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• In vivo SPME-based methods are developed to determine neonicotinoids in bananas. • The R s values depend on the sample matrix, sampling durations, and individuals. • Equilibrium calibration shows better stability, repeatability and sensitivity. • Equilibrium calibrated in vivo SPME is superior to the QuEChERS pretreatment. • It is a talented on-site strategy for detection of pesticides in undamaged samples. In vivo solid-phase microextraction (in vivo SPME) is an emerging fascinating sample pretreatment technique, but its quantitative correction method is different from the traditional correction methods, which has become a bottleneck limiting its development. At present, the sampling-rate calibration and equilibrium calibration are mainly used, however, their characteristics and applicability are not clear. In this study, the sampling-rate calibration and equilibrium calibration were evaluated in the case of the determination of neonicotinoids in bananas by in vivo SPME. The factors that affect the sampling rate (R s), such as the matrix states, sampling durations, and individual differences were studied, and they all had impacts on R s. Conversely, the equilibrium distribution coefficient (K fs) remained constant after extraction equilibrium and the individual differences were smaller. The highest accuracy and precision were achieved by equilibrium calibration, and the relative recoveries were in the range of 83.2 %-104.3 % with the relative standard deviations below 8.1 % compared to a standard QuEChERS-based method. The lower limits of quantification for 4 neonicotinoids in bananas were below 5 ng g−1, lower than the standard method and the maximum residue levels in China and the European Union. This work clarifies the characteristics, rules and performance of the sampling-rate calibration and equilibrium calibration, which is of crucial importance for the development and application of in vivo SPME. The developed method is convenient, sensitive, and accurate for the determination of pesticide residues, which is of great significance to guide the safe use of pesticides in the field and prevent products with excessive pesticide residues from entering the market. [ABSTRACT FROM AUTHOR]