Characterization of gelsevirine metabolites in rat liver S9 by accurate mass measurements using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Gelsemium elegans Benth. belongs to the family Loganiaceae and is widely distributed in northern America, east Asia, and southeast Asia. It has attracted wide attention for its diverse biological effects and complex architectures. Gelsevirine is one of the major components in G. elegans. Compared with other alkaloids from G. elegans, gelsevirine exhibits equally potent anxiolytic effects but with less toxicity. However, the metabolism of gelsevirine has not been clearly elucidated. METHODS: The metabolism of gelsevirine was investigated using liver S9 fractions derived from rat liver homogenates by centrifugation at 9000 g. A rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS) method was applied to characterize the gelsevirine metabolites. RESULTS: We discovered a total number of four metabolites of gelsevirine. The metabolic pathways of gelsevirine consisted of hydrogenation, N-demethylenation and oxidation in rat liver S9. CONCLUSIONS: This is the first study on the metabolism of gelsevirine. We proposed possible metabolic pathways of gelsevirine. These findings may warrant future studies of the in vivo metabolism of gelsemine in animals.

Identification of sanguinarine metabolites in pig liver preparations by accurate mass measurements using electrospray ionization hybrid ion trap/time-of-flight mass spectrometry.

RATIONALE: Sanguinarine (SA) is currently used in veterinary medicine for animal husbandry as a natural component of feed additive Sangrovit. To date, SA metabolism in food-producing animals has not yet been reported. Therefore, the purpose of the present study was to investigate the metabolism of SA in pig liver microsomes and cytosol. METHODS: The SA incubations mixtures of microsomes and cytosol were processed by trichloroacetic acid (TCA) and acetonitrile. Then, the samples were analyzed using a sensitive and reliable method based on liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC-IT/TOFMS). The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and product ions generated from precursor ions with those of the parent drug. RESULTS: Seven metabolites were identified in pig liver preparations. Dihydrosanguinarine (DHSA, m/z 334) was the main metabolite formed in liver microsomes and the only one in cytosol. One oxidative metabolite and two O-demethylenated metabolites of SA (m/z 320) were found in the TCA-treated microsomal samples. However, SA pseudobase and two additional O-demethylenated metabolites of DHSA (m/z 322) were found only in the acetonitrile-treated microsomal samples. CONCLUSIONS: It was demonstrated that different metabolites of SA were identified depending on the acidic or neural extraction conditions. A metabolic pathway of SA in pig was tentatively proposed based on these characterized metabolites and early reports.

An integrated method for degradation products detection and characterization using hybrid ion trap/time-of-flight mass spectrometry and data processing techniques: Application to study of the degradation products of danofloxacin under stressed conditions.

A new strategy using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography and post-acquisition data mining techniques was developed and applied to the detection and characterization of degradation products of danofloxacin. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Accurate full-scan MS data were acquired in the first run and processed with the combination of extracted ion chromatograms and LC-UV chromatograms. These processes were able to find accurate molecular masses of possible degradation products. Then, the accurate MS/MS data acquired through data-dependent analysis mode in another run facilitated the structural elucidations of degradation products. As a result, a total of 11 degradation products of danofloxacin were detected and characterized using the developed method. Overall, this analytical strategy enables the acquisition of accurate-mass LC/MS data, search of a variety of degradation products through the post-acquisition processes, and effective structural characterization based on elemental compositions of degradation product molecules and their product ions. The ability to measure degradation products via tandem mass spectrometry coupled with accurate mass measurement, all in only two experimental runs, is one of the most attractive features of this methodology. The results demonstrate that use of the LC/MS-IT-TOF approach appears to be rapid, efficient and reliable in structural characterization of drug degradation products.

The metabolism of gelsevirine in human, pig, goat and rat liver microsomes.

Gelsemium is a small genus of flowering plants from the family Loganiaceae comprising five species, three of which, Gelsemium sempervirens (L.) J. St.-Hil., G. elegans Benth and G. rankinii Small, are particularly popular. Compared with other alkaloids from G. elegans, gelsemine, gelsevirine and koumine exhibit equally potent anxiolytic effects and low toxicity. Although the pharmacological activities and metabolism of koumine and gelsemine have been reported in previous studies, the species differences of gelsevirine metabolism have not been well studied. In this study, the metabolism of gelsevirine was investigated by using liver microsomes of humans, pigs, goats and rats by means of HPLC-QqTOF/MS. The results indicated that the metabolism of gelsevirine in liver microsomes had qualitative and quantitative species differences. Based on the results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. Investigation of the metabolism of gelsevirine will provide a basis for further studies of the in vivo metabolism of this drug.

The metabolism of olaquindox in rats, chickens and pigs.

Olaquindox is a growth-promoting feed additive for food-producing animals. Its toxicities were reported to be closely related to the metabolism. To provide the interpretation of toxicities in animals, this study explored the metabolism of olaquindox in rats, chickens and pigs of different genders by qualitative metabolite profiling. Animals were fed olaquindox in an oral dose, and then their urine, plasma, feces, liver, kidney and muscle were collected. Liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used for structural investigation and identification of metabolites. The structures of metabolites were elucidated based on the accurate MS² spectra and comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite. A total of 18, 18 and 16 metabolites of rats, chickens and pigs were identified, respectively. Among the identified metabolites, 8 known metabolites were confirmed as an early study had stated, and 15 metabolites were found for the first time in vivo. The major metabolic pathways of olaquindox were proposed to be N-O reduction and oxidation of hydroxyl to carboxylic acid followed by N-O reduction. The qualitative species difference on the metabolite profiles of olaquindox among the three species was observed. However, metabolite profiles of olaquindox appeared to be qualitatively similar between female and male for the same species. The proposed metabolic pathways of olaquindox in animals will provide comprehensive data to clarify the metabolism of olaquindox among different species, and will give scientific explanation for toxicities and residues of olaquindox.

Metabolites and JAK/STAT pathway were involved in the liver and spleen damage in male Wistar rats fed with mequindox.

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275 mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N→O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change.

ROS mediated cytotoxicity of porcine adrenocortical cells induced by QdNOs derivatives in vitro.

Quinoxaline 1,4-dioxides (QdNOs) derivatives, the potent synthetic antibacterial group used in food-producing animals, are assumed to have pro-oxidant properties. However, how oxidative stress mediated their adrenal toxicity is far from clear. The aim of this study was to assess the ability of three QdNOs, i.e. olaquindox (OLA), mequindox (MEQ), and cyadox (CYA), to produce reactive oxygen species (ROS) and oxidative cell damage in porcine adrenocortical cells. Multiple approaches such as cell activity assay, biochemical detectation, flow cytometry and fluorescent were used to study the integrated role of ROS homeostasis, mitochondrial redox metabolism and cell apoptosis as well as chemical stability of these drugs. The results showed that OLA and MEQ treatment evoked a significant dose and time-dependent cell damage in adrenocortical cells, well CYA displayed much less toxicity. As for the intracellular ROS production, OLA irritated a persistent and utmost release of ROS while MEQ made a similar but weaker reaction. CYA, however, had a short and unstable release of intracellular ROS. On the other hand, quinoxalinine-2-carboxylie acid (QCA), one of the metabolites of OLA and MEQ, did not cause any significant production of ROS and showed relatively lower toxicity than its parents. Moreover, an imbalance in the redox metabolism and mitochondrial membrane damage has been implicated in adrenal toxicity of QdNOs. ROS scavengers partially reversed QdNOs-induced mitochondrial damage, indicating that mitochondria may be a major target and critical for ROS-mediated cell death. In a word, these results suggested that ROS is a key mediator of QdNOs-induced cell death via mitochondria-dependent pathway in adrenocortical cells. The results provide a mechanism approach in understanding the characterize of adrenal damage caused by QdNOs in vitro, which would in turn, help in designing the appropriate therapeutic strategies of these kind of feed additives.