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Liquid chromatography-mass spectrometry (LC-MS) with generic gradient elution for a large number of chemically different compounds is a common approach in drug development, used to acquire a large amount of data in a short time frame for drug candidates. The analysis with non-optimized parameters however may lead to a poor method performance for many compounds, and contains a risk of losing important information. Here, generic electrospray time of flight (ESI-TOF) MS methods in various pH conditions were tested for 55 chemically diverse compounds (10 acids, 25 bases, 17 neutrals, and 3 amphoterics), aiming to find best analytical conditions for each compound, for studies of in vitro metabolic properties in liver preparations. The effect of eluent pH and elution gradient strength on chromatographic performance and electrospray MS ionization efficiency were examined for each compound. The data are evaluated how well the best generic approach could cover the analysis of test compounds and how many compounds would still need completely different analytical conditions after that. Aqueous mobile phase consisting of 0.05% acetic acid and 5 mM ammonium acetate (pH 4.4) showed the best general suitability for the analyses, showing adequate performance for metabolite profiling for 41 out of 55 compounds either in positive or negative ion mode. In positive ion mode, the main limitation of performance in various pH conditions was generally not the lack of ionization, but rather the poor chromatographic performance (inadequate retention or poor peak shape), suggesting that more emphasis should be put in finding conditions providing best chromatographic performance, rather than highest ionization properties. However, a single generic approach for a large number of different compounds is not likely to produce good results for all compounds. Preferably, at least two or three different conditions are needed for the coverage of a larger number of structurally diverse compounds.

In vitro methods to produce metabolic information have increasingly been applied in toxicity risk assessment. In the current contract project of JRC/ECVAM In vitro-Toxicology Unit, 55 organic chemicals, mostly drugs and pesticides, most belonging to ECVAM/ICCVAM validation compounds, expected to be analyzable by LC-MS technique, were subjected to a feasibility study. The simple experimental setup consisted of one concentration of a chemical (25 muM), enzyme preparation (human or rat liver homogenate or microsomes), a set of cofactors (NADPH, UDPGA, PAPS, GSH), 4 time points (0, 15, 30, 60 min, including cofactor-less tubes). Metabolites produced were analyzed and tentatively identified by LC-MS techniques. Most of the chemicals were metabolized and metabolites were tentatively identified by TOF-MS analysis. For some chemicals, about 10 or even more metabolites were detectable (e.g. thioridazine, verapamil, amitriptyline). Altogether 11 out of 55 did not display any metabolites under the experimental conditions of this study. Regarding the metabolites formed, there were mostly quantitative differences, but about 20 substances displayed also species-dependent qualitative differences, i.e. a major metabolite was formed in one species, but not in the other. For most chemicals, differences between microsomes and homogenates were relatively modest at least in the initial analysis. The results demonstrate that LC-MS approach is feasible and rather efficient in providing useful metabolic data from a simple experimental setup. More complex analyses, e.g. quantitative assessment of differences between species or biological preparations, or in vitro-in vivo extrapolations, require more complex approaches and a collection of appropriate, preferably curated, data bases of in vivo characteristics of the studied chemicals.