Yan C C, Huxtable R J
Department of Pharmacology, College of Medicine, University of Arizona, Tucson 85724, USA.
J Chromatogr B Biomed Appl. 1995 Oct 20;672(2):217-24. doi: 10.1016/0378-4347(95)00226-9.
The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic gamma-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivatization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and gamma-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at -20 degrees C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic gamma-glutamylcysteine synthetase in the same liver preparation was found to be 4.85 +/- 0.47 nmol min-1 mg-1 protein by the OPA method and 4.42 +/- 0.52 nmol min-1 mg-1 protein by the MB method. GSH concentrations were found to be 90.4 +/- 6.5 nmol/mg protein for the OPA method and 92.5 +/- 3.4 for the MB method.
荧光邻苯二甲醛（OPA）衍生物的反相高效液相色谱分离法已应用于肝脏γ-谷氨酰半胱氨酸和谷胱甘肽（GSH）水平的测定以及产生这些肽的酶的测定。该方法已与使用单溴代双马来酰亚胺（MB）作为衍生剂的测定方法进行了比较。OPA方法具有衍生化速度更快、无需调节pH值、等度分离以及对GSH和γ-谷氨酰半胱氨酸具有选择性的优点。MB方法在衍生化后需要调节pH值并采用梯度洗脱色谱法。MB也没有选择性，会产生所有生物硫醇的荧光衍生物，并且色谱图上的干扰峰更多。基于MB的分析每个样品的成本也大约高60倍。MB在光照下会产生荧光降解产物。OPA加合物在-20℃下储存时可稳定长达10天。OPA检测对样品中12.5 pmol的物质敏感，信噪比为2.5。两种方法相关性良好。通过OPA方法测定同一份肝脏制剂中的肝脏γ-谷氨酰半胱氨酸合成酶为4.85±0.47 nmol min-1 mg-1蛋白质，通过MB方法测定为4.42±0.52 nmol min-1 mg-1蛋白质。OPA方法测得的GSH浓度为90.4±6.5 nmol/mg蛋白质，MB方法测得的为92.5±3.4。
