High performance liquid chromatographic determination of thromboxane B2 in human serum as a methoxime-panacyl ester derivative

R.H. Pullen , J.A. Howell , J.W. Cox
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引用次数: 3

Abstract

A high performance liquid chromatographic (HPLC) method was developed to measure thromboxane B2 (TxB2) levels in human serum. Serum samples (2 mL) were extracted using solid phase extraction columns in a C18/silica mode sequencing approach. The internal standard, 6-ketoprostaglandin F was added to the serum extracts. The eicosanoids were doubly derivatized, first with panacyl bromide, then with methoxyamine to form methoxime-panacyl ester derivatives. The eicosanoid derivatives were chromatographed using a reverse phase HPLC system with UV detection (254 nm). Assay linearity was demonstrated with fortified TxB2 standards in 3% bovine serum albumin over a range of 25 to 500 ng/ml (r>0.994). There was no significant interday difference or bias in assay results for pooled standards at 75, 226 and 376 ng/mL concentrations (p > 0.05). Pooled estimates of precision at these levels indicate an assay relative standard deviation 6–9%. The HPLC assay was used to quantitate TxB2 levels in human serum. Results were consistent with previously published values when drug-free serum was analyzed to assess ex vivo TxB2 formation.

高效液相色谱法测定人血清中甲氧肟-panacyl酯衍生物血栓素B2
建立了高效液相色谱法测定人血清中血栓素B2 (TxB2)含量的方法。血清样品(2ml)采用固相萃取柱,C18/二氧化硅模式测序法提取。血清提取物中加入内标6-酮前列腺素F1α。二十烷类化合物被双重衍生,首先与甲酰基溴,然后与甲氧基胺形成甲氧基-甲氧基酯衍生物。采用反相高效液相色谱法(254 nm)对类二十烷衍生物进行色谱分析。在3%牛血清白蛋白中,强化TxB2标准在25 ~ 500 ng/ml范围内呈线性(r>0.994)。在75、226和376 ng/mL浓度的混合标准中,检测结果没有显著的日间差异或偏差(p >0.05)。在这些水平上对精密度的汇总估计表明测定的相对标准偏差为6-9%。采用高效液相色谱法测定人血清TxB2水平。结果与先前发表的分析无药血清以评估体外TxB2形成的结果一致。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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