固相萃取-液相色谱-串联质谱法测定水稻中17种细胞分裂素

Analysis of 17 cytokinins in rice by solid phase extraction purification and liquid chromatographytandem mass spectrometry

建立了利用固相萃取-液相色谱-串联质谱同时测定水稻中17种细胞分裂素含量的分析方法。水稻样品经冷冻研磨，用甲醇-水（80∶20，v／v）溶液浸提，经聚合物阳离子交换树脂（PCX）纯化，采用 ZORBAX Extend-C18色谱柱，以甲醇和5 mmol／L甲酸铵水溶液为流动相进行梯度洗脱。采用电喷雾正离子模式电离，选择反应监测模式扫描，外标法定量。优化了色谱分离条件，研究了不同提取溶剂对17种细胞分裂素的提取效率，并考察了 PCX 固相萃取柱的纯化效果。结果表明，17种细胞分裂素在线性范围内的相关系数（ r）均大于0.9984，方法检出限为0.01～0.05 ng／g。水稻根、茎和叶基质在0.2、1和5 ng／g 3个添加水平的平均回收率为60.2％～125.4％（ n＝6），相对标准偏差（ RSD）为5.4％～29.7％。在水稻样品中检出5种细胞分裂素，其含量为0.02～0.93 ng／g。本方法纯化效果好、灵敏度高，可用于水稻中多种痕量细胞分裂素的同时分析。

A method was developed for the determination of 17 cytokinins in rice by solid phase extraction and liquid chromatography-tandem mass spectrometry. The rice samples were cryo-genically ground under liquid nitrogen and extracted with methanol-water（80∶20,v/v）at 4 ℃for 16 h. The supernatant was purified on a column packed with polymer cation exchange resin （ PCX）. The samples were transported and eluted on an analytical column ZORBAX Extend-C18 （100 mm×2. 1 mm,1. 8 μm）by methanol and 5 mmol/L ammonium formate aqueous solution. All the analytes were detected in selected reaction monitoring（ SRM）mode under positive elec-trospray ionization（ ESI＋）and quantified by external standard method. The separation condi-tions were optimized in order to achieve the sufficient separation for the several isomers of cytokinins,such as trans-zeatin-7-glucoside（ tZ7G）,trans-zeatin-9-glucoside（ tZ9G）,and trans-zeatin-O-glucoside（ tZOG ）. Moreover,the extraction efficiency of different extraction solvents and clean-up effects of PCX cartridge for each analyte were further investigated. The results showed that the correlation coefficients were not less than 0. 998 4 in the linear range, and the limits of detection were ranged from 0. 01 to 0. 05 ng/g. The mean recoveries of the 17 cytokinins at three spiked levels of 0. 2,1 and 5 ng/g were from 60. 2% to 125. 4% with the rela-tive standard deviations（RSDs）of 5. 4%-29. 7%（n=6）. Finally,five endogenous cytokinins＆nbsp;were successfully quantified in real sample,and their contents were between 0. 02 and 0. 93 ng/g. It means that this method is reliable for quantitative analysis of cytokinins in rice.