液相色谱-串联质谱法测定人血浆和全血中内源性还原型谷胱甘肽浓度

Determination of endogenous reduced glutathione in human plasma and whole blood by LC-MS/MS

目的建立快速测定人血浆和全血中还原型谷胱甘肽浓度的液相色谱-串联质谱法。方法以沉淀法快速处理全血及血浆样品,采用阿昔洛韦为内标,SymmetryShieldTM RP18色谱柱,流动相为乙腈-0.1%甲酸水溶液(6∶94,V∶V),流速为0.3 mL·min-1,进样量为10μL;采用电喷雾(ESI)离子源,正离子电离模式,多反应离子监测(MRM):还原型谷胱甘肽,[M+H]+m/z 308.2→179.1;阿昔洛韦,[M+H]+m/z 226.2→152.0。结果还原型谷胱甘肽血浆浓度线性范围为0.050～2.00 mg·L-1和全血浓度线性范围为50.0～500 mg·L-1,血浆和全血中的最低定量限分别为0.050 mg·L-1和50.0 mg·L-1;提取平均回收率在90%以上,批内、批间精密度均小于15%,准确度均在85%～115%范围内。采集的全血样品在4℃可保持1 h稳定,样品处理后在-70℃可保存1 wk左右待测。结论本实验建立的方法操作简单快速、灵敏度高、准确度好,临床应用方便,可用于人血浆和全血中还原型谷胱甘肽浓度的测定。

AIM To establish a quick and specific LC-MS/MS method for measurement of reduced glutathione in human plasma and whole blood. METHODS Plasma and whole blood samples were processed quick deproteinized precipitation with acyclovir as the internal standard （IS） , SymmetryShieldTM RP18 as the column and the mobile phase of acetonitrile -0.1% formic acid （6 ： 94, V/V） at a flow rate of 0.3 mL·min-1 under an incoming volume of 10 μL. ESI was used for MS with positive ionization model. In multiple reaction monitoring （MRM） mode, the [M＋H]^＋ ions of m/z 308.2→179.1 and m/z 226.2→152.0 were used to qualify the reduced glutathione and the IS, respectively. RESULTS The linear plasma and whole blood concentration ranges of reduced glutathione were 0.050 - 2.00 mg·L^-1 and 50.0 - 500 mg·L^-1, with the minimum quantitative limit of reduced glutathione as 0.050 mg·L^-1 and 50.0 mg·L^-1, respectively. The average recovery was above 90%. The precision discrepancy of inter and intra-batch were below 15% with simultaneous accuracy within the range of 85% - 115%. The sample quanlity was stable after been collected for 1 h at 4 ℃ and about one week after pre-processed at -70 ℃. CONCLUSION LC-MS/MS method is simple, quick, high sensitive, excellent accuracy in clinical practical and suitable for clinical monitoring of reduced glutathione in plasma and whole blood.