果树学报2017，Vol.34Issue(10)：1294-1300,7.DOI:10.13925/j.cnki.gsxb.20160298
甜樱桃果实GA3的测定技术研究
A study of the measurement method of gibberellic acid in sweet cherry
摘要
Abstract
[Objective] Some varieties of sweet cherry are not self-fertile,so pollinizer varieties are needed.Inappropriate temperature and rainfall during bloom affect pollination and fertilization,resulting in flower and fruit drop.Plant growth regulator (mainly GA) treatment during full bloom can significantly increase fruit set in cherry.In this study,we developed a method for the determination of gibberellic acid in sweet cherry fruit using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS),which provides support for studying roles of GA in fruit set.[Methods]5.00 g cherry sample was accurately weighed and put into a 50 mL polypropylene centrifuge tube,to which.25 mL acetonitrile and 2.0 g sodium chloride were added.After capped,the sample was vortex mixed for 1 min,and the suspension was centrifuged for 10 min at 4 000 r· min-1.The supernatant acetonitrile layer was transferred into a flask.The extraction procedure was repeated twice and the supernatant acetonitrile layers from the centrifuges were combined,which was then evaporated to dryness in a rotary evaporator at 45 ℃.The residue was dissolved in 10 mL sulphuric acid-water (pH 2.5) solution and added with 20 mL ethyl acetate and transferred into a 50 mL centrifuge tube.The centrifuge tube was then vortex mixed for 1 min before centrifuged for 10 min at 4 000 r· min-1.The ethyl acetate layer was transferred into a flask.The extraction was repeated twice and the ethyl acetate fractions were combined.Then 10 mL phosphate buffer solution (pH 7.5) was added into extracted ethyl acetate extract and shaken vigorously for 30 s before vortex mixed for 1 min.The suspension was centrifuged for 10 min at 4 000 r· min-1.The phosphate buffer solution layer was transferred into a centrifuge tube.The extraction was repeated twice and the extracted phosphate buffer fractions were combined and adjusted to pH 2.5 using sulphuric acid-water (1∶1).Then 20 mL ethyl acetate was added and vortex mixed for 1 min.The suspension was centrifuged for 10 min at 4 000 r· min-1.The ethyl acetate layer was transferred into a flask.The extraction was repeated twice and the ethyl acetate extracts were combined and evaporated to almost dryness in a rotary evaporator at 45 ℃.The dried extract was reconstituted in 10 mL methanol-water (1∶1,v/v),vortex mixed for 60 s,and forced through a 0.45 μm filter.Then the sample solution was analyzed with HPLC-MS/MS with SRM mode.[Results] Initial chromatographic experiments were carried out to verify the possibility of proposing a LC-MS/MS method for the determination of gibberellic acid.In the positive ion mode,the molecular ion peak [M+NH4]+ was determined to be m/z 364.Separation of the analyte was achieved using a Waters Atlantis T3 column and a mobile phase consisting of 0.l％(v/v) formic acid 10 mmol·L-1 ammonium formate solution (A)/with pure methanol (B).The gibberellic acid standard showed a good linearity in the range of 5.0-100.0 μg· L-1 with a correlation coefficient of 0.999 3.Limits of detection (LOD) and quantification (LOQ) at a signal-to-noise ratio of 3 (S/N=3) and 10 (S/N=10),respectively,were estimated to be 1.21 μg· kg-1 and 4.03 μg·kg-1 (sample size of 5.00 g).Method accuracy was evaluated by additive recovery experiments,with the spiked levels set at 0.001 mg·kg-1,0.002 mg·kg-1 and 0.01 mg· kg-1.It was shown that,recovery rate of gibberellic acid under the 3 spiked levels (n=6) was 89.69％,91.84％ and 95.37％,with a relative standard deviation of 7.46％,6.21％ and 4.85％ respectively,and the uncertainty was 0.13 μg· kg-1.The method was fi nally applied to determine the level of GA3 in 5 sweet cherry samples.The determination results showed that,the content of gibberellic acid in the 5 samples was in a range of 21.3-71.1 μg· kg-1.[Conclusion]By optimizing the HPLC chromatography conditions and mass spectrometer conditions,a method was established for the determination of gibberellin acid (GA3) in sweet cherry,using high performance chromatography-tandem mass spectrometry (HPLC-MS/MS).It is characterized by high sensitivity,simple operation,low detection limit,good stability and reliability,which meets requirements for gibberellin residue analysis.The HPLC-MS/MS detection technology also showed good sensitivity and reproducibility for the detection of trace plant hormones.Gibberellin is one of the plant hormones widely and exists in nature.In Japan,the regular limit of gibberellin in 42 fruits is 0.2 mg· kg-1 and the maximum residue limit in EU is 5.0 mg · kg-1.While at present,residue limitation of gibberellin in fruit has not been constituted in China.It is necessary to strength tracking the residues of growth regulators and set standards of residue limitations.关键词
甜樱桃/赤霉酸/高效液相色谱-串联质谱/定性定量检测Key words
Sweet cherry/Gibberellic acid/High performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)/Qualitative and quantitative detection分类
农业科技引用本文复制引用
张倩,贝峰,孙欣,王明林,辛力..甜樱桃果实GA3的测定技术研究[J].果树学报,2017,34(10):1294-1300,7.基金项目
山东省现代农业产业技术体系水果创新团队建设(SDAIT-06-13) （SDAIT-06-13）
山东省农业重大应用技术创新项目——樱桃采后商品化处理设备研制与试验示范 （）
泰安市科技计划重大专项(201340629) （201340629）
