Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS.

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-((14)C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs.

A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary.

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry.

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-D-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.