A study of the measurement method of gibberellic acid in sweet cherry

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 inflower 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 insweet 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 ac⁃curately 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 suspensionwas 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 centrifugeswere combined, which was then evaporated to dryness in a rotary evaporator at 45 ℃. The residue was dis⁃solved 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 wasrepeated 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 for1 min. The suspension was centrifuged for 10 min at 4 000 r·min^-1. The phosphate buffer solution layerwas 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 a0.45 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 meth⁃od 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 columnand a mobile phase consisting of 0.1%(v/v) formic acid 10 mmol·L^-1 ammonium formate solution (A)/withpure 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-tonoiseratio 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 spikedlevels 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 finally applied to determine the level of GA3 in 5 sweet cherry samples. The determination results showedthat, the content of gibberellic acid in the 5 samples was in a range of 21.3-71.1 μg·kg^-1.【Conclusion】Byoptimizing 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 chromatogra⁃phy-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 de⁃tection of trace plant hormones. Gibberellin is one of the plant hormones widely and exists in nature. InJapan, the regular limit of gibberellin in 42 fruits is 0.2 mg·kg^-1 and the maximum residue limit in EUis 5.0 mg·kg^-1. While at present, residue limitation of gibberellin in fruit has not been constituted inChina. It is necessary to strength tracking the residues of growth regulators and set standards of residuelimitations.