Identification of the in vitro and in vivo metabolites of chrysotoxine using liquid chromatography combined with benchtop orbitrap high resolution mass spectrometry

Identification of the in vitro and in vivo metabolites of chrysotoxine using liquid chromatography combined with benchtop orbitrap high resolution mass spectrometry | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 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Data may be preliminary. 1 August 2025 V1 Latest version Share on Identification of the in vitro and in vivo metabolites of chrysotoxine using liquid chromatography combined with benchtop orbitrap high resolution mass spectrometry Authors : Chengzhen Guan , Yiqiang , and Meiling Wan 0009-0005-2434-7759 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175403368.80902379/v1 Published Rapid Communications in Mass Spectrometry Version of record Peer review timeline 232 views 159 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rationale: Chrysotoxine, a bibenzyl derivative from the stems of Dendrobium medicinal herbs, has recently emerged as a promising therapeutic candidate for cervical cancer. This study aimed to characterize chrysotoxine metabolites across multiple hepatocyte species and in rat urine. Methods: Metabolites were identified and characterized using liquid chromatography coupled with benchtop Orbitrap high-resolution mass spectrometry (LC–Orbitrap–MS/MS). Structural elucidation relied on accurate mass measurements (mass error <5 ppm) and comprehensive MS 2 fragmentation pattern interpretation. Results: Twelve distinct metabolites were structurally identified. Among these, M4, M6, M7, M8, M10, M11, and M12 are newly reported. Metabolic transformations occurred via five principal pathways: hydroxylation, demethylation, glucuronidation, sulfation, and glutathione (GSH) conjugation. Cross-species analysis of hepatocytes revealed direct glucuronidation as the predominant metabolic reaction. Urinary excretion profiles in rats identified hydroxylated ( M9 ) and glucuronidated ( M11 ) metabolites as the major elimination products. During the metabolism, chrysotoxine can be metabolized into quinone methide and ortho quinone intermediates that can be conjugated with GSH, forming the adducts M1, M2, M3, and M5. Conclusions: This study delineates chrysotoxine metabolites in vitro and in vivo , providing critical insights for further pharmacokinetic and toxicity assessments. Identification of the in vitro and in vivo metabolites of chrysotoxine using liquid chromatography combined with benchtop orbitrap high resolution mass spectrometry Chengzhen Guan 1 ( Email: [email protected] ), Yiqiang An 2 ( Email: [email protected] ), Meiling Wan 1 ( Email: [email protected] ) 1. Department of Pharmacy, the Affiliated Hospital of Xuzhou Medical University, No. 99, Huaihai Road, Xuzhou 221006, China 2. The Health Policy Research Institute of Xuzhou Medical University, No. 209 Tongshan Road, Xuzhou 221004, China, Meiling Wan, Department of Pharmacy, the Affiliated Hospital of Xuzhou Medical University, No. 99, Huaihai Road, Xuzhou 221006, China Email: [email protected] ABSTRACT Rationale: Chrysotoxine, a bibenzyl derivative from the stems of Dendrobium medicinal herbs, has recently emerged as a promising therapeutic candidate for cervical cancer. This study aimed to characterize chrysotoxine metabolites across multiple hepatocyte species and in rat urine. Methods: Metabolites were identified and characterized using liquid chromatography coupled with benchtop Orbitrap high-resolution mass spectrometry (LC–Orbitrap–MS/MS). Structural elucidation relied on accurate mass measurements (mass error <5 ppm) and comprehensive MS 2 fragmentation pattern interpretation. Results: Twelve distinct metabolites were structurally identified. Among these, M4, M6, M7, M8, M10, M11, and M12 are newly reported. Metabolic transformations occurred via five principal pathways: hydroxylation, demethylation, glucuronidation, sulfation, and glutathione (GSH) conjugation. Cross-species analysis of hepatocytes revealed direct glucuronidation as the predominant metabolic reaction. Urinary excretion profiles in rats identified hydroxylated ( M9 ) and glucuronidated ( M11 ) metabolites as the major elimination products. During the metabolism, chrysotoxine can be metabolized into quinone methide and ortho quinone intermediates that can be conjugated with GSH, forming the adducts M1, M2, M3, and M5. Conclusions: This study delineates chrysotoxine metabolites in vitro and in vivo , providing critical insights for further pharmacokinetic and toxicity assessments. Keywords: Dendrobium, chrysotoxine , metabolite characterization, LC-Orbitrap-MS/MS, 1. Introduction Shi-Hu is one of the well-known herb medicines derived from different species of Dendrobium , including D. chrysotoxum Lindl., D. nobile Lindl., and D. fimbriatum Hook. This herb medicine has been reported to exhibit antioxidant, immunomodulatory, anticancer, and antisenescence activities [1]. Bibenzyl derivatives such as moscatilin, erianin, gigantol and chrysotoxine were identified as the most predominant bioactive compounds contributing to its pharmacological effects [2, 3]. For example, moscatilin, has been reported to have anti-inflammatory [4] and anti-cancer [5, 6] effects. In recent years, chrysotoxine has attracted our research interest due to its anti-cancer activity. Chrysotoxine effectively suppresses the cell proliferation, migration, invasion and apoptosis in cervical cancer through regulating ferroptosis and the PI3K/AKT/mTOR pathway [7]. Chrysotoxine can also inhibit 6-hydroxydopamine induced apoptosis in SH-SY5Y cell via mitochondria protection and NF- κ B modulation and it serves as a candidate in protection against neurodegeneration in Parkinson’s disease [8]. Drug metabolism, which involves both qualitative and quantitative analysis of metabolites in biological matrices, plays a pivotal role in drug discovery and development. It would be meaningful to know the metabolic fate for better understanding on when, where and how a drug can be effective. Current research emphasizes that certain metabolites exhibit pharmacological activity or toxicity, thereby contributing to the overall therapeutic and adverse effects of a drug [9, 10]. Notably, highly reactive metabolites capable of covalent binding to macromolecules have been implicated in idiosyncratic adverse drug reactions [11], as exemplified by the case of sitaxentan [12]. Early identification of such reactive metabolites enables strategic intervention to mitigate their formation during drug development. Hepatocytes, which retain most hepatic drug-metabolizing enzymes, have emerged as a physiologically relevant in vitro model for metabolic studies. Given the interspecies variations in drug metabolism due to differences in enzyme expression and activity, comprehensive characterization of human metabolic pathways early in development is essential for effective therapeutic design. The structural elucidation of unknown metabolites remains challenging due to unpredictable biotransformation pathways. Modern analytical strategies employing liquid chromatography coupled with high-resolution Orbitrap mass spectrometry (LC-Orbitrap-MS/MS) have significantly enhanced metabolite identification capabilities through accurate mass measurements and MS 2 spectral data analysis [13]. Data post-processing techniques—including background subtraction, mass defect filtering (MDF), and extracted ion chromatogram (EIC)—effectively improve metabolite chromatographic peak identification in biological samples by selectively removing the majority of interfering peaks [14]. Combining structure-related accurate mass measurements with fragmentation pattern interpretation enables the precise localization of metabolic reaction sites. Hence, the aim of this work was to 1) investigate in vitro metabolism of chrysotoxine across five species (mouse, rat, dog, monkey, and human) using hepatocyte models and in vivo metabolism after oral administration at 20 mg/kg; 2) to identify the metabolites using advanced LC-Orbitrap-MS/MS combined with multiple data processing approaches; 3) to propose the metabolic pathways of chrysotoxine. The resulting interspecies metabolic map of chrysotoxine provides critical insights for rational drug development and enhances our understanding of its pharmacological behavior across preclinical models and humans. 2. Materials and methods 2.1. Chemicals and Reagents Chrysotoxine (purity >98%) and moscatilin (purity Ltd. (Shanghai, China). Cryopreserved hepatocytes from CD-1 mice, Sprague-Dawley rats, beagle dogs, cynomolgus monkeys, and humans were obtained from Bioreclamation IVT (Baltimore, MD, USA). HPLC-grade acetonitrile was supplied by Tedia Company Inc. (Fairfield, OH, USA). Ultrapure water was generated using a Milli-Q Integral water purification system (Millipore, Billerica, MA, USA). All other analytical-grade chemicals and reagents were commercially available. 2.2. Analytical conditions Chromatographic separation was performed using a Thermo Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a quaternary pump, online degasser, auto-sampler, and temperature-controlled column compartment. Analytes were resolved on a Waters ACQUITY BEH C 18 column (2.1 × 100 mm, 1.7 μm; Waters Corporation, Milford, MA, USA) maintained at 40°C. The mobile phase consisted of (A) aqueous 0.1% formic acid and (B) acetonitrile, delivered at 0.3 mL/min with the following gradient program: 5% B (0–1 min), 5–30% B (1–3 min), 30–65% B (4–6 min), 65–90% B (6–8 min), isocratic 90% B (8–10 min), and re-equilibration to 5% B (0–12 min). Mass spectrometric detection was conducted using a Q-Exactive Orbitrap system (Thermo Fisher Scientific) with heated electrospray ionization (HESI) in positive ion mode. Source parameters were optimized as follows: spray voltage, 3.0 kV; sheath gas, 40 arbitrary units (arb); auxiliary gas, 10 arb; S-lens RF level, 50 V; and capillary temperature, 300°C. Full-scan MS coupled with data-dependent MS² (dd-MS²) acquisitions was implemented with stepped collision energies of 10, 15, and 18 eV. Mass spectra ( m/z 50-1000 Da) were recorded in centroid mode with a resolution of 70000 FWHM for MS 1 and 17500 FWHM for MS 2 . Instrument operation and data collection were managed using Xcalibur™ software (version 4.3, Thermo Fisher Scientific), while metabolite characterization was executed via Compound Discoverer TM software (version 3.2, Thermo Fisher Scientific). 2.3. Animal experiment and in vivo metabolism Six male Sprague-Dawley rats (190–210 g) were obtained from the Laboratory Animal Center of Anhui Medical University (Hefei, China). The animals were housed in a specific pathogen-free (SPF) breeding room under controlled environmental conditions (temperature: 25 ± 1°C; humidity: 55–65%; 12 h light/dark cycle), and were fed with standardized rodent chow and filtered water ad libitum . Before drug administration, the animals were fasted for 12 h but free access to water. The study adhered to the Chinese National Standards for Laboratory Animal Welfare (GB/T 35892-2018) and was approved by the Institutional Animal Care and Use Committee (IACUC) of Anhui Medical University (Hefei, China). Rats were randomly assigned to the control and the treatment groups (n = 3 for each group) using metabolic cages. Chrysotoxine formulated in 0.5% aqueous Tween-80 was orally administered to rats at a single dose of 20 mg/kg for the treatment group and 0.5% aqueous Tween-80 for the control group. Urine samples were collected cumulatively over the following intervals: 0–4, 4–8, and 8–24 h. All bio-samples were stored at −80°C pending further analysis. 2.4. In vivo sample pretreatment Urine metabolites were extracted using a methanol: acetonitrile (1:1, v/v ) solvent system. For each sample, 400 μL of pooled biological matrix was combined with 1.2 mL of ice-cold extraction solvent. Following thorough vortex mixing (2 min) to achieve complete protein precipitation, the mixture was centrifuged at 25,000× g for 10 min at 4°C. The resulting protein-free supernatant was quantitatively transferred and evaporated to dryness under a controlled nitrogen stream at 40 °C. Dried extracts were reconstituted in 150 μL of acetonitrile/water (1:3, v/v ) with 30 s of vortex agitation prior to LC-MS analysis. 2.5. Incubation with hepatocytes A chrysotoxine stock solution (50 mM) was prepared in methanol. Hepatocyte incubation systems (200 μL) were established in Williams E medium containing 20 μM chrysotoxine and 2×10 6 cells/mL hepatocytes, with organic solvent content maintained below 0.5% ( v/v ). Blank controls were prepared using equivalent medium without chrysotoxine. Incubations proceeded at 37°C in a humidified 5% CO 2 atmosphere. Reactions were terminated at 0 and 2 h by adding 600 μL ice-cold acetonitrile. Samples were centrifuged (25,000× g , 15 min, 4°C), and supernatants were transferred, evaporated under nitrogen gas at 40°C, and reconstituted in 150 μL acetonitrile/water (1:3, v/v ). After re-centrifugation (25,000× g , 15 min), 2 μL aliquots were submitted to LC-MS for analysis. 3. Results and discussion 3.1. LC-Orbitrap-MS/MS analysis of chrysotoxine Metabolite identification necessitates comprehensive characterization of the parent compound’s chromatographic and mass spectrometric properties. Under the established analytical conditions, chrysotoxine was eluted at 9.11 min, exhibiting a protonated molecular ion [M+H]⁺ at m/z 319.1541 (mass error: 0.3 ppm) corresponding to the molecular formula C 18 H 23 O 5 . MS 2 fragmentation of this ion generated four diagnostic fragment ions (Figure 1a) . The fragments at m/z 151.0755 (C 9 H 11 O 2 ) and 167.0701 (C 9 H 11 O 3 ), were originated from the breakage of benzyl C–C bond. The fragment at m/z 181.0860 (C 10 H 13 O 3 ) was formed via loss of the di-methoxyphenyl group and the fragment at m/z 165.0916 (C 10 H 13 O 2 ) was derived from 2,6-dimethoxyphenol moiety elimination. The proposed fragmentation pathways (Figure 1b) demonstrated these diagnostic cleavages. Fragmentation of the parent drug based on HRMS afforded valuable structure-related diagnostic ions, enabling efficient metabolite profiling by providing comparative benchmarks for spectral interpretation. Through systematic comparative analysis of the fragment ion profiles between the parent compound and its metabolites, the sites of biotransformation could be logically inferred with enhanced confidence. 3.2. Characterization of chrysotoxine metabolites by LC-Orbitrap-MS/MS The LC-Orbitrap-MS/MS data were analyzed using Compound Discoverer and Xcalibur software. Figure 2 displays the metabolic profiles of chrysotoxine derived from in vitro hepatocyte incubations and in vivo rat urine samples collected after a single-dose administration (20 mg/kg). Table 1 comprehensively catalogs the structural and analytical characteristics of the 12 metabolites identified across both biological matrices. Comparative metabolites analysis with blank controls revealed chrysotoxine-derived metabolites originating from six distinct biotransformation pathways: (i) GSH conjugation (M1, M2, M3 and M5); (ii) direct glucuronidation (M11); (iii) hydroxylation (M9); (iv) sequential hydroxylation-glucuronidation (M4, M7 and M10); (v) sulfation (M12); and (vi) demethylation coupled with glucuronidation (M6 and M8). Notably, the glucuronide conjugate M11 constituted the predominant metabolite across all experimental systems. Structural elucidation of metabolites was achieved through multi-parameter validation, including derivation of molecular formulas (<5 ppm mass error), diagnostic fragment ion patterns via MS 2 , and comparative spectral alignment with the parent compound. Critically, the absence of these metabolites in vehicle-treated controls confirmed their enzymatic origin, excluding non-enzyme degradation. 3.2.1 Metabolite M1 M1 eluted at 3.29 min and exhibited a protonated molecular ion [M+H]⁺ at m/z 474.1545, corresponding to the tentative molecular formula C 19 H 28 N 3 O 9 S + (mass error: 0.8 ppm). Its MS² fragmentation profile (Figure 3a) revealed two diagnostic ions: m/z 345.1117 and 167.0704, generated through neutral losses of the glutamyl moiety (-129 Da) and GSH, (-307 Da), respectively. Additionally, characteristic GSH-derived fragment ions were observed at m/z 308.0913, 233.0581, and 179.0487, consistent with established GSH fragmentation patterns [15, 16]. Based on these structural hallmarks, M1 was conclusively identified as a GSH conjugate. 3.2.2. Metabolites M2 and M5 M2 and M5, eluting at 4.45 and 4.75 min respectively, shared an identical protonated molecular ion [M+H]⁺ at m/z 624.2228, corresponding to the empirical formula C 28 H 38 N 3 O 11 S + (mass error: 1.0 ppm). This molecular signature confirmed both metabolites as GSH conjugates. The MS 2 spectrum of M2 (Figure 3b) featured a diagnostic ion at m/z 317.1379, arising from neutral loss of the GSH moiety (-307 Da), alongside canonical GSH-derived fragments at m/z 308.0910, 233.0589, and 179.0485, consistent with established fragmentation pathways [15, 16]. In contrast, M5 exhibited a distinct fragmentation pattern (Figure 3c) , yielding two diagnostic ions at m/z 549.1903 and 495.1799 via neutral loss of glycinyl (-75 Da) and glutamyl (-129 Da) moieties, respectively. Notably, the presence of the ion at m/z 211.0423 confirmed GSH adduction at the 2,6-dimethoxyphenol substructure of chrysotoxine. 3.2.3. Metabolite M3 M3, eluted at 4.61 min, exhibited a protonated molecular ion [M+H] + at m/z 610.2065. Its chemical formula was postulated to be C 27 H 36 N 3 O 11 S + (mass error: -1.0 ppm), indicating a metabolic pathway involving demethylation followed by GSH conjugation. MS 2 spectrum (Figure 4a) displayed two GSH-associated neutral losses: glycinyl (-75 Da) and glutamyl (-129 Da), confirming the GSH conjugation motif. The diagnostic fragment ins at m/z 197.0268 (C 9 H 9 O 3 S + , mass error: 0.5 ppm) and 151.0753 (C 9 H 11 O 2 + , mass error: -0.7 ppm) demonstrated the demethylation followed by GSH conjugation at 2,6-dimethoxyphenol substructure of chrysotoxine. 3.2.4. Metabolites M4, M7 and M10 M4, M7 and M10 eluted at 4.65, 4.90, and 5.15 min, respectively. All three metabolites exhibited an identical ammonium adduct ion [M+NH 4 ] + at m/z 528.2078 (C 24 H 34 NO 12 + , mass error : 0.8 ppm), suggesting a metabolic pathway involving hydroxylation followed by glucuronidation. M7 and M10 showed the same MS 2 spectrum (Figure 4b) , which displayed a characteristic glucuronide-associated neutral loss of -176 Da (glucuronyl group), yielding a fragment ion at m/z 352.1754 (C 18 H 26 NO 6 + , mass error: 0.0 ppm). The diagnostic ion at m/z 197.0812 (C 10 H 13 O 4 + , mass error: -3.0 ppm) demonstrated hydroxylation at 2,6-dimethoxyphenol substructure of chrysotoxine. Additional ions at m/z 165.0909 and 151.0751 suggested the intact of di-methoxyphenyl moiety. MS 2 spectrum of M4 (Figure 4c) featured two key ions at m/z 181.0855 and 167.0.696, consistent with hydroxylation occurring directly on the di-methoxyphenyl moiety. 3.2.5. Metabolites M6 and M8 M6 and M8 eluted at 4.85, and 4.94 min, respectively. Both metabolites exhibited an identical ammonium adduct ion [M+NH 4 ] + at m/z 498.1975 (C 23 H 32 NO 11 + , mass error : -0.4 ppm), suggesting a metabolic pathway involving demethylation followed by glucuronidation. Both metabolites showed the same MS 2 spectrum (Figure 5a) , which displayed a characteristic glucuronide-associated neutral loss of glucuronyl (-176 Da), yielding a fragment ion at m/z 322.1643 (C 17 H 24 NO 5 + , mass error: -1.9 ppm). The diagnostic ions at m/z 151.0754 (C 10 H 13 O 4 + , mass error: -3.3 ppm) and 137.0596 (C 10 H 13 O 4 + , mass error: -3.0 ppm) demonstrated demethylation at di-methoxyphenyl substructure of chrysotoxine. 3.2.6. Metabolite M9 M9 was observed at 5.00 min, which showed a protonated molecular ion at m/z 335.1488 (C 18 H 23 O 6 + , mass error: 0.6 ppm). The mass shift suggested that M9 was hydroxylation product of chrysotoxine. MS 2 spectrum (Figure 5b) displayed a diagnostic ion at m/z 197.0810 (C 10 H 13 O 4 + , mass error: -2.0 ppm), indicating hydroxylation at 2,6-dimethoxyphenol substructure of chrysotoxine. 3.2.7. Metabolite M11 M11 was observed at 5.42 min, which showed an ammonium adduct ion at m/z 512.2126 (C 24 H 34 NO 11 + , mass error: -1.2 ppm). The mass shift suggested that M11 was glucuronidation product of chrysotoxine. The MS 2 spectrum (Figure 6a) revealed a diagnostic neutral loss of glucuronyl (-176 Da), forming the fragment ion at m/z 336.1801 (C 18 H 26 NO 5 + , mass error: -1.2 ppm). The other fragment ions at m/z 181.0852, 165.0904 and 151.0756 were identical to those of the parent, confirming retention of the core chrysotoxine scaffold. 3.2.8. Metabolite M12 M12 observed at 6.13 min, showed an ammonium adduct ion at m/z 416.1373 (C 18 H 26 NO 6 S + , mass error: -0.2 ppm), indicative of a sulfation product of chrysotoxine. The MS 2 spectrum (Figure 6b) displayed a diagnostic neutral loss of sulfate group (-SO 3 , -80 Da), forming the fragment ion at m/z 336.1801 (C 18 H 26 NO 5 + , mass error: -1.2 ppm). The other fragment ions at m/z 181.0859, 165.0910 and 151.0756 were identical to those of the parent, confirming retention of the core chrysotoxine scaffold. 3.3. Metabolite profiles of chrysotoxine In this study, we proposed an HRMS-based analytical strategy to characterize the metabolites of chrysotoxine across multiple hepatocyte models (mouse, rat, dog, monkey, and human) and rat urine. Through comprehensive mass spectral interpretation, twelve distinct metabolites were structurally elucidated, with proposed metabolic pathways illustrated in Figure 7 . As a phenolic compound, chrysotoxine predominantly undergoes Phase II biotransformation pathways, particularly glucuronidation and sulfation. This observation aligns with previous reports on structurally analogous Dendrobium-derived phenolic compounds, moscatilin [17] and erianin [18], where glucuronidated metabolites constituted the major metabolic species. In the present study, the identified metabolic pathways of chrysotoxine comprised glucuronidation, sulfation, hydroxylation, demethylation and GSH conjugation. The glucuronidated metabolite M11 emerged as the dominant species across all test matrices, particularly in rat urine where it accounted for the primary clearance pathway. In contrast, the sulfated metabolite M12 demonstrated limited metabolic significance and was undetectable in urinary specimens. In rat urine, a hydroxylation metabolite M9 was detected with significant amount, suggesting that hydroxylation may be one of the predominant metabolic pathways of chrysotoxine. Chrysotoxine’s para-positioned phenolic hydroxyl and methylene groups create an electrophilic scaffold susceptible to cytochrome P450-mediated oxidation, generating reactive quinone methide intermediates. GSH can attack the aromatic ring or exocyclic methylene carbon, resulting the corresponding adducts, M5 and M2, respectively. GSH adduction at the aromatic ring (M5) manifests characteristic neutral losses of glycinyl (75 Da) and glutamyl (129 Da) moieties during collision-induced dissociation (CID), whereas exocyclic methylene carbon conjugation (M2) preferentially exhibits complete glutathione moiety loss (307 Da) without diagnostic amino acid fragments. Notably, methide-directed GSH attack induces spontaneous dimethoxyphenyl cleavage, producing metabolite M1, a transformation pattern previously documented for moscatilin [17]. Chrysotoxine also underwent demethylation to form demethylation metabolite, which can conjugate with glucuronide, forming M6 and M8. The desmethyl metabolite was also ready to oxidized to ortho quinone intermediate, which was susceptible to GSH nucleophilic attack, forming M3. This finding was identical to a recent study, which demonstrated that chrysotoxine and its hydroxylation can undergo oxidation to form reactive species in liver microsomes [19]. The formation of electrophilic intermediates raises critical safety concerns [11]. Quinoid species may covalently modify hepatic proteins through Michael addition, potentially triggering idiosyncratic toxicity as observed with sitaxentan [20] and nefazodone [21]. Furthermore, mechanism-based inactivation of P450 enzymes via intermediate adduction could precipitate clinically significant drug-drug interactions. These findings underscore the necessity for thorough evaluation of chrysotoxine’s metabolic activation potential, including detailed studies on enzyme inactivation kinetics and hepatic protein adduct formation. It should be noted that, chrysotoxine and its hydroxylation metabolites are typical substrates for UGT-glucuronosyltransferases, with glucuronidation dominating their elimination in hepatocytes. M11 was identified as the predominant metabolite while GSH conjugates were minor. These findings demonstrated that chrysotoxine preferentially undergoes glucuronidation over oxidative pathways within hepatocytes, thereby minimizing the formation of reactive metabolites. 4. Conclusions This study employed an LC-Orbitrap-MS/MS-based approach to systematically characterize metabolites of chrysotoxine, a bioactive constituent derived from Dendrobium chrysotoxum Lindl. Structural elucidation of metabolites was achieved through accurate mass measurements and comprehensive MS² fragmentation pattern interpretation. Rational interpretation of HRMS data revealed 12 metabolites, comprising one phase I metabolite and 11 phase II metabolites. Identified biotransformation pathways included hydroxylation, demethylation, glucuronidation, sulfation, and GSH conjugation. Cross-species analysis demonstrated direct glucuronidation as the predominant metabolic reaction. Notably, hydroxylated (M9) and glucuronidated (M11) metabolites emerged as the most abundant species in rat urine. To our knowledge, this investigation represents the first comprehensive metabolic study of chrysotoxine in hepatocytes across five species and rat urine. These findings establish a critical foundation for subsequent pharmacokinetic evaluations and toxicity assessments of this phytochemical. Data Availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of interest The authors declared no conflict of interest. References [1]. Ng TB, Liu J, Wong JH, Ye X, Wing Sze SC, Tong Y, Zhang KY. Review of research on Dendrobium, a prized folk medicine, Appl. Microbiol. Biotechnol. 2012, 93(5): 1795-1803. https://doi.org/ 10.1007/s00253-011-3829-7 . [2]. Li PY, Li L, Wang YZ. Traditional uses, chemical compositions and pharmacological activities of Dendrobium: A review, J. Ethnopharmacol. 2023, 310: 116382. https://doi.org/10.1016/j.jep.2023.116382. [3]. Wang YH. 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Kalgutkar AS, Vaz AD, Lame ME, Henne KR, Soglia J, Zhao SX, Abramov YA, Lombardo F, Collin C, Hendsch ZS, Hop CE. Bioactivation of the nontricyclic antidepressant nefazodone to a reactive quinone-imine species in human liver microsomes and recombinant cytochrome P450 3A4, Drug Metab. Dispos. 2005, 33 (2): 243-253. https://doi.org/10.1124/dmd.104.001735. Figure Legends Figure 1. MS 2 spectra (a) and tentative fragmentation patterns (b) of chrysotoxine Figure 2. LC-HRMS chromatograms of chrysotoxine and its metabolites from mouse, rat, dog, monkey and human hepatocytes, and rat urine following a single dose of 20 mg/kg chrysotoxine Figure 3. MS 2 spectra and tentative fragmentation patterns of M1 (a) , M2 (b) and M5 (c) Figure 4. MS 2 spectra and tentative fragmentation patterns of M3 (a) , M7/M10 (b) and M4 (c) Figure 5. MS 2 spectra and tentative fragmentation patterns of M6/M8 (a) and M9 (b) Figure 6. MS 2 spectra and tentative fragmentation patterns of M11 (a) and M12 (b) Figure 7. Proposed metabolic pathways of chrysotoxine Supplementary Material File (figure.docx) Download 5.01 MB File (table.docx) Download 21.85 KB Information & Authors Information Version history V1 Version 1 01 August 2025 Peer review timeline Published Rapid Communications in Mass Spectrometry Version of Record 10 Sep 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Rapid Communications in Mass Spectrometry Keywords chrysotoxine dendrobium lc-orbitrap-ms/ms metabolite characterization Authors Affiliations Chengzhen Guan Xuzhou Medical University Affiliated Hospital Department of Anaesthesiology View all articles by this author Yiqiang Xuzhou Tongshan District Hospital of Traditional Chinese Medicine View all articles by this author Meiling Wan 0009-0005-2434-7759 [email protected] Xuzhou Medical University Affiliated Hospital Department of Anaesthesiology View all articles by this author Metrics & Citations Metrics Article Usage 232 views 159 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Chengzhen Guan, Yiqiang, Meiling Wan. Identification of the in vitro and in vivo metabolites of chrysotoxine using liquid chromatography combined with benchtop orbitrap high resolution mass spectrometry. Authorea . 01 August 2025. 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