杨树嫩茎质外体中脯氨酸含量测定方法的建立

Determination of Proline Content in Apoplast of Poplar Stems

采用高效液相色谱法HPLC对微透析液进行痕量脯氨酸的检测,研究了痕量脯氨酸柱前衍生化的方法。采用苯异硫氰酸酯PITC-乙腈溶液和三乙胺-乙腈溶液对透析液中痕量脯氨酸进行衍生化后,通过环己烷萃取,得到透析液中痕量脯氨酸衍生化的最适方法,无需后续处理可直接进样,并确定了HPLC检测条件：岛津Inertsil ODS-SP色谱柱（250 mm ×4.6 mm,5μm）；柱温：31℃；流动相A：0.05 mol·L-1醋酸钠溶液；流动相 B：乙腈-流动相 A（1：1）；洗脱条件：A：B（50：50）；流速：1.0 mL· min-1；检测波长：254 nm；进样量：20μL。脯氨酸加标回收率为95.1%~104.2%。同时,对3种杨树质外体透析液中的脯氨酸进行了测量。

Based on microdialysis experience in apoplast of poplar stems, the trace of proline in plant was detected by using the High -performance liquid chromatography （ HPLC ） method, precolumn derivatization method was studied. Phenyl isothiocyanate PITC- acetonitrile solution and triethylamine acetonitrile solution were applied to derivate the proline. Cyclohexane was used to extract proline, the sample could be directly injected in HPLC without subsequent pro-cessing. The detection conditions were determined： the SHIMADZU Inertsil ODS-SP column （250 mm × 4. 6 mm, 5 μm）;column temperature：31 ℃;mobile phase A：0. 05mol·L-1 so-dium acetate solution, mobile phase B： acetonitrile-mobile phase A （1：1）; Elution condi-tions：A：B （50：50）;flow rate：1. 0 mL·min-1;detection wavelength：254nm;the injection volume was 20μL. The recovery rate of proline was 95. 1 -104. 2%. At last the proline con-tents of three poplar apoplast dialysis were detected by this method.