How can unknown substances be identified?

The identification of unknown substances in complex samples is one of the greatest challenges in analytical chemistry. Liquid chromatography coupled with high-resolution mass spectrometry (LC/HRMS) has established itself as a powerful method for overcoming this challenge. Using this technique, it is possible to analyze a wide range of compounds without prior knowledge of the target substances. This approach is known as non-target screening (NTS).
 

The NTS forms the basis for the identification of unknown substances. All detectable signals of a sample are recorded and then compared with databases and legal requirements in a data analysis in order to identify potential analytes.

The main steps include

1. Data acquisition: 
   Acquisition of MS and MS/MS data for all detectable compounds.
    
2. Data processing: 
   Use of software tools for extracting and filtering relevant signals.
    
3. Database synchronization: 
   Comparison of the mass spectra obtained with databases of known compounds.

Smart Suspect Screening is an extension of the NTS. Known transformation products are generated and analyzed in the laboratory. This data can then be used to identify similar compounds in environmental samples. This increases the probability of identifying relevant substances while reducing the number of false-positive results.

The identification of unknown substances using LC/HRMS is associated with several challenges:

1. Data complexity: 

   • Large amounts of data: LC/HRMS generates an enormous amount of data, as each sample contains numerous signals and peaks. Processing and interpreting this data requires powerful software and experienced analysts.

   • Signal overlay: In complex samples, signals from different substances can overlap, which makes identification more difficult.
      

2. Data quality: 

   • Blank values: High-resolution systems are sensitive to contamination. Even minor impurities in chemicals and materials can generate signals that distort the analysis.

   • Purity of the materials: Differences in the purity of vials and analytical columns can influence the quality of the results.
      

3. Databases and reference materials:

   • Incomplete databases: The identification of unknown substances requires extensive and well-maintained databases. However, these databases are often incomplete or do not contain all possible compounds.

   • Missing reference materials: For many unknown substances there are no commercially available reference materials, which makes it difficult to confirm the identity.
      

4. Method development: 

   • Optimization of the separation conditions: The choice of mobile and stationary phase as well as the optimization of the gradient are decisive for the separation performance and sensitivity of the method.

   • Sample preparation: Sample preparation must be carried out carefully to ensure a good peak shape and stable retention times.
      

5. Interpretation of the results: 

   • Structure elucidation: The structural elucidation of unknown compounds requires detailed knowledge of fragmentation patterns and chemical properties.

   • False-positive results: The large number of signals can lead to false-positive results. These must be minimized by additional analyses and confirmations.