Profiling and Identification of Novel Tiletamine Metabolites in Human Urine and Hair by Liquid Chromatography Coupled With Quadrupole‐Orbitrap Mass Spectrometry

ABSTRACT

Rationale

Tiletamine, an animal tranquilizer increasingly misused by humans, lacks comprehensive metabolic characterization in authentic biological matrices. To date, tiletamine phase II metabolism remains poorly characterized. This study investigated the metabolic fate of tiletamine in human urine and hair by identifying novel metabolites and elucidating biotransformation pathways relevant to forensic toxicological monitoring.

Methods

Authentic human urine (n = 3) and hair (n = 2) samples from individuals with documented tiletamine exposure were analyzed using liquid chromatography coupled with Q Extractive HF hybrid quadrupole‐Orbitrap high‐resolution mass spectrometry (LC‐QE‐HF‐MS). Metabolites were detected and identified based on full‐scan MS and data‐dependent MS/MS (ddMS²) fragmentation patterns. Structural elucidation, including the assignment of hydroxylation sites, was achieved through a diagnostic fragment ion analysis.

Results

A total of 14 urinary metabolites were identified, 11 of which (4 phase I and 7 phase II) were previously unreported. Hydroxylation was the predominant phase I process, whereas glucuronidation predominated among phase II reactions. Methylation and glucuronidation were identified as previously unreported metabolic pathways. In hair samples, tiletamine and four metabolites, including three reduced metabolites and T1, were identified for the first time. In addition, three metabolic transformations, namely, reduction, hydroxylation, and methylation, were newly identified in this matrix. Notably, T1 exhibited substantially higher signal intensities than all other metabolites across all urine samples.

Conclusions

This study substantially expands the known urinary metabolome by identifying novel phase I and II metabolites. The consistently high abundance of T1 in urine suggests that it is a promising urinary biomarker for monitoring tiletamine use. These findings broaden the range of analytical targets available for toxicological screening and confirmational analysis, thereby improving the detection and monitoring of tiletamine exposure.

1. Introduction

Tiletamine (Figure 1) is a dissociative anesthetic that acts as a noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist. Structurally related to phencyclidine (PCP) and ketamine, it exerts its pharmacological effects by inhibiting glutamatergic neurotransmission [1]. This mechanism underlies its analgesic, anesthetic, and dissociative effects. Classified as a dissociative anesthetic, tiletamine is used primarily in veterinary medicine and most commonly in a fixed 1:1 combination with zolazepam, a positive allosteric modulator of the γ‐aminobutyric acid type A (GABAAA) receptor marketed as Telazol (or Zoletil) [2, 3, 4]. At low doses, NMDA receptor antagonism produces anticonvulsant effects, whereas at higher doses, tiletamine may induce convulsions, potentially through mechanisms involving non‐NMDA receptors [5, 6]. In addition to NMDA receptor antagonism, tiletamine binds to PCP recognition sites in the brain, contributing to its distinct pharmacological profile relative to those of other related dissociative anesthetics. Tiletamine also exhibits significant toxicity. In humans, its misuse is associated with severe neurological manifestations, including coarse tremors, ataxia, dysarthria, and dissociative symptoms. Individuals who misuse tiletamine may also experience chest tightness, palpitations, anxiety, and depression. Chronic misuse has been associated with suicidal ideation, persistent psychosis, and hallucinations [7, 8]. Due to its psychoactive properties, tiletamine has a substantial potential for misuse and dependence. It is frequently co‐administered with other central nervous system depressants, such as alcohol or etomidate, which can potentiate its toxic effects and increase the risk of fatal overdose [9, 10]. Polydrug use involving tiletamine may exacerbate withdrawal‐associated neuroexcitotoxicity and increase the risk of cross‐dependence [1, 11]. Chronic misuse has been associated with irreversible neurological damage, multi‐organ dysfunction, and severe psychological deterioration. In the United States, tiletamine is classified as a Schedule III controlled substance under the Controlled Substances Act [12, 13]. In South Korea, tiletamine is similarly regulated as a controlled substance [7, 14]. In China, regulatory controls have been progressively strengthened. Since 15 July 2024, tiletamine hydrochloride has been classified as a veterinary prescription anesthetic and is explicitly prohibited for human use [15]. More recently, tiletamine has emerged as a novel psychoactive substance (NPS) and has been increasingly detected in electronic cigarette (e‐cigarette) products [16].

FIGURE 1.

Chemical structures of tiletamine and its 14 metabolites (T1–T14) in human urine and hair samples (* represents the chiral carbon).

Because tiletamine is used primarily as a veterinary anesthetic, its metabolism has been extensively investigated in animals. Studies of biological samples from monkeys, cats, pigs, tigers, and horses have identified several metabolites, including reduced tiletamine, desethyl tiletamine, and reduced desethyl tiletamine [10, 17, 18, 19]. Fenwick et al. identified five tiletamine metabolites in equine liver microsomes using liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) with an LTQ‐Orbitrap device. In addition to desethyl tiletamine, the major metabolite, four monohydroxylated metabolites were identified; however, the hydroxylation sites could not be definitively determined [20]. In contrast, only four studies have investigated tiletamine metabolism in humans. Tang et al. used liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS) to detect tiletamine and desethyl tiletamine in urine samples from clinical patients [21]. Cai et al. [22] developed and validated an ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method for the quantitative determination of tiletamine and its metabolite, desethyl tiletamine, in human biological samples for forensic applications. In two authentic forensic cases, tiletamine was detected at concentrations of 0.37 μg/mL (blood), 0.15 μg/g (liver), and 0.11 μg/g (kidney) in case 1 and 8.75 ng/mL (blood) in case 2. Desethyl tiletamine was also detected in the blood samples from both cases. He et al. [23] used gas chromatography‐quadrupole time‐of‐flight mass spectrometry (GC‐QTOF‐MS) to detect tiletamine, zolazepam, and their metabolites in samples from specific drug sexual assault incidents. Tiletamine and three metabolites (one desethylated metabolite and two reduced metabolites) were identified in urine samples from authentic case specimens. Wang et al. [24] applied a validated high‐performance liquid chromatography–tandem mass spectrometry method to screen 469 clinical specimens (433 hair and 36 urine samples). The targeted analysis revealed that tiletamine was detected in 33 hair specimens, whereas the parent drug was not detected in any urine samples. Prior to this study, four metabolites (reduced tiletamine, desethylated tiletamine, a desethylation/dehydrogenation product, and a reduction/hydroxylation product involving the nitrate ester group) had been identified using ultra‐high‐performance liquid chromatography–high‐resolution mass spectrometry with both in vitro experiments and authentic reference materials. Among these, three novel metabolites were identified: the desethylamine derivative, the dehydrogenated/reduced product, and the hydroxylated metabolite formed at the nitrate ester moiety. All four of these metabolites were detected in human hair. To date, five metabolic pathways have been reported for tiletamine: hydroxylation, reduction, desethylation, dehydrogenation, and desethylation.

In the present study, the tiletamine metabolic profiles were investigated using liquid chromatography coupled with a hybrid quadrupole‐Orbitrap mass spectrometer (LC‐QE‐HF‐MS) to enable accurate mass measurements. Analyses were conducted on three human urine specimens and two human hair samples. A total of 14 putative metabolites were detected, of which 11 were identified as novel metabolites via LC‐QE‐HF‐MS (Figure 1). Additionally, hydroxylated and methylated metabolites (T1) in human hair samples are reported here for the first time.

2. Experiment

2.1. Chemicals and Reagents

The Fada Institute of Forensic Medicine & Science provided the standard solution of tiletamine in methanol at a concentration of 100 ng/mL. All solvents and additives were of LC–MS grade, including methanol, ammonium formate, and formic acid (Thermo Fisher Scientific, Waltham, MA, USA). Ultrapure water (resistivity: 18.2 MΩ·cm) was prepared using a Milli‐Q Advantage A10 system (Merck Millipore, Billerica, MA, USA). Solid‐phase extraction (SPE) was performed using Oasis HLB cartridges (3 cc, 60 mg; Waters Corporation, Beijing, China).

2.2. Instrument (LC/Quadrupole‐Orbitrap MS) Conditions

Chromatographic separation was performed using a Thermo Vanquish LC system coupled with a Thermo Hypersil GOLD C18 column (2.1 mm × 100 mm, 1.9 μm particle size; Thermo Fisher Scientific, Waltham, MA, USA). The column temperature was maintained at 30°C. The mobile phase was composed of solvent A (water containing 0.1% formic acid) and solvent B (methanol containing 0.1% formic acid). The gradient elution program was set as follows: initial 10% B and held for 5 min; ramped to 30% B within 2 min and held for 4 min; and then ramped to 95% B within 4 min and held for 4 min (total run time: 19 min). Finally, the column was re‐equilibrated to 10% B for 2 min. The flow rate was set to 0.3 mL/min.

A high‐resolution mass spectrometer (Q Extractive HF, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization (HESI) source was used for the mass analysis. Full‐scan (mass range: 100–1000) and data‐dependent tandem mass spectrometry (ddMS²) analyses were performed in positive ion mode, where the system automatically selected the top 7 precursor ions for the product ion analysis. Key parameters were established as follows: collision gas pressure at 0.2 Pa (99.9995% nitrogen), automatic gain control (AGC) target at 1 × 10⁶, maximum injection time (MIT) at 100 ms, isolation window at 2 Da, and stepped normalized collision energy (NCE) values of 20, 40, and 60. The sheath gas and auxiliary gas (99.9% purity) flow rates were set to 40 and 10 arbitrary units, respectively. The vaporizer temperature and capillary temperature were both set to 350°C, the spray voltage was set to +4000 V, and the NCE range was established between 30 and 70. The data acquisition resolution (R) was 60 000 for the full‐scan mode and 30 000 for the ddMS² mode (measured as full width at half maximum (FWHM) at m/z 195.0882).

2.3. Data Processing and Metabolite Identification

Compound Discoverer 3.3 was used for the feature extraction, with precursor and product ion tolerances of ±5 and ±10 ppm, respectively. An inclusion list based on the predicted biotransformations (oxidation, reduction, demethylation, glucuronidation, and sulfation) guided the MS² acquisition. A signal was considered a tiletamine metabolite only if it met all of the following criteria: (i) present in treated samples and absent in controls; (ii) mass accuracy of ≤ 5 ppm; (iii) at least two diagnostic MS² fragments consistent with the tiletamine scaffold; and (iv) reproducible retention time and chromatographic behavior.

2.4. Acquisition of Authentic Urine and Hair Samples

The forensic investigation included a series of cases involving the misuse and distribution of tiletamine, which is frequently administered through e‐cigarette products. Two representative cases were examined: one involving entertainment use and another involving a specific drug sexual assault incident. Urine and hair specimens were collected from all cases, with detailed case descriptions provided in Sections 2.4.1 and 2.4.2. Ethical review was conducted, and the study was approved by the Ethical Committee of the Institute of Evidence Law and Forensic Science, China University of Political Science and Law. Upon collection, all samples were stored at −20°C. They were then transported to the laboratory in a frozen state. Urine and hair samples used as “blank” matrices were obtained from drug‐free healthy volunteers (the authors).

2.4.1. Case 1 (Entertainment Use)

The primary suspect was a 25‐year‐old male who admitted to selling and using e‐cigarettes containing tiletamine. He distributed these devices to multiple acquaintances he met at bars and nightclubs. Through this supply channel, authorities identified a 26‐year‐old secondary suspect. Both suspects confessed to using the substance multiple times over the past 2 months and provided biological samples (urine and hair) within 8 h for toxicological analysis.

2.4.2. Case 2 (Suspected Specific Drug Sexual Assault Incident)

A 21‐year‐old woman was lured by her boyfriend into smoking an e‐cigarette and was sexually assaulted after losing consciousness. Upon regaining consciousness, she contacted the police, and her urine sample was collected within 24 h of exposure.

2.5. Sample Preparation

The preparation of urine samples utilized solid‐phase extraction (SPE) for purification. The specific steps were as follows: The SPE cartridge was activated with 2 mL of methanol and then equilibrated with 2 mL of purified water. After high‐speed centrifugation, 2 mL of the supernatant was loaded onto the cartridge. The cartridge was washed with 2 mL of purified water, and elution was performed with 2 mL of methanol. Following elution, the extract was dried under nitrogen and reconstituted in 200 μL of the initial mobile phase, and a 40 μL aliquot was injected into the liquid chromatography‐high resolution mass spectrometry system.

The preparation of hair samples relied primarily on ultrasonic extraction. The steps were as follows: Two hair samples were each subjected to ultrasonic washing three times with water and twice with methanol. After air‐drying, 20 mg of each hair sample was collected, mixed with 1 mL of methanol, and wet‐ground using a ball mill (3 min of grinding followed by 1 min of rest, repeated three times). The samples were then ultrasonicated for 5 min and centrifuged at high speed for 10 min. The supernatant was collected and evaporated to dryness under a nitrogen stream. The residue was reconstituted in 150 μL of the initial mobile phase, and a 40‐μL aliquot was injected for the analysis.

3. Results and Discussion

3.1. Selection of the Chromatographic Condition and the Parent Drug Analysis

Due to the high polarity of tiletamine, the use of acetonitrile as mobile phase B resulted in inadequate baseline separation of many metabolites. Therefore, methanol was selected as the organic mobile phase to improve chromatographic resolution. To maintain peak symmetry, methanol was supplemented with an equal proportion of 0.1% formic acid. A relatively shallow elution gradient was used to achieve adequate separation of the metabolite peaks, resulting in a retention time of 9.03 min for the parent compound (Figure 2). Because tiletamine contains a chiral center, reduction of the carbonyl group followed by hydroxylation can increase the number of chiral centers in its metabolites to as many as three. Through optimization of chromatographic conditions, multiple stereoisomeric metabolites were successfully separated (Figure S1).

FIGURE 2.

Extracted ion chromatography (EIC) and mass spectrum and proposed fragmentation pattern of tiletamine.

In the positive product ion analysis, two main fragment ions at m/z 179.05209 (C₁₀H₁₁OS, Δppm: −2.33) and 151.05731 (C₉H₁₁S, Δppm: −1.89) originated from the loss of an ethylamine group (45 Da) from the [M + H]⁺ ion (m/z 224.11036), followed by a neutral loss of CO (28 Da). The fragmentation pattern of tiletamine involved retention of the core cyclohexanone parent structure while losing the ethylamine side chain, and the primary dissociation occurred at the C–N bond (Figure 2).

3.2. Identification of Potential Metabolites

3.2.1. Urine Samples

The authentic urine specimens included three samples collected from two different time periods: One female urine sample was collected within 24 h after e‐cigarette use, and two male urine samples were collected within 8 h after the individuals were apprehended. The male subjects reported multiple instances of using tiletamine‐containing e‐cigarettes over the preceding 2 months. A total of 11 novel metabolites were identified in these urine samples (Table 1). The structural elucidation of these metabolites is described below.

TABLE 1.

Information on 11 tiletamine metabolites and their related metabolic patterns, including retention time (in minutes), elemental composition, experimentally determined accurate mass (m/z), mass measurement error (ppm), and characteristic fragment ions employed for identification.

Metabolite ID	Metabolic pattern	Retention time (min)	Elemental composition	Measured accurate mass (m/z)	Mass error (ppm)	Diagnostic product ions
T1	Hydroxylation Methylation	8.41	C13H19NO2S	254.12074	0.72	208 180 165
T2	Di‐hydroxylation Methylation	9.54	C13H19NO3S	270.11600	0.58	252 224.196
T3	Di‐hydroxylation Methylation	11.40	C13H19NO3S	270.11572	−0.43	252 224.196
T4	Di‐hydroxylation Methylation	9.06	C13H19NO3S	270.11572	−0.43	252 224.196
T5	Reduction Glucuronidation	9.49	C18H27NO7S	402.15802	−0.2	181 163
T6	Reduction Glucuronidation	9.62	C18H27NO7S	402.15765	0.2	181 163
T7	Reduction Glucuronidation	10.01	C18H27NO7S	402.15762	−1.1	181 163
T8	Reduction Glucuronidation	10.48	C18H27NO7S	402.15787	−0.57	181 163
T9	Hydroxylation Glucuronidation	6.40	C18H25NO8S	416.13676	−1.47	240 222 204
T10	Hydroxylation Reduction Glucuronidation	7.75	C18H27NO8S	418.15283	−0.4	197 179 151
T11	Hydroxylation Reduction Glucuronidation	9.89	C18H27NO8S	418.15265	−0.87	238 181 163

T1 exhibited an [M + H]⁺ ion at m/z 254.12074 (C₁₃H₁₉NO₂S, Δppm: −0.72), suggesting hydroxylation and methylation. In the product ion analysis, a neutral loss of 46 Da (–CH₂CH₃OH) from the [M + H]⁺ ion to the m/z 208.07921 ion (C₁₁H₁₄NOS, Δppm: 0.70) was observed (Figure 3), indicating that the ethylamine group underwent hydroxylation. Subsequently, a neutral loss of 28 Da (CO) from the m/z 208.07921 ion to the m/z 180.08420 ion (C₁₀H₁₄NS, Δppm: 0.28) indicated the elimination of the carbonyl group. Furthermore, a loss of 15 Da (–CH₃) from the m/z 180.08420 ion to the m/z 165.06081 ion (C₉H₁₁NS, Δppm: 0.81) revealed that methylation also occurred on the ethylamine group. Therefore, T1 was identified as a hydroxylated and methylated derivative of tiletamine.

FIGURE 3.

Mass spectra and proposed fragmentation patterns of 14 tiletamine metabolites (T1–T14).

Three metabolites (T2–T4) with retention times of 9.54, 11.40, and 9.06 min exhibited the same [M + H]⁺ ion at m/z 270.11600 (C₁₃H₁₉NO₃S, Δppm: 0.58), suggesting di‐hydroxylation and methylation. Their fragmentation patterns yielded similar product ion spectra, with the primary difference being that the signal intensities of the dehydration peaks at m/z 252.10541 (C₁₃H₁₈NO₂S, Δppm: 0.52) for T2 and T3 were considerably lower than that for T4 (Figure 3). Compared with the T4 product ion mass spectra, the ions at m/z 224.07368 (C₁₁H₁₄NO₂S, Δppm: −1.3), 196.07903 (C₁₀H₁₄NOS, Δppm: −0.18), and 181.05556 (C₉H₁₁NOS, Δppm: −0.16) were each 16 Da higher than the corresponding T1 product ions at m/z 208.07921, 180.08420, and 165.06079, respectively. This indicated that these ions contained one additional oxygen atom compared to their T1 counterparts. The presence of a dehydration peak at m/z 252.10541 suggested that hydroxylation occurred on the cyclohexanone ring. Therefore, T2–T4 were identified as di‐hydroxylated and methylated tiletamine metabolites.

Four metabolites (T5–T8) with retention times of 9.51, 9.62, 10.01, and 10.48 min all displayed the same [M + H]⁺ ion at m/z 402.15802, corresponding to the molecular formula C₁₈H₂₈NO₇S (Δppm: −0.2). This indicated reduction followed by tiletamine glucuronidation. The product ion spectra of these four metabolites closely resembled those of three reduced metabolites (Figure S2), suggesting the presence of four optical isomers. Accordingly, T5–T8 (Figure S3) were identified as reduced and glucuronidated tiletamine metabolites.

The [M + H]⁺ ion of T9 was observed at m/z 416.13676, corresponding to the molecular formula C₁₈H₂₆NO₈S (Δppm: −1.47). This suggested the hydroxylation and glucuronidation of tiletamine. Three major product ions were generated upon fragmentation of T9: m/z 240.10513 (C₁₂H₁₈NO₂S, Δppm: −0.6), 222.09473 (C₁₂H₁₆NOS, Δppm: 0.07), and 204.08418 (C12H14NS, Δppm: 0.17). The ion at m/z 240.10513 resulted from the neutral loss of 176 Da (C₆H₈O₆) from the precursor ion, while the two dehydration ions at m/z 222.09473 and 204.08418 further indicated that hydroxylation occurred on the cyclohexanone ring. Accordingly, T9 was identified as a hydroxylated and glucuronidated metabolite of tiletamine.

T10 and T11 both exhibited an [M + H]⁺ ion at m/z 418.15283 (C₁₈H₂₈NO₈S, Δppm: −0.4, RT: 7.75 and 9.88 min), suggesting the reduction, hydroxylation, and glucuronidation of tiletamine. The T10 fragmentation pattern yielded product ions at m/z 197.06306 (C₁₀H₁₃O₂S, Δppm: −0.06), 179.05252 (C₁₀H₁₁OS, Δppm: −0.05), and 151.05763 (C₉H₁₁S, Δppm: 0.23). The ion at m/z 197.06306 was 16 Da higher than the characteristic product ion at m/z 181.06796, indicating that it contained one additional oxygen atom. The remaining fragment ions were similar to those observed for tiletamine, suggesting that hydroxylation occurred on the carbon adjacent to the original hydroxyl‐bearing carbon (i.e., the α‐carbon). Following the loss of a water molecule, the ion at m/z 197.06306 formed an enol intermediate that subsequently rearranged into a carbonyl structure (Figure 3). This fragmentation behavior was consistent with that of tiletamine, which was dissociated to produce two characteristic product ions at m/z 179.05252 and 151.05763. Fragmentation of T11 generated three major product ions at m/z 238.09192 (C₈H₁₆O₇N, Δppm: −0.88), 181.06818 (C₁₀H₁₃OS, Δppm: −0.08), and 163.05771 (C₁₀H₁₁S, Δppm: −0.68). The ion at m/z 238.09192 originated from the loss of the (2‐thienyl) cyclohexanone moiety from the [M + H]⁺ ion, retaining the glucuronic acid unit and the ethylamine group. The other two ions at m/z 181.06818 and 163.05771 were identical to those observed for the three reduced metabolites (Figure S2).

3.2.2. Hair Samples

In hair samples, tiletamine and four metabolites, including three reduced tiletamine metabolites and T1 (hydroxylated and methylated tiletamine), were identified in two hair specimens (Figure 1). Tiletamine and the three reduced tiletamine metabolites were detected in all samples, and the tiletamine response was substantially higher than that of the reduced tiletamine metabolites in each sample. Hydroxylated and methylated tiletamine T1 was detected in only two samples, and the response was lower than that of tiletamine and the reduced tiletamine metabolites.

3.2.3. Proposed Metabolic Pathways and Comparative Analysis

According to the available literature, previously reported tiletamine metabolic pathways include three phase I reactions—hydroxylation, reduction, and desethylation—whereas no phase II metabolites have been identified to date. In the present study, a total of 14 metabolites (11 novel metabolites) were identified in urine samples, involving two phase I reactions (hydroxylation and reduction) and two phase II reactions (methylation and glucuronidation), both of which represent novel metabolic pathways for tiletamine. Hydroxylation was the predominant phase I pathway, while glucuronidation was the major phase II pathway. Although hydroxylation had been documented, the specific sites of hydroxylation were not identified in earlier studies. In this investigation, in addition to hydroxylation occurring on the cyclohexanone ring, hydroxylation sites were also localized on the methyl group of the ethylamine moiety (metabolites T1, T2–T4), the nitrogen atom (T11), and the carbon atom adjacent to the carbon bearing the original hydroxyl group (i.e., the α‐carbon, T10). In hair samples, four metabolites were detected, primarily involving three metabolic pathways: hydroxylation, reduction, and methylation, with reduction being the dominant pathway. This study also provides the first comparative analysis of tiletamine metabolic profiles in human urine and hair samples. Notably, the parent drug was detected in hair samples but not in the three authentic urine samples, and its presence in hair further supported the urinary analytical findings. Given that the response of metabolite T1 in all three authentic urine samples was significantly higher than those of the other metabolites, T1 can be considered a potential biomarker for monitoring tiletamine misuse.

3.2.4. Proposed Fragmentation Pattern

The cleavage of tiletamine primarily occurred at the C–N bond, resulting in the retention of the cyclohexanone ring and the loss of the ethylamine group. Neutral loss was also the predominant fragmentation pathway for tiletamine, identified by the loss of 45 Da (HNCH₂CH₃) and 46 Da (CH₂CH₃OH). These losses generated two characteristic fragment ions at m/z 181.06818 and 179.05252 that served as important markers for identifying tiletamine metabolites. Additionally, the loss of 15 Da was identified as a key indicator of N‐methylation. For the glucuronidated metabolites, a neutral loss of 176 Da, corresponding to the loss of glucuronic acid, was typically observed and served as a major diagnostic glucuronidation feature.

3.3. Limitations and Future Work

It should be noted that the e‐cigarette liquids were not analyzed for impurities or thermal degradation products; hence, potential interference from byproducts or pyrolysis artifacts could not be completely excluded. Nevertheless, this limitation did not compromise the definitive identification of tiletamine itself in the biological specimens. Currently, the structural characterization of the identified tiletamine metabolites is restricted by the inherent limitations of LC‐HRMS, particularly in differentiating isomeric hydroxylated and reduced species. Future work should prioritize the large‐scale synthesis or biosynthetic production of these metabolites, with special emphasis on the potential urinary biomarker T4, which is critical for tracing tiletamine misuse. This will enable the application of orthogonal analytical techniques, such as nuclear magnetic resonance spectroscopy and tandem high‐resolution mass spectrometry with ion mobility, for an unequivocal structural assignment and differentiation of positional isomers. Furthermore, subsequent studies should involve the chemical synthesis of authentic reference standards to systematically evaluate matrix effects, determine limits of detection and quantification, and assess the stability of both the parent drug and metabolites under relevant biological matrices (e.g., human urine, whole blood, and liver microsomes). These endeavors are indispensable for translating this metabolic profiling approach into practical applications in clinical toxicology and forensic casework laboratories.

4. Conclusion

In this study, the metabolic profiles of tiletamine in human urine and hair samples were investigated using LC‐QE‐HF‐MS. The metabolic profile of tiletamine in human hair was identified for the first time based on analyses of three authentic urine samples and two hair samples. A total of 14 urinary metabolites were identified based on their fragmentation patterns obtained from full‐scan MS and ddMS² experiments. Among them, 11 metabolites, including 6 phase I and 7 phase II metabolites, had not been previously reported. Two phase I metabolic reactions (hydroxylation and reduction) and two phase II reactions (methylation and glucuronidation) were observed, with methylation and glucuronidation being unreported metabolic pathways. Hydroxylation was the primary phase I pathway, and glucuronidation was the primary phase II pathway in human urine. The hydroxylation sites of six hydroxylated metabolites (T1, T2–T4, T10, and T11) were also identified for the first time based on their characteristic fragment ions. In hair samples, tiletamine itself and three metabolites, comprising two reduced metabolites and T1, are reported here for the first time. In addition, methylation was identified as a previously unreported metabolic pathway in hair. Given that T1 exhibited a substantially higher response than the other metabolites in the three authentic urine samples, it may represent a promising candidate biomarker for monitoring tiletamine exposure.

Author Contributions

Shiyang Qin: conceptualization, methodology, software. Guibin Bi: investigation. Yuanfeng Wang: supervision. Bing Liu: investigation, validation, project administration. Zhenjia Cui: project administration, validation, investigation. Xuxiao Zhao: validation. Xiaopei Wu: methodology, validation. Jing Jing: formal analysis. Jianghai Lu: conceptualization, methodology, validation, project administration, writing – review and editing, writing – original draft.

Supporting information

Figure S1: Extracted ion chromatography (EIC) of 14 tiletamine tabolites (T1–T14).

Figure S2: Product ion mass spectra of three tiletamine reduced metabolites.

Figure S3: Product ion mass spectra of novel metabolites (T6, T7, T8).

Data Availability Statement

Research data are not shared.