to pre-clinical studies.



中文翻译:

LC-MS / MS对大鼠和猴血浆中的7α-羟基-4-胆甾烯-3-酮(C4)进行定量

7α-羟基-4-胆甾烯-3-酮(C4)是通过胆固醇7α-羟化酶(也称为胆固醇7-α-单加氧酶或细胞色素P450 7A1(CYP7A1)的酶)进行胆固醇代谢的氧化酶产物。C4是胆汁酸生物合成的限速途径中的稳定中间体。先前的研究表明血浆C4水平与CYP7A1的酶活性相关,并且可以作为胆汁酸合成的生物标志物。在这里,我们开发并鉴定了一种简单可靠的高通量方法,该方法使用液相色谱与串联质谱(LC-MS / MS)结合定量大鼠和猴血浆中的C4。由于C4是内源性化合物,因此该方法使用50/50的校准标准:乙腈/水(v / v)。为了模拟所产生的样品,在真实血浆中制备了质量控制样品。7)用作内标。用于分析的样品体积为20μL,样品制备方法为使用乙腈进行蛋白质沉淀。来自10个不同批次的大鼠和猴血浆的平均内源C4浓度分别为53.0±16.5 ng / mL和6.8±5.6 ng / mL。基于这些观察到的内源C4水平,分别用于大鼠测定和猴子测定的校准曲线范围分别定为1–200 ng / mL和0.5–100 ng / mL。该方法以可接受的准确性,精密度,线性和特异性合格。还评估了台式,冻融和长期冷冻保存的基质效应,回收率和血浆稳定性。该方法已成功应用于临床前研究。

更新日期:2017-09-05
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