Feussner A, Rolinski B, Weiss N, Deufel T, Wolfram G, Roscher A A
Medizinische Poliklinik, Ludwig-Maximilians-Universität München, Klinikum Innenstadt, Germany.
Eur J Clin Chem Clin Biochem. 1997 Sep;35(9):687-91. doi: 10.1515/cclm.1997.35.9.687.
A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.
本文描述了一种用于测定人血浆中总同型半胱氨酸的简单、灵敏且精确的等度高效液相色谱法。通过用三正丁基膦还原，从血浆蛋白中释放出硫醇化合物，并用硫醇特异性荧光标记物7-氟苯并-2-恶唑-1,3-二氮杂环丁烷-4-磺酸盐进行衍生化。使用Superspher 100 RP-18柱作为固定相，通过反相高效液相色谱在7分钟内对等度分离衍生物。采用这种方法，每天可检测200多个样品中的总同型半胱氨酸。该方法在高达100μmol/L时呈线性，检测限为0.1μmol/L，缓冲液中同型半胱氨酸的可靠定量下限为0.5μmol/L，证明具有灵敏性。在同型半胱氨酸浓度为10μmol/L时，批内和批间变异系数均<4%。在0.5至100μmol/L的同型半胱氨酸浓度范围内获得了相似的结果。这些浓度的分析回收率在94.9%至117.0%之间。与迄今为止发表的其他方法相比，这种改进的方法不太复杂，但同样灵敏且可重复，并且能够在常规条件下快速测定人血浆中的总同型半胱氨酸和半胱氨酸。
