Residues and dissipation dynamics of phthalanilic acid in apple

Abstract：【Objective】Phthalanilic acid is a new type of plant growth regulator. In recent years, it has been widely used in fruit trees and vegetables. To evaluate the residual level and dissipation dynamics of 20% aqueous solutions of phthalanilic acid in apples, and ensure the safe use of 20% aqueous solutions of phthalanilic acid in apples, the residue test of 20% aqueous solutions of phthalanilic acid on apples was carried out.【Methods】Field trials of 20% aqueous solutions of phthalanilic acid were carried out in research stations at three different locations, including Penglai of Shandong province, Huaibei of Anhui province and Changshun of Guizhou province, in 2017 and 2018. Both dissipation kinetics and residue determination experiments were carried out. The field trials involved a total of 4 treatments that consisted of 3 treatments at different doses and a blank control. There were three two-trees repeated for each treatment. The apple were applied with pesticides during young fruit for the degradation dynamic trials and the final residue trials. The 20% aqueous solutions of phthalanilic acid were evenly sprayed at a dose of 1.5 times of the recommended maximum dosage (375 mg · kg^{- 1} ) once for the degradation dynamic trials by using knapsack sprayer and the blank control area was sprayed with water. The apple samples were collected at the 2nd hour, 1, 3, 7, 14, 21, 28, 35 and 50 day after the application of pesticides. The 20% aqueous solutions of phthalanilic acid were evenly sprayed at 250(the recommended maximum dosage) and 375 mg · kg^-1 (1.5 times of the recommended maximum dosage) once for the final residue trials. The apples were sampled at harvest time after the pesticide application. The collected samples were cut into small pieces that were less than 1 cm. The evenly mixed samples were dispensedinto two sets of 150 g sample by using the coning and quartering method, and loaded into sample bags with proper labeling. All labeled samples were stored at -20±2 ℃ in a low-temperature freezer. We employed the QuEChERS pre-treatment method, where the samples were extracted with 0.1% (φ) formic acid in acetonitrile and purified with PSA and anhydrous magnesium sulfate, followed by gradient elution in Eclipse plus C18 (3.5 μm, 2.1 mm×100 mm) column with mobile phase consisted of formic acid (0.1%, φ) solution-acetonitrile. The instrument used for the analysis was an Agilent 1260-6420 high-performance liquid chromatography- tandem mass spectrometry (HPLC- MS/MS) system equipped with electrospray ionization (ESI) source. The samples were measured in multiple reaction monitoring (MRM) modes. In order to calculate the recovery rate of spike and coefficient of variation (CV), the blank samples of apple were spiked with 0.01, 0.1 and 1 mg · kg- 1 of phthalanilic acid. Each spiked amount was measured in parallel for 5 times. The samples were extracted with method as above described and tested with instrument conditions as described.【Results】The linear correlation of the method was assessed with curves constructed by measuring a series of concentrations of phthalanilic acid (0.005, 0.01, 0.05, 0.1, 0.5, and 1 mg ·L^-1 ) standards in the apple matrix, The concentration was plotted on the horizontal or x-axis whereas its corresponding peak area was plotted on the vertical or y-axis to obtain a linear regression equation (Y=131 806X - 553), the measurement of phthalanilic acid in apple matrices exhibited a linear correlation (R² =0.992 9) within the range of 0.005 mg · kg^-1 to 1 mg ·L^-1 . We employed the matrix-based standard curve to calculate the concentration in the samples from the field in order to obtain phthalanilic acid residue data. When the spiking levels of phthalanilic acid in the samples were 0.01-1.00 mg·kg^-1 , mean recovery values achieved for phthalanilic acid residues ranged from 79 to 91, and the relative standard deviations (RSD) were 2.5%-4.1%. The limit of detection of the analytical method was 2.5×10-11 g, and the limit of quantification was 0.01 mg · kg-1 in apple. This method has a good sensitivity, accuracy and precision that meeting the requirements for pesticide residue detection. We used the method developed to determine the residue dynamics and final residues of 20% aqueous solutions of phthalanilic acid in apple. The relationship between the residual concentration of phthalanilic acid in apple and the time interval after the pesticide application was consistent with the equation describing the first-order kinetics: Ct=C0e- kt. The half-life of phthalanilic acid in apple from Shandong, Anhui, and Guizhou were 3.7- 4.7 days, 4.3- 4.8 days and 5.7- 5.8 days, respectively. The residues of phthalanilic acid in apple gradually reduced over time with up to more than 86.7% of degradation rate at 14 day. The final residue tests showed that 20% aqueous solutions of phthalanilic acid was sprayed for one time at application dose of 250 375 mg · kg^-1 , at harvest,the final residues of phthalanilic acid in apple were all ＜ 0.01 mg·kg^-1 . Phthalanilic acid residue was not detected in apple samples from control areas.【Conclusion】The residues of phthalanilic acid in apple were determined by HPLC-MS/MS, this method has a good sensitivity, accuracy, and precision. The half-lives of phthalanilic acid in apple were 3.7- 5.8 d, phthalanilic acid was rapidly degradated in apples. At harvest, the final residues of phthalanilic acid in apple were all ＜ 0.01 mg·kg^-1 . It is safe to used 20% aqueous solutions of phthalanilic acid at recommended dosage on apple trees.