Menzies School of Health Research, Rocklands Drive, Tiwi, Darwin, NT, Australia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jan 1;878(1):8-12. doi: 10.1016/j.jchromb.2009.10.035.
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 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 100microL of plasma.
人们越来越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。这凸显了需要一种准确的高效液相色谱 (HPLC) 方法来检测小体积血液中的不对称二甲基精氨酸 (ADMA) 和对称二甲基精氨酸 (SDMA)。本文描述了一种准确、精密的 HPLC 方法，用于测定血浆中 ADMA、SDMA、同型精氨酸和精氨酸浓度。固相萃取后，用 AccQ-Fluor 衍生化，在 Gemini-NX 柱上在 pH 9 下进行反相分离。通过紫外可见和荧光检测器同时检测可提供额外的验证。这种固相萃取方法使 ADMA 和 SDMA 的绝对回收率超过 85%，ADMA 和 SDMA 的相对回收率分别为 102%和 101%。ADMA 和 SDMA 的日内相对标准偏差分别为 2.1%和 2.3%，日间相对标准偏差分别为 2.7%和 3.1%。该方法的优点包括使用异丙醇在固相萃取中提高所有分析物的回收率；使用高 pH 流动相得到尖锐、分辨率良好的色谱峰；使用非内源性内标 n-丙基 L-精氨酸；以及仅从 100μL 血浆中准确、精密地测定甲基化精氨酸浓度。
