Metabolic profiling of pharmaceuticals

Metabolic transformation of small molecules

Metabolism is the enzymatic conversion of one chemical compound into another (= metabolite). During metabolic transformation, polar bodies are either introduced or unmasked, which results in (more) polar metabolites of the original chemicals. The modification of xenobiotics facilitates their removal from the body. When metabolites are pharmacologically inert, metabolism deactivates the administered dose of parent drug and this usually reduces the effects on the body. Metabolites may also be pharmacologically active, sometimes more so than the parent drug.

Identification of metabolites using mass spectrometric methods

Beside other techniques, mass spectrometry (MS) plays an invaluable role in the elucidation of biotransformation pathways of drugs by identifying circulatory and excretory metabolites. Especially liquid chromatography (LC) hyphenated to electrospray ionization (ESI) MS has become a powerful tool for the rapid detection, structure elucidation, and quantification of drug-derived species within various biological specimens. LC enables the cleanup and fraction of compounds prior to mass spectrometric analysis. Thus, adverse effects on the mass spectrometric detectability of metabolites caused by coionization with matrix compounds or transformation products are reduced which facilitates the identification of possible metabolites. Furthermore, based on varying chromatographic properties LC can be used to separate and thus to identify isomeric species. MS can be operated in different scan modes. For targeted approaches varying combinations of product ion, precursor ion and neutral loss scans are applied to screen a sample for predicted metabolic transformation products. Targeted analysis is specific and sensitive, but suffers from the problem that uncommon or unexpected transformation pathways might be ignored. Full scan MS represents a more versatile acquisition strategy which should enable the detection of most ionisable metabolites. Low- and high-resolution MS can be applied for the detection of transformation products by analyzing test and control samples. For structure elucidation of tentative metabolites full scan tandem mass spectrometric (MS/MS) data is acquired and interpreted.

Use of similarity search to prove relatedness between a metabolite and the precursor drug

For structure elucidation of tentative metabolites full scan tandem mass spectrometric (MS/MS) data is acquired and interpreted. Typically, a lot of expert knowledge in gas-phase fragmentation chemistry is necessary to unequivocally prove the structural relatedness of a metabolite and its precursor drug. Thus, metabolite identification is often a difficult to accomplish and time consuming process. Alternatively, the 'similarity search' approach for the fast and unequivocal differentiation between metabolites and non-drug-related species. 'Similarity search' relies on the match of MS/MS spectra of a putative metabolite to a spectral library. The library does not contain any spectrum specific for a metabolite. Thus, direct identification of a metabolite via 'identity search' is impossible. However, the database system can be used to find entries which possess structures or structural features that are similar to that of the query compound. Typically, only selected parts of a compound's structure are changed in the course of a metabolic transformation process. Thus, a large number of identical fragment ions might be observed in the fragment ion mass spectra obtained from a compound and its metabolite. Only those fragment ions that contain the transformed moiety will differ in mass. Library search with a tandem mass spectrum of a metabolite often yields a high score for the corresponding precursor drug. Conversely, if for a tandem mass spectrum of a putative metabolite the parent drug or a similar compound is obtained as library search result structural relatedness between the tentative metabolite and the drug would have been proven.

Unravelling the metabolic transformation of tetrazepam to diazepam

The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C16H17ClN2O, average mass: 288.772) were studied by analyzing human plasma and urine samples collected from healthy volunteers. Accurate molecular mass measurements in full scan mode were used to survey the collected samples for putative metabolic transformation products. Full scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to our tandem mass spectral library to prove the structural relatedness of a tentative metabolite to tetrazepam. The performed 'similarity search' approach represented a rapid and powerful tool to exclude non-drug-related species even without knowledge of fragmentation chemistry. Only for the elucidation of the site of transformation interpretation of tandem mass spectrometric data was necessary. Possible metabolic routes from tetrazepam to diazepam (C16H13ClN2O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to the common doctrine this distinct transformation reaction might be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.

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