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High-performance liquid chromatography was used in combination with infrared ion spectroscopy for the identification of positional isomers of hydroxy-atorvastatins, the primary metabolites of the drug atorvastatin. The results demonstrate the direct applicability of infrared ion spectroscopy in the field of drug metabolism and, more generally, its promising role in state-of-the-art analytical laboratories for the identification of small molecules buried in complex mixtures. In combination with chromatographic separation, infrared spectroscopy of mass-selected ions provides a promising new route for the identification of the molecular structures of unknown m/z peaks in a mass spectrum. We demonstrate that currently existing experimental protocols allow the measurement of an IR spectrum from less than 10 ng of sample obtained in a collected HPLC fraction.

Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable “heteroelement” (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups....

During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel ((14)C or (3)H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisible, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach. However, the method lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with "reverse" online isotope dilution ("reverse" online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a (81)Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and "mixed" species, based on their isotopic "fingerprint". A good repeatability (relative standard deviation of 4.2%) and limit of detection (0.35 mg of drug compound L(-1) of feces extract), of the same order of magnitude as those observed for "normal" online ID HPLC/ICP-MS and HPLC/radiodetection, were achieved.