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Characteristics and Differences Between Targeted and Untargeted Metabolomics

Metabolomics is a research tool that comprehensively analyzes biological responses of organisms to internal (genetic) or external (diet, environment, etc.) stimuli. Metabolomics can be divided into targeted metabolomics and untargeted metabolomics based on the research objectives.

Targeted metabolomics involves the multiple analyses of known metabolites, including absolute qualitative and quantitative analysis of the test substance using standard substances. Isotope internal standards are also used to improve sensitivity and accuracy of substance qualitative and quantitative analysis and to reduce false-positive results. However, the coverage of substances is limited. On the other hand, untargeted metabolomics aims to collect as much information on substances as possible and has a more extensive coverage of substances. However, due to the lack of standard substances, it may produce many false-positive signals and lacks absolute qualitative and quantitative data of substances. Today, we will share with you the characteristics and differences between targeted and untargeted metabolomics to help us better understand these two research methods.

Ⅰ. Performance of targeted and untargeted metabolomics analysis methods

1. Performance of targeted analysis methods

Firstly, QqQHILIC and QqQFI were used to analyze 181 metabolites in NIST plasma for targeted analysis, and the accuracy of the method was evaluated by comparing with the reference concentration values in NIST plasma (evaluated by calculating RSD). Compared with the reference values in NIST SRM 1950 plasma, the RSD averages of substances analyzed by QqQHILIC and QqQFI were 7.8% and 10.9%, respectively. From the perspective of precision, both QqQHILIC and QqQFI are suitable for targeted metabolomics analysis. In practical applications, QqQHILIC is generally chosen for targeted metabolomics analysis because it is equipped with a chromatographic column that allows for pre-separation of substances, making experimental operation and data analysis simpler and more convenient than QqQFI.

2. Performance of untargeted analysis methods

Untargeted metabolomics mainly uses UPLC-Orbitrap-MS/MS to analyze three different types of samples (fish liver, fish brain, NIST plasma) and compare different ionization modes (ESI+ and ESI-) and stationary phases (HILIC and C18). The study found that under different ionization modes, ESI+ produces more characteristic ions than ESI-, approximately 133%-145% more, which highlights the fact that ESI+ ionization mode has a larger coverage of substances. Among different stationary phases, it was found that HILIC can produce more ion characteristics than C18, up to 104%-236%, indicating that HILIC has a larger coverage of substances. By combining the analysis of ionization mode and stationary phase, it was found that the combination of ESI+ and HILIC would produce the largest number of chromatographic features.

Ⅱ. Comparison of targeted and untargeted metabolomics methods

By comparing the accuracy of substance detection between targeted and untargeted analysis methods through technical repetition and inter-batch validation experiments of biological samples (NIST plasma, fish liver, fish brain), it was found that almost all substances had higher accuracy in QqQHILIC than in OrbiHILIC, regardless of whether it was technical repetition or inter-batch analysis. Secondly, QqQFI was used to analyze the samples, and the results showed that QqQHILIC had a higher detection rate for target substances than QqQFI. Overall, targeted metabolomics has higher sensitivity and accuracy than untargeted metabolomics, but the latter has a wider coverage of substances.