HPLC analysis of asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH
HPLC analysis of asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH
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DOI：
10.1016/j.jchromb.2009.10.035
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发表时间：
2010-01-01
影响因子：
3
通讯作者：
McNeil, Yvette R.
中科院分区：
文献类型：
--
作者：
Jones, Catherine E.;Darcy, Christabelle J.;McNeil, Yvette R.
越来越多的人认识到内源性一氧化氮合酶抑制剂在危重病中的临床重要性。这突出了需要一个准确的高效液相色谱法（HPLC）检测不对称二甲基精氨酸（ADMA）和对称二甲基精氨酸（SDMA）在小体积的血液。本文描述了用于测定血浆中ADMA、SDMA、高精氨酸和精氨酸浓度的准确、精密HPLC方法的验证。固相萃取后用AccQ-Fluor（TM）衍生化，并在Gemini-NX柱上在pH 9下进行反相分离。同时检测紫外可见和荧光检测器提供额外的验证。ADMA和SDMA的绝对回收率均大于85%，相对回收率分别为102%和101%。ADMA和SDMA的批内相对标准偏差分别为2.1%和2.3%，批间相对标准偏差分别为2.7%和3.1%。该方法的优点包括在固相萃取中使用异丙醇改善了所有分析物的回收率;使用高pH移动的相获得了清晰、分辨率良好的色谱峰;非内源性内标物正丙基L-精氨酸;以及仅从100 μ L血浆中准确和精密测定甲基化精氨酸浓度。(C)2009 Elsevier B. V.保留所有权利。
There is increasing recognition of the clinical importance of endogenous nitric oxide synthase inhibitors in critical illness. This has highlighted the need for an accurate high performance liquid chromatography (HPLC) method for detection of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in small volumes of blood. Here, the validation of an accurate, precise HPLC method for the determination of ADMA, SDMA, homoarginine and arginine concentrations in plasma is described. Solid phase extraction is followed by derivatisation with AccQ-Fluor (TM) and reversed phase separation on a Gemini-NX column at pH 9. Simultaneous detection by both UV-vis and fluorescence detectors affords extra validation. This solid phase extraction method gives absolute recoveries of more than 85% for ADMA and SDMA and relative recoveries of 102% for ADMA and 101% for SDMA. The intra-assay relative standard deviations are 2.1% and 2.3% for ADMA and SDMA, respectively, with inter-assay relative standard deviations of 2.7% and 3.1%, respectively. Advantages of this method include improved recovery of all analytes using isopropanol in the solid phase extraction; sharp, well-resolved chromatographic peaks using a high pH mobile phase; a non-endogenous internal standard, n-propyl L-arginine: and accurate and precise determination of methylated arginine concentrations from only 100 mu L of plasma. (C) 2009 Elsevier B.V. All rights reserved.