LC‐HRMS‐Based Metabolic Profiling of the REV‐ERB Agonist SR9009 in Camel Liver Homogenate and Cunninghamella elegans for Anti‐Doping Analysis

ABSTRACT

Rationale

SR9009 is a synthetic REV‐ERB agonist with potential performance‐enhancing properties and is included on the World Anti‐Doping Agency (WADA) Prohibited List for human sport. Its potential misuse has also raised concerns in animal sports, particularly equine and camel racing. Although SR9009 metabolism has been investigated in human and equine models, its metabolic fate in racing camels ( Camelus dromedarius ) remains unclear. Characterizing its biotransformation pathways and identifying suitable biomarkers are essential for developing reliable anti‐doping screening strategies for camel racing.

Methods

An in vitro metabolic study was conducted using camel liver homogenate and the fungal model Cunninghamella elegans. Metabolites were characterized using liquid chromatography‐high‐resolution mass spectrometry (LC‐HRMS). Putative structural elucidation was achieved through accurate mass measurements of precursor and product ions. Diagnostic fragmentation pathways were established using collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS), enabling the systematic identification of metabolic modifications based on characteristic fragment ions and neutral losses.

Results

Seventeen Phase I metabolites of SR9009 were identified. Major biotransformation pathways included N‐dealkylation, mono‐hydroxylation, di‐hydroxylation, and combinations thereof. The N‐dealkylated metabolite was the predominant metabolite detected in both camel liver homogenate and Cunninghamella elegans, indicating its suitability as a primary analytical target for anti‐doping screening. No Phase II metabolites were detected under the experimental conditions employed. Putative structural assignments were supported by characteristic high‐resolution tandem mass spectral fragmentation patterns.

Conclusions

This study provides the first comprehensive metabolic profile of SR9009 in a camel‐specific in vitro model. The identification of 17 Phase I metabolites, particularly the predominant N‐dealkylated derivative, provides valuable analytical targets for doping control. These findings establish a metabolic foundation for future in vivo studies to determine pharmacokinetics and detection windows in racing camels.

1. Introduction

Circadian rhythms are endogenous biological clocks that generate approximately 24‐h cycles and regulate a wide range of physiological processes, including metabolism, energy homeostasis, and glucose regulation [1, 2, 3]. These processes are tightly controlled by the circadian timing system, which coordinates multiple metabolic pathways. Disruption of circadian regulation has been strongly associated with metabolic disorders such as obesity and insulin resistance [4, 5].

The nuclear receptors REV‐ERBα and REV‐ERBβ play a central role in the regulation of circadian rhythms and metabolic homeostasis [6]. Synthetic agonists such as SR9009 and SR9011 have gained significant attention due to their ability to modulate metabolic pathways. The chemical structure of the REV‐ERB agonist SR9009 is shown in Figure 1. Preclinical studies have reported that SR9009 produces diverse physiological effects, including reductions in body weight and lipid levels [7, 8], alterations in sleep–wake cycles [9], and anti‐inflammatory and anxiolytic effects [10, 11, 12, 13]. Although many of these effects are attributed to REV‐ERB activation, emerging evidence suggests that SR9009 may also exert REV‐ERB‐independent actions in the regulation of metabolic and cellular processes [14, 15]. Notably, studies in mice have demonstrated that SR9009 enhances exercise capacity, potentially through increased mitochondrial biogenesis in skeletal muscle [16].

FIGURE 1.

Chemical structure of the REV‐ERB agonist SR9009.

The potential ergogenic properties of SR9009 and related compounds are a significant concern in anti‐doping. Consequently, these substances are included on the World Anti‐Doping Agency (WADA) Prohibited List [17], highlighting the need for robust detection strategies. Their ability to influence lipid and glucose metabolism, as well as mitochondrial function, further supports their potential for performance enhancement [7].

To address this critical issue, a comprehensive understanding of the metabolic fate of the REV‐ERB agonist SR9009 is essential for the development of reliable anti‐doping strategies. SR9009 metabolism has previously been investigated in vitro using human and equine liver preparations, resulting in the identification of multiple Phase I metabolites [18, 19, 20, 21]. However, species‐specific metabolic data relevant to camel racing are currently lacking, and no studies have yet explored the in vitro biotransformation of SR9009 using camel‐derived liver systems.

Camel liver homogenate provides a species‐relevant in vitro system for assessing metabolic transformations in camels [22], while Cunninghamella elegans has been widely reported to mimic mammalian Phase I metabolic transformation through cytochrome P450‐like enzymatic systems and has been used as a microbial model for predicting the metabolism of xenobiotics, including performance‐enhancing substances and emerging compounds [23, 24]. Together, these complementary models enable a more comprehensive characterization of SR9009 metabolism and support the development of sensitive analytical strategies for anti‐doping control in camel racing.

In this context, the present study aimed to investigate the in vitro metabolism of SR9009 using camel liver homogenate and Cunninghamella elegans. By employing these complementary biological models, the metabolic profiles of SR9009 were compared between mammalian and microbial systems to evaluate similarities and differences in metabolite formation and to identify analytically relevant biomarkers for mass spectrometric detection.

2. Materials and Methods

2.1. Chemicals and Reagents

SR9009 was purchased from Toronto Research Chemicals Inc. (North York, Ontario, Canada). LC–MS grade acetonitrile (LiChrosolv hyper grade, ≥ 99.9%), acetone (LiChrosolv, gradient grade ≥ 99.9%), ammonium formate (≥ 99%), formic acid (≥ 99%), and acetic acid (≥ 99%) were obtained from Merck KGaA (Darmstadt, Germany). Uridine‐5′‐diphospho‐α‐d‐glucuronic acid trisodium salt (UDPGA), nicotinamide adenine dinucleotide phosphate (NADPH), 1,4‐saccharic acid lactone, sodium dihydrogen phosphate monohydrate, sodium hydrogen phosphate, and magnesium chloride were purchased from Sigma‐Aldrich (St. Louis, Missouri, United States). 3′‐Phosphoadenosine 5′‐phosphosulfate (PAPS), a sulfate donor for phase II sulfation reaction, was obtained from Merck Millipore (Burlington, Massachusetts, United States). Cunninghamella elegans Lendner (ATCC‐9245) was obtained from ATCC (Manassas, Virginia, United States). Sabouraud Dextrose Agar and Sabouraud Dextrose Broth were procured from HiMedia (Mumbai, India). Ultrapure water was generated using a Barnstead Smart2Pure water purification system (Thermo Fisher Scientific, Waltham, Massachusetts, United States).

2.2. Preparation of the Camel Liver Homogenates

Fresh camel liver was collected, sectioned into uniform slices (approximately 1 cm thickness) under sterile conditions, and immediately stored at −80°C to preserve enzymatic activity. Prior to incubation, the tissue was subdivided (~1 cm³) and homogenized at 4°C in ice‐cold incubation buffer to minimize enzymatic degradation and maintain metabolic competence.

2.3. Phase I In Vitro Metabolism Using Camel Liver Homogenates

The incubation mixture consisted of NADPH (10 mM in phosphate buffer, 50 μL) and the test compound (1 mM, 5 μL), which were added to phosphate buffer (100 mM, 432 μL, pH 7.4) in an Eppendorf tube [25]. The mixture was vortexed and incubated at 37°C for 5 min; 1 cm³ homogenized camel liver was added to initiate the reaction, and the incubation was continued for 60 min with continuous shaking. The metabolic reaction was terminated by the addition of ice‐cold acetone (200 μL) to precipitate proteins. Samples were centrifuged at 12575×g for 10 min at 4°C (Mikro 200R, Hettich, Germany). The supernatant was carefully transferred to vials and analyzed by LC‐HRMS.

All incubations were performed in duplicate. Control experiments included incubation without NADPH to assess non‐enzymatic degradation, as well as substrate blanks, cofactor blanks, and enzyme blanks to ensure specificity of metabolite formation.

2.4. Phase II In Vitro Metabolism Using Camel Liver Homogenates

Metabolic incubations were carried out according to a standardized experimental protocol. Briefly, the incubation mixture was prepared in a 2‐mL Eppendorf tube by combining 1 mL of phosphate buffer (100 mM, pH 7.4) containing NADPH (10 mM), 1,4‐saccharic acid lactone (5 mM), and MgCl₂ (5 mM) with 5 μL of a 1 mM SR9009 solution [26]. The mixture was vortex‐mixed and pre‐incubated at 37°C for 5 min before the addition of homogenized camel liver. Incubation was then continued at 37°C under continuous agitation for 30 min. Subsequently, UDPGA (5 mM, 50 μL) and PAPS (5 mM, 50 μL), prepared in phosphate buffer, were added to initiate Phase II metabolism, and the samples were further incubated for 2.5 h.

Metabolic activity was terminated by adding 250 μL of ice‐cold acetone. Following centrifugation at 12575×g for 10 min (Mikro 200R, Hettich), the precipitated proteins were removed, and the supernatant was transferred to a Kimble tube. The extract was diluted with 1 mL of deionized water and subjected to two consecutive liquid–liquid extractions using 4 mL of tert‐butyl methyl ether (TBME). The combined organic extracts were evaporated to dryness, and the residue was reconstituted in 100 μL of a 9:1 (v/v) mixture of 0.2% formic acid in water and acetonitrile prior to LC‐HRMS analysis.

To ensure experimental validity, Phase I and Phase II metabolic assays were conducted separately, with each analytical series including a substrate, co‐factor, and enzyme blanks as control.

2.5. In Vitro Metabolic Study Using the Fungus Cunninghamella elegans

Cunninghamella elegans (ATCC 9245) was cultured on Sabouraud dextrose agar plates incubated at 26°C for 5 days. Following incubation, the fungal biomass was harvested and suspended in sterile saline (10 mL) using an inoculation loop. The suspension was homogenized for 5 min to obtain a uniform inoculum and stored at 2°C–8°C [26, 27]. An aliquot (1.5 mL) of the fungal suspension was inoculated into 30 mL of sterile Sabouraud dextrose broth in a Schott flask. The cultures were then incubated in an orbital shaker (GRANT‐BIO ES80, Cambridgeshire, United Kingdom) at 28°C for 48 h with continuous agitation at 100 rpm. After sufficient fungal growth had been achieved, 50 μL of SR9009 solution (10 mg/mL prepared in methanol) was added to the culture flask, and incubation was continued under the same conditions. Aliquots (5 mL) were withdrawn at 24‐h intervals up to 192 h. The metabolic reactions were immediately quenched by adding 2 mL of acetonitrile to each collected sample. Approximately 1.5 mL of each culture was transferred into Eppendorf tubes and centrifuged at (12 000×g) for 10 min. The supernatant was then transferred to a Kimble tube and extracted with tert‐butyl methyl ether (TBME, 4 mL). The organic layer was separated, evaporated under a stream of nitrogen, and reconstituted in an appropriate solvent for LC‐HRMS analysis. Control incubations were performed under identical conditions, including (i) fungal cultures without SR9009 (biological blank) and (ii) culture medium containing SR9009 without fungus (chemical control), to distinguish between biotic and abiotic transformations.

2.6. LC‐HRMS Analysis

2.6.1. Liquid Chromatography

The chromatographic separation was performed using Agilent Zorbax Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) on a Vanquish UHPLC system (Thermo Scientific, Bremen, Germany). The mobile phase consisted of acetonitrile (solvent A) and 0.2% formic acid in water (solvent B). The gradient elution program began with 100% solvent B (0% solvent A) at 0 min, at a flow rate of 0.6 mL/min. By 4 min, the composition was linearly adjusted to 95% solvent A and 5% solvent B, maintained until 10 min. Thereafter, the mobile phase was returned to the initial condition of 100% solvent B and allowed to re‐equilibrate prior to the next injection until 14 min.

2.6.2. Mass Spectrometry

Mass spectrometric analysis was performed on a Q Exactive exploris 120 high‐resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI) source for the identification and characterization of SR9009 and its in vitro metabolites. The system was operated in positive electrospray ionization (+ESI) mode with the S‐lens value set to 50. Capillary voltage was set to +_3.5 kV, and the capillary temperature was maintained at 320°C. Sheat gas and auxiliary gas were set to 45 and 15 arbitrary units (AU), respectively. Mass spectrometry data were acquired at a resolution of 60 000 over a mass range of m/z 50–750. For MS2 experiments, the resolution was set to 30 000. Product ion (MS²) experiments were performed to characterize the fragmentation patterns of SR9009 and its metabolites. The normalized collision energy (NCE) was optimized individually for each metabolite to obtain the most informative product ion spectra. The isolation window was m/z 2, and the maximum injection time was set to 100 ms. Automatic gain control (AGC) targets for MS and MS2 experiments were set to 1e6 and 5e5, respectively. The acquired data were analyzed using Thermo Xcalibur software (version 4.4).

3. Results and Discussion

3.1. Characterization of SR9009 Metabolites

In LC‐HRMS analysis, the drug SR9009 yielded a parent protonated molecule [M + H]⁺ at m/z 438.1239 that was generated corresponding to the molecular formula (C₂₀H₂₄ClN₃O₄S) and eluted at a retention time (RT) of 10.01 min (Figure 2). In the MS² experiment acquired at a normalized collision energy (NCE) of 30%, the dominant base peak was detected at m/z 125.0149, corresponding to the (4‐chlorophenyl)methyl moiety, while a less intense ion at m/z 141.9954 was attributed to the (5‐nitrothiophen‐2‐yl)methyl moiety. The other fragment ions are at m/z 392.08089 (C₁₈H₁₉ClN₃O₃S) resulting from cleavage at the pyrrolidine‐carboxamide moiety with the loss of the ethoxy group.

FIGURE 2.

Extracted ion chromatogram and ESI product ion mass spectrum of SR9009 (C₂₀H₂₄ClN₃O₄S).

Following LC‐HRMS characterization of the parent drug and its metabolites, camel liver homogenate generated metabolites M1‐M17, whereas Cunninghamella elegans produced metabolites M1, M2, M3, M4, M11, M12, M15, M16, and M17. The accurate masses, empirical formulas, retention times (RT), relative abundances, and mass errors of the parent compound and its metabolites are summarized in Table 1. The metabolite M1 was formed through N‐dealkylation of the ethoxycarbonyl‐pyrrolidine‐methyl group from SR9009 with a protonated molecule [M + H]⁺ at m/z 283.0301 (C₁₂H₁₁ClN₂O₂S) and eluted at RT 6.45 min. In the MS² experiment acquired at a normalized collision energy (NCE) of 15%, metabolite M1 was characterized by a predominant fragment ion at m/z 125.0150 (C₇H₆Cl), resulting from cleavage of the (4‐chlorophenyl)methyl moiety from the precursor ion as shown in (Figure 3). Another fragment ion at m/z 141.9954 (C₅H₄NO₂S) was observed, arising from dissociation of the (5‐nitrothiophen‐2‐yl)methyl group.

TABLE 1.

Summary of the metabolic modifications of SR9009 and its metabolites.

Analyte ID	Biotransformations	Molecular formula	RT (min)	Exact mass [M + H]+	Measured mass [M + H]+	Mass error (ppm)	Product ions (m/z)	Relative abundance (%)
	SR9009	C20H24ClN3O4S	10.01	438.1249	438.1239	−2.28	125.0149 141.9954	NA
M1	N‐dealkylation	C12H11ClN2O2S	6.45	283.0303	283.0301	−0.70	141.9954 125.0149	6.00
M2	N‐dealkylation	C15H21ClN2O2	6.27	297.1364	297.1356	−2.69	251.0939 125.0149	100
M3	N‐dealkylation	C13H19N3O4S	5.95	314.1169	314.1163	−1.91	268.0744 141.9954	45.75
M4	N‐dealkylation	C17H20ClN3O2S	7.31	366.1037	366.1029	−2.18	141.9954 125.0149	3.02
M5	N‐dealkylation+hydroxylation	C15H21ClN2O3	5.81	313.1313	313.1311	−0.63	154.0417 125.0152	0.29
M6	N‐dealkylation+hydroxylation	C15H21ClN2O3	7.19	313.1313	313.1317	1.27	125.0152 154.0417	0.32
M7	N‐dealkylation+hydroxylation+oxidation	C15H19ClN2O3	7.64	311.1157	311.1147	−3.21	265.0734 125.0149	0.12
M8	N‐dealkylation+hydroxylation+oxidation	C15H19ClN2O3	7.78	311.1157	311.1142	−4.82	265.0734 125.0149	0.20
M9	N‐dealkylation+dehydrogenation	C15H19ClN2O2	6.02	295.1207	295.1216	3.04	125.0154 154.0423	3.91
M10	N‐dealkylation+dehydrogenation	C15H19ClN2O2	6.3	295.1207	295.1218	3.72	125.0154 154.0423	0.38
M11	N‐dealkylation	C12H17ClN2	7.24	225.1153	225.1158	2.22	84.0810, 125.0155	0.06
M12	N‐dealkylation+hydroxylation	C13H19N3O5S	5.41	330.1118	330.1123	1.51	141.9960 268.0759	0.07
M13	N‐dealkylation+hydroxylation+oxidation	C17H18ClN3O3S	8.68	380.0830	380.0819	−2.89	295.0298 125.0149	0.34
M14	Dehydrogenation	C20H22ClN3O4S	9.05	436.1092	436.1089	−0.68	295.0301 125.0151	2.61
M15	Hydroxylation	C20H24ClN3O5S	9.32	454.1198	454.1190	−1.76	295.0296 125.0149	0.80
M16	Hydroxylation	C20H24ClN3O5S	9.65	454.1198	454.1176	−4.84	295.0296 125.0149	0.22
M17	Dihydroxylation	C20H24ClN3O6S	8.73	470.1147	470.1131	−3.40	188.0912 125.0149	0.09

Note: Measured m/z values correspond to the protonated molecular ions [M + H]⁺ obtained from full‐scan HRMS data.

FIGURE 3.

Extracted ion chromatogram and ESI product ion mass spectra of N‐dealkyalted metabolite M1 (C₁₂H₁₁ClN₂O₂S), M2 (C₁₅H₂₁ClN₂O₂), M3 (C₁₃H₁₉N₃O₄S) in positive ion mode.

Metabolite M2 was formed via the loss of the (5‐nitrothiophen‐2‐yl)methyl moiety from the precursor ion, yielding a protonated molecule [M + H]⁺ at m/z 297.13566 corresponding to the molecular formula (C₁₅H₂₁ClN₂O₂) and eluted at 6.27 min (Figure 3). M2 was identified as the predominant metabolite of SR9009. In the MS² experiment acquired at an NCE of 30%, metabolite M2 exhibited a base peak at m/z 125.0149 (C₇H₆Cl) from the (4‐chlorophenyl)methyl moiety. A product ion at m/z 251.0939 (C₁₃H₁₆ClN₂O) was formed by the loss of an ethoxy group. Another fragment ion at m/z 142.0859 (C₇H₁₂NO₂) was observed, attributed to an ethoxycarbonyl‐pyrrolidine derived fragment.

Metabolite M3 was formed via N‐dealkylation involving cleavage of the (4‐chlorophenyl)methyl moiety from SR9009, corresponding to a protonated molecule [M + H]⁺ at m/z 314.1163 with the molecular formula (C₁₃H₁₉N₃O₄S) and eluted at 5.95 min (Figure 3). In the MS² experiment acquired at an NCE of 20%, the metabolite M3 exhibited a base peak at m/z 268.0744 (C₁₁H₁₄N₃O₃S), corresponding to a neutral loss of an ethoxy group from the precursor ion. An additional fragment ion at m/z 141.9954 (C₅H₄NO₂S), attributed to the (5‐nitrothiophen‐2‐yl)methyl moiety, was observed. Consistent with the findings of Cutler et al. [25], three N‐dealkylated metabolites (M1, M2, and M3) were identified following incubation with both camel liver homogenate and Cunninghamella elegans.

Metabolite M4 was characterized by a protonated molecule [M + H]⁺ at m/z 366.1029 corresponding to the molecular formula (C₁₇H₂₀ClN₃O₂S) and eluted at 7.31 min (Figure S1 [supporting information]). M4 was formed via N‐dealkylation by the loss of ethoxycarbonyl moiety from the parent compound. In the MS² experiment acquired at an NCE of 30%, the metabolite M4 exhibited a characteristic fragment ion at m/z 330.1269 (C₁₇H₁₉N₃O₂S), corresponding to the neutral loss of HCl from the precursor ion. Additional fragment ions were observed at m/z 141.9954 (C₅H₄NO₂S), attributed to a fragment derived from the (5‐nitrothiophen‐2‐yl)methyl moiety, and at m/z 125.0149 (C₇H₆Cl), corresponding to the (4‐chlorophenyl)methyl moiety.

Metabolites M5 and M6 were formed via hydroxylation of metabolite M2, yielding protonated molecules [M + H]⁺ at m/z 313.1313, corresponding to the molecular formula (C₁₅H₂₁ClN₂O₃). These metabolites eluted at retention times of 5.81 and 7.19 min, respectively (Figure S2 [supporting information]). In the MS² experiment acquired at an NCE of 30%, both metabolites exhibited a base peak at m/z 125.0149 (C₇H₆Cl) attributed to the (4‐chlorophenyl)methyl moiety. A fragment ion observed at m/z 295.1203 (C₁₅H₂₀ClN₂O₂) corresponded to the loss of water, which is consistent with proton rearrangement and protonation at the nitrogen atom of the metabolite, as previously described in the literature [18]. The fragment ion at m/z 154.0417 (C₈H₉ClN) was further attributed to dissociation of the hydroxymethyl pyrrolidine moiety. The fragment ion at m/z 267.0892 (C₁₃H₁₆ClN₂O₂) observed in metabolite M6 was formed by the neutral loss of the ethoxy group from the precursor ion.

Metabolites M7 and M8 were formed via oxidation of metabolites M5 and M6, yielding protonated molecules [M + H]⁺ at m/z 311.1147, corresponding to the molecular formula (C₁₅H₁₉ClN₂O₃). These metabolites eluted at retention times of 7.64 and 7.78 min (Figure 4), respectively. In the MS² experiment acquired at an NCE of 20%, a characteristic fragment ion at m/z 265.0734 (C₁₃H₁₄ClN₂O₂) was formed by cleavage of the ethoxy group from the precursor ion. An additional fragment ion was observed at m/z 125.0149 (C₇H₆Cl), corresponding to cleavage of the (4‐chlorophenyl)methyl moiety. The presence of the product ion at m/z 125.0149, together with the absence of the ion at m/z 141.9954, suggested loss of the (5‐nitrothiophen‐2‐yl)methyl moiety and indicated oxidation had occurred on the pyrrolidine ring. Although M7 and M8 exhibited the same accurate mass and generated the same major product ions, their MS/MS spectra differed in the relative abundances of these fragments, supporting their assignment as putative isomeric metabolites. Specifically, the fragment ion at m/z 125.0149 was the dominant product ion for M7 but was of lower relative abundance in M8. In contrast, the fragment ion at m/z 265.0734 was of low abundance in M7 and became a prominent product ion in M8. These differences in fragmentation behavior likely reflect structural differences affecting the preferred fragmentation pathways of the two isomers. Interestingly, Mazzarino et al. [19] reported only a single metabolite with the same accurate mass, whereas the present study resolved two chromatographically distinct metabolites (M7 and M8). This discrepancy may be attributed to interspecies differences in SR9009 metabolism, as well as differences in chromatographic separation and LC‐HRMS/MS analytical conditions, which enabled improved characterization of isomeric metabolites in the present study.

FIGURE 4.

Extracted ion chromatogram and ESI product ion mass spectra of metabolite M7, M8 (C₁₅H₁₉ClN₂O₃) in positive ion mode.

Metabolites M9 and M10 were formed via dehydrogenation of M2 with a protonated molecule [M + H]⁺ at m/z 295.1216 (C₁₅H₁₉ClN₂O₂), and eluted at retention times of 6.02 and 6.30 min, respectively (Figure S3 [supporting information]). In the MS² experiment acquired at an NCE of 30%, both metabolites exhibited a base peak ion at m/z 125.0149 (C₇H₆Cl) attributed to the (4‐chlorophenyl)methyl group. An additional fragment ion was observed at m/z 154.0420 (C₈H₉ClN) corresponding to the chlorophenylmethyl iminium ion. Furthermore, fragment ions at m/z 249.0797 (C₁₃H₁₄ClN₂O), formed by cleavage of the ethoxy group from the precursor ion, and at m/z 206.0737 (C₁₂H₁₃ClN), generated following cleavage of the pyrrolidine ring, were also detected.

Metabolite M11 was identified as a minor metabolite and was formed via N‐dealkylation of metabolite M2, yielding a protonated molecule [M + H]⁺ at m/z 225.1158, corresponding to the molecular formula (C₁₂H₁₇ClN₂), and eluting at a retention time of 7.24 min (Figure S4 [supporting information]). At an NCE of 35%, M11 fragmented to yield a base peak ion at m/z 125.0150 (C₇H₆Cl), attributed to the (4‐chlorophenyl)methyl moiety. Additional fragment ions were observed at m/z 84.0810 (C₅H₁₀N), corresponding to methylpyrrolidine, and at m/z 208.0891 (C₁₂H₁₅ClN) resulting from cleavage of the pyrrolidine moiety from the precursor ion.

Metabolite M12 was identified as a minor metabolite and was formed via hydroxylation of M3 yielding a protonated molecule [M + H]⁺ at m/z 330.1123 (C₁₃H₁₉N₃O₅S), and eluted at a retention time of 5.41 min (Figure 5). In the MS² experiment acquired at an NCE of 35%, metabolite M12 exhibited a base peak ion at m/z 141.9954 (C₅H₄NO₂S) attributed to a fragment originating from the (5‐nitrothiophen‐2‐yl)methyl moiety. An additional fragment ion at m/z 268.0759 (C₁₁H₁₄N₃O₃S) was observed, corresponding to the loss of a hydroxylated ethoxy moiety from the precursor ion.

FIGURE 5.

Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M12 (C₁₃H₁₉N₃O₅S) in positive ion mode.

Metabolite M13 was generated via hydroxylation and oxidation of M4, yielding a protonated molecule [M + H]⁺ at m/z 380.0819, corresponding to the molecular formula (C₁₇H₁₈ClN₃O₃S), and eluting at a retention time of 8.68 min (Figure 6). In the MS² experiment acquired at an NCE of 20%, metabolite M13 exhibited a fragment ion at m/z 125.0149 (C₇H₆Cl), attributed to the (4‐chlorophenyl)methyl moiety. A fragment ion at m/z 333.0813 (C₁₇H₁₈ClN₂OS) corresponded to the neutral loss of a nitro group (–NO₂) from the precursor ion. An additional fragment ion at m/z 141.9954 (C₅H₄NO₂S) was also detected and was attributed to the (5‐nitrothiophen‐2‐yl)methyl moiety.

FIGURE 6.

Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M13 (C₁₇H₁₈ClN₃O₃S) in positive ion mode.

The metabolite profile also included diverse modifications of the parent compound. Metabolite M14, for instance, was formed via dehydrogenation of the parent drug, yielding a protonated molecule [M + H]⁺ at m/z 436.1089 corresponding to the molecular formula (C₂₀H₂₂ClN₃O₄S), and eluting at a retention time of 9.05 min (Figure S5 [supporting information]). In the MS² experiment acquired at an NCE of 30%, M14 exhibited a base peak at m/z 125.0151 (C₇H₆Cl), attributed to the (4‐chlorophenyl)methyl moiety. Additional fragment ions were observed at m/z 295.0301(C₁₃H₁₂ClN₂O₂S), corresponding to the loss of an ethoxycarbonylpyrrolidine moiety from the dehydrogenated precursor ion. A fragment ion at m/z 154.0861(C₈H₁₂NO₂) was attributed to a dehydrogenated ethoxycarbonylpyrrolidine fragment, consistent with the formation of a double bond within the pyrrolidine ring.

Metabolites M15 and M16 were formed via mono‐hydroxylation of the parent drug, resulting in a mass increase of 16 Da. In the MS² experiment acquired at an NCE of 15%, these hydroxylated metabolites exhibited a protonated molecule [M + H]⁺ at m/z 454.1176, corresponding to the molecular formula (C₂₀H₂₄ClN₃O₅S), and eluted at retention times of 9.32 and 9.65 min, respectively (Figure 7). The mass spectra of M15 and M16 showed a major fragment ion at m/z 295.0296 (C₁₃H₁₂ClN₂O₂S), corresponding to the loss of a hydroxylated ethoxycarbonylpyrrolidin‐3‐yl moiety. Additional fragment ions were observed at m/z 125.0149(C₇H₆Cl) attributed to the (4‐chlorophenyl)methyl moiety, and at m/z 408.0775 (C₁₈H₁₉ClN₃O₄S), formed by cleavage of the hydroxylated ethoxycarbonyl moiety.

FIGURE 7.

Extracted ion chromatogram and corresponding ESI product ion spectra of metabolite M15, M16 (C₂₀H₂₄ClN₃O₅S) in positive ion mode.

Metabolite M17 was detected as a minor metabolite and was formed via di‐hydroxylation of the parent drug, resulting in a mass increase of 32 Da. In the MS² experiment acquired at an NCE of 20%, M17 was characterized by a protonated molecule [M + H]⁺ at m/z 470.1131 corresponding to the molecular formula (C₂₀H₂₄ClN₃O₆S), and eluted at a retention time of 8.73 min (Figure 8). Fragment ions were detected at m/z 424.0714 (C₁₈H₁₉ClN₃O₅S), arising from cleavage of the ethoxy group (‐OC₂H₅), and at m/z 188.0912 (C₈H₁₄NO₄), attributed to the dihydroxylated pyrrolidine‐1‐carboxylate moiety. Additional characteristic fragment ions were observed at m/z 141.9954 (C₅H₄NO₂S), resulting from cleavage of the (5‐nitrothiophen‐2‐yl)methyl moiety, and at m/z 125.0149 (C₇H₆Cl), corresponding to the (4‐chlorophen‐2‐yl)methyl moiety. The metabolic pathway of SR‐9009 is shown in (Figure 9).

FIGURE 8.

Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M17 (C₂₀H₂₄ClN₃O₆S) in positive ion mode.

FIGURE 9.

Proposed metabolic pathway of SR9009 in camel liver homogenate and fungus Cunninghamella elegans.

Several metabolites identified in camel liver homogenates were consistent with those previously reported in other mammalian in vitro systems. Mazzarino et al. [19] identified 13 phase I metabolites in human liver microsomes, whereas Cutler et al. [20] and Kwak et al. [21] reported 6 and 12 metabolites, respectively, in equine liver microsomes. The N‐dealkylated metabolites M1, M2, and M3 were consistently detected in camel liver homogenates as well as in both human and equine in vitro systems. In addition, hydroxylated and further oxidized metabolites, including M4, M5, M6, M9, M10, M13, M14, M15, and M17, were detected in at least one of the previously investigated mammalian system. In contrast, M8, M12, and M16 were identified only in camel liver homogenates, suggesting species‐dependent differences in SR9009 metabolism.

These differences may also reflect the distinct experimental models used, as previous studies employed liver microsomes, whereas the present study used camel liver homogenates containing both microsomal and cytosolic enzymes. This broader enzymatic composition may contribute to the expanded metabolite profile observed in camel liver homogenates.

Cunninghamella elegans produced metabolites M1, M2, M3, M4, M11, M12, M15, M16, and M17, several of which overlapped with those detected in camel liver homogenates. These findings demonstrate the ability of Cunninghamella elegans to generate mammalian‐like metabolic transformations of SR9009 and highlight its potential as a complementary model for metabolite identification. The combined use of camel liver homogenates and Cunninghamella elegans provides broader metabolic coverage and supports the identification of candidate biomarkers for SR9009 detection in anti‐doping analysis.

4. Conclusion

In conclusion, this study provides the first LC‐HRMS‐based characterization of SR9009 metabolism using camel liver homogenate, with Cunninghamella elegans as a complementary in vitro model. The identified metabolites revealed that SR9009 undergoes multiple phase I biotransformations involving N‐dealkylation, hydroxylation, and oxidation pathways. The most abundant N‐dealkylated metabolite M2 represents a potential biomarker for SR9009 detection in camel racing anti‐doping analysis. The combined use of camel liver homogenate and Cunninghamella elegans expands the available metabolic information for SR9009 and supports the development of improved analytical screening strategies. Further in vivo investigations are required to confirm metabolite excretion profiles and detection windows.

Author Contributions

Ansar Babu Palathinkal: conceptualization, methodology, validation, writing – original draft, formal analysis. S. Farook Basha: supervision. Saraswathy Laya: supervision. Jahfar Nalakath: writing – review and editing. O. K. Praseen: formal analysis. Marina Joseph: formal analysis. Shantymol V. Jose: formal analysis.

Funding

The authors have nothing to report.

Ethics Statement

The authors have not conducted any research involving human or live animal participants for this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M4 (C₁₇H₂₀ClN₃O₂S) in positive ion mode.

Figure S2: Extracted ion chromatogram and ESI product ion mass spectra of metabolite M5, M6 (C15H21ClN2O3) in positive ion mode.

Figure S3: Extracted ion chromatogram and ESI product ion mass spectra of metabolite M9, M10 (C15H19ClN2O2) in positive ion mode.

Figure S4: Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M11 (C12H17ClN2) in positive ion mode.

Figure S5: Extracted ion chromatogram and ESI product ion mass spectrum of metabolite M14 (C20H22ClN3O4S) in positive ion mode.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.