The variation of qualitative information among different types of mainstream hyphenated instruments of ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) makes data sharing and standardization, and further comparison of results consistency in metabolite annotation not easy to attain. In this work, a quantitative study of correlation and difference was first achieved to systematically investigate the variation of retention time (tR), precursor ion (MS1), and product fragment ions (MS2) generated by three typical UPLC-HRMS instruments commonly used in metabolomics area. In terms of the findings of systematic and correlated variation of tR, MS1, and MS2 between different instruments, a computational strategy for integrated metabolite annotation was proposed to reduce the influence of differential ions, which made full use of the characteristic (common) and non-common fragments for scoring assessment. The regular variations of MS2 among three instruments under four collision energy voltages of high, medium, low, and hybrid levels were respectively inspected with three technical replicates at each level. These discoveries could improve general metabolite annotation with a known database and similarity comparison. It should provide the potential for metabolite annotation to generalize qualitative information obtained under different experimental conditions or using instruments from various manufacturers, which is still a big headache in untargeted metabolomics. The mixture of standard compounds and serum samples with the addition of standards were applied to demonstrate the principle and performance of the proposed method. The results showed that it could be an optional strategy for general use in HRMS-based metabolomics to offset the difference in metabolite annotation. It has some potential in untargeted metabolomics.