Selectivity of substance identification by HPLC-DAD in toxicological analysis using a UV spectra library of 2682 compounds

The UV spectra and relative retention times (RRT) of 2682 toxicologically relevant substances were measured by high-performance liquid chromatography with diode array detection (HPLC-DAD) in an acetonitrile phospate buffer (pH 2.3) mixture on an RP8 column and were arranged in a database. A complete survey of the molecular structures of all database entries showed the presence of 1650 different chromophores or chromophore combinations. The specificity of the UV spectrum for substance identification was determined by calculation of the similarity indices (SI) of all possible substance pairs within the database with an SI > 0.9990, which indicated spectral identity. In a similar way, the RRT was evaluated for all possible pairs: two compounds were declared indistinguishable because the RRT of at least one of them fell into the RRT error window of the other. Although the use of the RRT alone produced unsatisfactory identification results, 1619 substances (60.4%) were unambiguously identified by their UV spectrum only. This rate was increased to 84.2% by the combination of spectrum and RRT. The selectivity parameters discrimination power (DP) and mean list length (MLL) were calculated (DP = 0.9999, MLL = 1.253) and compared with literature data, which proved HPLC-DAD to be one of the most reliable methods for substance identification in toxicological analysis. The practical relevance of the results for systematic toxicological analysis is demonstrated by the example of a multidrug intoxication and in the context of sample preparation methods routinely used.