**1. Introduction**

78 Chromatography – The Most Versatile Method of Chemical Analysis

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The study of the metabolic fate of drugs is an essential and important part of the drug development process, research of drug metabolism pathways, drug-drug interactions, drugherb interactions, influence of genetic polymorphisms and other factors that influence the phase I and/or II metabolism of a drug. Different *in vitro* methods, from subcelullar to organ range, and *in vivo* studies are applied for the clarification of drug metabolism. The analysis of metabolites in complex biological matrices is a challenging task therefore several analytical methods for qualification and quantification of drug metabolites are used. Liquid chromatography coupled with mass spectrometry (LC-MS) has become the most powerful analytical tool for screening and identification of drug metabolites in biological matrices. However, adequate sample preparation is a key prerequisite aspect of successful quantitative and qualitative bioanalysis. Different approaches for metabolite quantification in biological samples from direct quantification, indirect quantification through parent drug after metabolite hydrolysis to quantification supported by using response factors between drug and their metabolites are often used. The most frequently used method for quantification is liquid chromatography coupled to different detectors such as mass spectrometer or UV detector. The LC-MS/MS methods are considered as most appropriate for determination of drugs and their metabolites and are also best suited for high throughput analysis. However, in LC-MS/MS assays, matrix effect and selection of suitable internal standards should be adequately addressed.
