Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard
Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard
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DOI：
10.1002/rcm.1112
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发表时间：
2003-01-01
影响因子：
2
通讯作者：
Whigan, DB
中科院分区：
文献类型：
--
作者：
Jemal, M;Schuster, A;Whigan, DB
开发并验证了用于测定人血浆和尿液中甲羟戊酸（胆固醇生物合成的中间体，因此是开发降胆固醇药物的有用生物标志物）的选择性、准确性和重现性液相色谱/串联质谱（LC/MS/MS）方法。甲羟戊酸的七氘代类似物用作内标。对于这两种方法，由于内源性甲羟戊酸水平过高，因此在水中而不是人血浆和尿液中制备校准标准品。在水中制备较低质量控制（QC）样品，而在生物基质中制备较高QC样品。为了从血浆和尿液基质中分离/纯化甲羟戊酸，首先将样品酸化以将酸分析物转化为其内酯形式。对于血浆样品，内酯分析物保留在聚合物固相萃取（SPE）吸附剂上，然后洗脱。对于尿液方法，将含有内酯分析物的样品通过C-18 SPE柱，该柱不保留分析物，随后分析物保留在聚合物SPE吸附剂上，然后洗脱。在极性封端的C-18分析柱上用含有0.5 mM甲酸的水/甲醇移动的流动相等度地实现色谱分离。采用负离子电喷雾串联质谱法进行检测。血浆方法的标准曲线范围为0.500-20.0 ng/mL，尿液方法的标准曲线范围为25.0-1000 ng/mL。在所有检测浓度水平下，两种方法均获得了极佳的准确度和精密度。值得注意的是，对于某些批次的尿液，当使用较大的样品体积进行分析时，观察到高度的基质效应，这不仅导致绝对响应值的衰减，而且导致分析物/内标物响应值比的变化。这表明，在某些条件下，使用稳定的同位素类似物内标物并不能保证分析物/内标物响应比的恒定性，而这是耐用的生物分析方法的先决条件。另一方面，在样品基质没有这种有害影响的条件下，我们发现稳定的同位素类似物可以作为替代（替代）分析物。因此，我们已经证明，使用通过用三氘代或四氘代甲羟戊酸加标血浆而不是甲羟戊酸本身（分析物）制备的校准标准品，可以成功分析含有甲羟戊酸的血浆QC样品，以准确和精确定量甲羟戊酸。替代分析物的使用提供了测量生物基质中制备的低浓度水平方法的日常性能的机会，否则由于生物基质中分析物的内源性浓度而无法实现。版权所有（C）2003约翰威利父子有限公司。
Selective, accurate, and reproducible liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of mevalonic acid, an intermediate in the biosynthesis of cholesterol and therefore a useful biomarker in the development of cholesterol lowering drugs, in human plasma and urine. A hepta-deuterated analog of mevalonic acid was used as the internal standard. For both methods, calibration standards were prepared in water, instead of human plasma and urine, due to unacceptably high levels of endogenous mevalonic acid. The lower quality control (QC) samples were prepared in water while the higher QC samples were prepared in the biological matrices. For the isolation/purification of mevalonic acid from the plasma and urine matrices, the samples were first acidified to convert the acid analyte into its lactone form. For the plasma samples, the lactone analyte was retained on and then eluted off a polymeric solid-phase extraction (SPE) sorbent. For the urine method, the sample containing the lactone analyte was passed through a C-18 SPE column, which did not retain the analyte, with the subsequent analyte retention on and then elution off a polymeric SPE sorbent. Chromatographic separation was achieved isocratically on a polar-endcapped C-18 analytical column with a water/methanol mobile phase containing 0.5 mM formic acid. Detection was by negative-ion electrospray tandem mass spectrometry. The standard curve range was 0.500-20.0 ng/mL for the plasma method and 25.0-1000 ng/mL for the urine method. Excellent accuracy and precision were obtained for both methods at all concentration levels tested. It was interesting to note that for certain batches of urine, when a larger sample volume was used for analysis, a high degree of matrix effect was observed which resulted not only in the attenuation of the absolute response, but also in a change of analyte/internal standard response ratio. This demonstrated that, under certain conditions, the use of a stable isotope analog internal standard does not, contrary to conventional thinking, guarantee the constancy of the analyte/internal response ratio, which is a prerequisite for a rugged bioanalytical method. On the other hand, under conditions where the sample matrix does not have such a deleterious effect, we have found that a stable isotope analog could serve as a surrogate (substitute) analyte. Thus, we have shown that using calibration standards prepared by spiking plasma with tri-deuterated or tetra-deuterated mevalonic acid, instead of mevalonic acid itself (the analyte), plasma QC samples that contain mevalonic acid can be successfully analyzed for the accurate and precise quantitation of mevalonic acid. The use of a surrogate analyte provides the opportunity to gauge the daily performance of the method for the low concentration levels prepared in the biological matrix, which otherwise is not achievable because of the endogenous concentrations of the analyte in the biological matrices. Copyright (C) 2003 John Wiley Sons, Ltd.