Raggi M A, Mandrioli R, Casamenti G, Musiani D, Marini M
Dipartimento di Scienze Farmaceutiche, Facoltà di Farmacia, Università di Bologna, Italy.
Biomed Chromatogr. 1998 Sep-Oct;12(5):262-6. doi: 10.1002/(SICI)1099-0801(199809/10)12:5<262::AID-BMC744>3.0.CO;2-J.
A simple and sensitive HPLC method is proposed for the determination of glutathione (GSH) in human mononuclear cells, based on the derivatization of the tripeptide with Ellman's reagent. The mobile phase was composed of a mixture of methanol and ammonium formate (10:90 v/v, with a flow rate of 1 mL/min). The stationary phase was a C18 (4.6 microns, 250 x 4 mm) reversed phase column. The detection of GSH was performed at 280 nm, resulting in a neat chromatographic peak at 5.8 min. A calibration curve showed good linearity over the concentration range 3 x 10(-6) - 6 x 10(-5) M, with a satisfactory precision. The method was found to yield a quantitative recovery of glutathione (96%), to be sensitive (down to 30 pmol of glutathione per injection) and to have a high precision (R.S.D.% approximately equal to 2). The proposed HPLC method allows for the separation and quantitation of cysteine and N-acetylcysteine, if present in biological samples. Furthermore, the method allows for the determination of total thiol present in human mononuclear cells.
基于用埃尔曼试剂对三肽进行衍生化，提出了一种简单灵敏的高效液相色谱法用于测定人单核细胞中的谷胱甘肽（GSH）。流动相由甲醇和甲酸铵的混合物（10:90 v/v，流速为1 mL/min）组成。固定相为C18（4.6微米，250×4毫米）反相柱。在280纳米处进行GSH检测，在5.8分钟时得到一个清晰的色谱峰。校准曲线在3×10⁻⁶ - 6×10⁻⁵ M的浓度范围内显示出良好的线性，精密度令人满意。该方法被发现谷胱甘肽的定量回收率为96%，灵敏（每次进样低至30皮摩尔谷胱甘肽）且精密度高（相对标准偏差%约为2）。所提出的高效液相色谱法能够分离和定量生物样品中存在的半胱氨酸和N - 乙酰半胱氨酸。此外，该方法还能测定人单核细胞中存在的总硫醇。
