柱前衍生化结合气质联用同时测定糖尿病肾病大鼠血浆中18种氨基酸含量

Simultaneous determination of 18 amino acids in the plasma of diabetic nephropathy rats using pre- column derivation coupled with GC -MS

目的 建立同时测定大鼠血浆中18种氨基酸的柱前衍生化结合气质联用（gas chromatography-mass spectrometry, GC-MS）分析方法。方法 血浆用氯甲酸乙酯衍生化后GC-MS分析，条件如下：Agilent HP-5MS UI（60 m × 0.25 mm × 0.25 μm）柱为固定相；程序升温；进样口温度：240℃；不分流模式进样；载气为高纯氦气，流速设为1.0 mL·min^-1。采用电子轰击离子源；选择离子监测模式；离子源温度230℃；电子能量70 eV；溶剂延迟时间5.45 min。结果 18种氨基酸得到较好分离, 且在1.6~2400 μmol·L-范围内其浓度与响应值具有良好的线性关系（r2＞0.99）；批内及批间精密度和准确度（RSD）均小于10%；样品平均加样回收率及基质效应分别在72.17%~111.4%和90.42%~114.6%之间；血浆室温放置8h、-80℃放置28d，血浆衍生化后4℃放置12h、-20℃放置24h均稳定；和正常大鼠相比，除组氨酸和脯氨酸外，糖尿病大鼠血浆中其他16种氨基酸含量均发生了显著变化。结论 该方法简便易操作、准确度高、特异性好，而且该衍生化产物稳定、线性范围宽、基质效应小, 可用于正常及糖尿病大鼠血浆中氨基酸的测定。

Objective To establish a method of pre - column derivation coupled with gas chromatography - mass spectrometry （ GC - MS） for simultaneous determination of 18 amino acids in the plasma of diabetic nephropathy rats. Methods Rat plasma samples were determined by GC - MS after being derived using ethyl chloroformate under the flowing conditions. The chromatographic column was Agilent HP-SMS UI （60 m × 0.25 mm× 0.25 μm）. The injection mode was in splitless mode and the injection port temperature was 240℃. He gas was used as the carrier gas and the flow rate was 1 mL/min. The EI source interface was operated and the detector voltage was 70 eV. The solvent delay time was 5.45 min. All the analytes were quantified in the selected ion monitoring mode. Results 18 amino acids in rat plasma were well separated and the compounds within the range of 1.6 - 2 400 μmol · L - 1 can be detected with a good linear response correlation coefficient （ r2 〉 0. 9985 ）. The relative standard deviation （ RSD ） values of intra - batch and inter - batch precision and accuracy were all less than 10%. The average recoveries and matrix effects of samples were in the range of 72.17% - 111.4% and 90.42% - 114.6% , respectively. The plasma samples were stable for 8 h at room temperature and 28 days at -80℃. The derivatized samples were stable for 12 h at 4℃ and 24 h at -20℃. Remarkable changes were found in the content of all amino acids in the plasma of diabetic nephropathy rats, except histidine and proline, compared with normal rats. Conclusions The method developed is simple and easy to use with good accuracy and specificity. The derivatized product is stable with a wide linear range and low matrix effects. This method can be used for determination of plasma amino acids in normal and diabetic nephropathy rats.