Drug Safety Sciences, Janssen Research & Development, 1400 McKean Road, Spring House, PA 19477, USA.
J Pharm Biomed Anal. 2013 Nov;85:145-54. doi: 10.1016/j.jpba.2013.07.016. Epub 2013 Jul 25.
A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5 μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500 ng/ml and 50 to 2000 μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9 μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis. The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.
建立并确证了一种液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）分析方法，可用于分析人及鼠血浆中的 4β-羟基胆固醇和胆固醇，检测限为 5μl 血浆中的 5500ng/ml 4β-羟基胆固醇和 502000μg/ml 胆固醇。方法中，4β-羟基胆固醇和胆固醇的内标物分别为其氘代稳定同位素标记物，而校准曲线和质控样品的制备则采用磷酸盐缓冲生理盐水与 4.2%（w/v）人血清白蛋白的混合物作为替代基质。该方法在考察了分析物的回收率、自动进样器稳定性以及化学氧化对 4β-羟基胆固醇生成的潜在影响后，对其准确度和精密度进行了评估，结果表明，该方法可对 4β-羟基胆固醇和胆固醇进行定量分析，检测下限分别为 5500ng/ml 和 502000μg/ml。在对样品进行碱水解后，分析物可被定量为游离形式和共轭形式，随后衍生化为电喷雾电离敏感的吡啶酰酯，经碰撞诱导解离后，以多反应监测方式进行选择性检测，可得到高质量的前体-产物离子对，以此实现对低浓度血浆样品中胆固醇的定量分析。由于在多反应监测检测中，使用反向相 16 分钟梯度洗脱、1.9μm 粒径、C18 柱可实现两种分析物的色谱分离，因此可克服其他等电荷血浆氧化固醇的干扰。本方法与胆固醇的正交酶法相比，除了一个样本之外，所有样本的检测值均在 10%以内。此外，根据已报道的对再分析样品的接受标准，该方法通过了人及鼠血浆中两种分析物的实际样品测试。两种分析物的定量分析可用于测定 CYP3A4/5 活性的内源性生物标志物 4β-羟基胆固醇与其与胆固醇的比值。LC-ESI-MS/MS 分析方法还成功地应用于人及未处理的鼠血浆样品中 4β-羟基胆固醇和胆固醇的定量分析，包括用人源化肝脏的 FRG™ KO C57Bl/6 嵌合鼠。初步数据表明，嵌合鼠及其他鼠的血浆 4β-羟基胆固醇浓度或其与胆固醇的比值均高于人。
