Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices.
For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.
Kiffe
Michael
M
Novartis Institutes for BioMedical Research, Metabolism and Pharmacokinetics, CH-4002 Basel, Switzerland. michael.kiffe@novartis.com
Graf
Daniel
D
Trunzer
Markus
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005243:Feces / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008968:Molecular Conformation; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000652:urine*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016482:Urinalysis / Q000379:methods*
D004364:Pharmaceutical Preparations