Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62–25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%–13.0%, and acceptable accuracy with recoveries in the range of 84.0%–119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse.

细胞分裂素（CKs）是一类重要的植物激素，在植物生命的大多数方面都得到了广泛研究。与其他植物激素相似，CK及其糖缀合物是亲水性的。它们在质谱（MS）检测中的电离效率相当差，而它们在反相液相色谱（RPLC）上的保留和分离常常令人不满意。已经为大多数其他植物激素开发了化学同位素标记LC-MS分析方法，从而增强了它们的LC分离度和定量灵敏度。但是，目前尚无化学标记的CK的报告。在这里，我们报告了一种新的化学同位素标记LC-MS分析方法，用于CK碱基及其糖缀合物的一锅衍生化，基于苄基溴及其氘同位素标记试剂对CKs的腺嘌呤骨架进行差异苄基标记。苯甲酰化可改变CK及其糖缀合物的亲水性，从而改善其在RPLC上的保留和分离。所开发的方法显示出更高的灵敏度，因为可以用0.62–25.9 pg / mL范围内的LOD分析CK及其糖缀合物。该方法还显示出良好的日内和日间精度，标准偏差在1.9％–13.0％范围内，并且可接受的精度在84.0％–119.9％范围内。所开发的方法用于定量测定鲜根中的CKs。所开发的方法显示出更高的灵敏度，因为可以用0.62–25.9 pg / mL范围内的LOD分析CK及其糖缀合物。该方法还显示出良好的日内和日间精度，标准偏差在1.9％–13.0％范围内，并且可接受的精度在84.0％–119.9％范围内。所开发的方法用于定量测定鲜根中的CKs。所开发的方法显示出更高的灵敏度，因为可以用0.62–25.9 pg / mL范围内的LOD分析CK及其糖缀合物。该方法还显示出良好的日内和日间精度，标准偏差在1.9％–13.0％范围内，并且可接受的精度在84.0％–119.9％范围内。所开发的方法用于定量测定鲜根中的CKs。从施肥和未施肥田中收集的黄芪。观察到肥料对内源性CKs代谢具有重要影响。因此，监测内源性CK及其代谢物可能有望控制肥料的滥用。