Dutov A A, Nikitin D A, Fedotov A A
Biomed Khim. 2010 Sep-Oct;56(5):609-15. doi: 10.18097/pbmc20105605609.
Isocratic HPLC determination of plasma/serum homocysteine and cysteine with separation on reversed-phase column and UV detection at 330 nm is proposed. The mobile phase consist of acetonitrile - 0.05 M citrate-phosphate buffer with pH 2.4 - isopropanol (15:85:1, v/v/v). Full separation of cysteine, cysteamine (IS), glutathione and homocysteine was achieved within less than 10 minutes. Reduction of thiols from disulfides was performed by 1,4-dithioerithreitol, and derivatization by with Ellman's reagent [5'5-dithiobis-(2-nitrobenzoic acid)]. After that plasma/serum, containing derivatives of thiols, is cleared and concentrated on cartridge packed with 10 mg of hypercross-linked polystyrene (Purosep-200). Elution from cartridge is made with water-organic solvent (without evaporation and concentration, but without dilution), as well as waterless solvents (with evaporation and concentration). Simplicity, reproducibility in combination with high cleanliness of extracts and sufficient sensitivity (0.4 ng for homocysteine, 2 ng for glutathione and 0.2 ng for cysteine and cysteamine at a signal/noise ratio > 3), make this method suitable for routine clinical application.
本文提出了一种等度高效液相色谱法，用于测定血浆/血清中的同型半胱氨酸和半胱氨酸，该方法采用反相柱分离，在330 nm处进行紫外检测。流动相由乙腈-0.05 M柠檬酸-磷酸盐缓冲液（pH 2.4）-异丙醇（15:85:1，v/v/v）组成。在不到10分钟的时间内实现了半胱氨酸、半胱胺（内标）、谷胱甘肽和同型半胱氨酸的完全分离。通过1,4-二硫苏糖醇将二硫化物中的硫醇还原，并用埃尔曼试剂[5,5'-二硫代双-(2-硝基苯甲酸)]进行衍生化。之后，含有硫醇衍生物的血浆/血清经过净化，并在装有10 mg超交联聚苯乙烯（Purosep-200）的柱上进行浓缩。用含水有机溶剂（不进行蒸发和浓缩，但不稀释）以及无水溶剂（进行蒸发和浓缩）从柱上洗脱。该方法具有简单、可重复性好、提取物清洁度高以及灵敏度足够（同型半胱氨酸为0.4 ng、谷胱甘肽为2 ng、半胱氨酸和半胱胺为0.2 ng，信噪比>3）等优点，适用于常规临床应用。
