Detomidine, a potent α2-adrenergic receptor agonist, is rapidly absorbed due to its high lipophilicity, rendering it more effective as a sedative and analgesic in horses than xylazine (Grimsrud et al. 2009; Mama et al. 2009). Additionally, detomidine is known for its powerful cardiovascular effects, including bradycardia, increased systemic and pulmonary vascular resistance, and the induction of a state of hypertension (Grimsrud et al. 2009; Knych and Stanley 2014).

The International Federation of Horseracing Authorities, which regulates the use and practices of prohibited substances in horse racing, prohibits the administration of detomidine during periods that may affect racing performance. To reasonably control the detection levels of therapeutic drugs that do not affect the performance of racehorses, they have established International Screening Limits (ISLs) at the screening stage (International Federation of Horseracing Authorities 2018). The adoption of ISLs is subject to the consent of each racing authority, and most of them apply these standards. Since detomidine is rapidly metabolized in the body, detection of the parent drug is only feasible for a short duration; hence, the ISL is set at 0.02 ng/mL for the plasma concentration of its metabolite, 3-hydroxy detomidine.

In doping control, metabolites are important evidence for positive results (Kwak and Choi 2023; Kwak et al. 2023a; 2024a; Lee et al. 2023). The biotransformation of detomidine involves the aliphatic hydroxylation of detomidine to generate 3-hydroxy detomidine. Subsequently, 3-hydroxy detomidine undergoes an additional dehydrogenation reaction to produce 3-carboxy detomidine (Grimsrud et al. 2009). When reviewing previously published papers on the administration of detomidine in horses and the resulting profiles in blood, an important observation emerges from the fact that 3-carboxy detomidine is detected following 3-hydroxy detomidine (Grimsrud et al. 2009). This result is considered significant as it may suggest a potential increase in the detection window for doping control.

This study aims to investigate the pharmacokinetics of detomidine and its metabolites in horses after intravenous (IV) administration, with a focus on the detection profile in plasma, to prevent drug abuse. For doping control of detomidine, the metabolites 3-hydroxy detomidine and 3-carboxy detomidine were monitored together to evaluate their potential usefulness.

Materials

The reference standards of detomidine hydrochloride, 3-hydroxy detomidine hydrochloride, 3-carboxy detomidine hydrochloride, and 3-hydroxy detomidine-d4 hydrochloride (internal standard, ISTD) were purchased from the Matreya LLC (Pleasant Gap, PA, USA). HPLC-grade acetonitrile (ACN), HPLC-grade methanol (MeOH), and HPLC-grade distilled water (DW) were purchased from J.T. Baker (Phillipsburg, NJ, USA). LC-MS-grade formic acid (FA) and LC-MS-grade ammonium formate were purchased from Fisher Scientific (Bremen, Germany).

Preparation of standard solutions

Stock solutions containing detomidine, metabolites (3-hydroxy detomidine, 3-carboxy detomidine), and the ISTD were prepared at a concentration of 10 mg/mL each in ACN and stored at –20°C until required. The stock solutions of detomidine and metabolites were individually diluted with ACN and combined to generate working standard solutions. The ISTD solution was diluted with ACN to formulate a working standard, achieving a final concentration of 3 µg/mL. The blank equine plasma was prepared by pooling equine plasma samples that were determined to be negative in the tests conducted on blood samples collected after the race. For the calibration standards, the working standard solutions were introduced to equine plasma, resulting in concentrations of 0.2, 10, 100, 500, and 1,000 ng/mL for detomidine and 0.5, 10, 100, 1,000, and 2,000 ng/mL for metabolites. Quality control (QC) samples were prepared by utilizing working solutions, using a final concentration of 50 ng/mL.

Protein precipitation of plasma

A plasma sample (100 µL) underwent the addition of 2 µL of the ISTD working solution. Following this, plasma proteins were precipitated by the addition of 200 µL of MeOH and vortexed for 30 sec. The resulting mixture was then centrifuged at 59,000 rcf for 5 min. The supernatant was subsequently transferred to a vial for LC-MS/MS analysis.

LC-MS ⁄MS instrumentation and conditions

Chromatographic separation utilized an Agilent 1200 series high-performance liquid chromatography (HPLC) system (Agilent Technologies, Palo Alto, CA), comprising a binary pump, an autosampler, and a column oven with the cooling system. The analytical column employed was a ZORBAX Eclipse XDB-C8 column (3.0 × 100 mm, 3.5 µm; Agilent Technologies), with the column oven temperature maintained at 30°C. The mobile phase consisted of 5 mM ammonium formate (pH 3.0) in DW (solvent A) and 0.1% FA in MeOH (solvent B). A gradient program was employed at a flow rate of 0.5 mL/min: starting with 20% solvent B for 1 min, transitioning to 20%-90% solvent B over 6 min, maintaining 90% solvent B for 2 min, returning to 20% solvent B over 1 min, and finally held at 20% solvent B for 3 min. The injection volume was set at 2.5 µL. The HPLC system was coupled with an API 4000 instrument (SCIEX, Toronto, Canada) featuring an electrospray ionization (ESI) source. ESI was conducted in positive mode with nitrogen serving as the curtain gas. The ion spray voltage, temperature, ion source gas 1, and ion source gas 2 were optimized at 5,400 V, 550°C, 55 psi, and 55 psi, respectively. Multiple reaction monitoring (MRM) detection was implemented, and the detection parameters for each target substance are detailed in Table 1. Data acquisition and processing were managed using Analyst software (AB SCIEX, Darmstadt, Germany).

Table 1 . Retention time and multiple reaction monitoring conditions for detomidine, its metabolites, and internal standard.

Compound	RT (min)	Polarity	Precursor ion (m/z)	Product ion (m/z)	DP (ms)	CE (V)
Detomidine	5.69	Positive	187.4	81.2	80	80
3-hydroxy detomidine	2.45	Positive	203.2	185.0	40	40
3-carboxy detomidine	2.41	Positive	217.1	199.1	40	40
3-hydroxy detomidine-d4 (ISTD)	2.45	Positive	207.2	81.2	40	40

RT, retention time; DP, declustering potential; CE, collision energy; MRM, multiple reaction monitoring..

Method validation

The validation method for the analysis was based on the methods described in previously published papers (Kwak et al. 2022a; 2022b; 2022c; 2023b; 2023c). Calibration curves were constructed utilizing five or six concentrations tailored to the target substances. The linearity of the curve was determined by plotting the peak area ratio of the analyte to the ISTD against the nominal analyte concentration. The lowest point on the calibration curve was defined as the lower limit of quantification (LLOQ), demonstrating satisfactory above signal-to-noise 9. Method precision and accuracy were appraised by measuring the concentrations of QC samples (n = 3) at 50 ng/mL within a single day. The precision values are expressed as the mean ± relative standard deviation (RSD). Evaluation of stability was conducted at –4°C for 7 days.

Pharmacokinetic study in horses

Approval for all animal procedures was granted by the Korea Racing Authority (IACUC-1803, AEC-1703). Two female thoroughbred horses, aged 10-12 years and weighing between 450-500 kg, received a single IV bolus of detomidine (Provet) at a dosage of 30 μg/kg. Blood samples (10 mL) were collected from the jugular vein into heparin tubes at various time points: 1, 2, 4, 6, 10, 15, 20, 30, 45 min, and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 18, 24, 36, and 48 h post-administration. The collected blood was centrifuged at 4,000 rcf for 5 min at 4°C, and the resulting plasma supernatant was transferred to a 15 mL conical tube. The plasma was then stored at –20°C until further pretreatment.

The kinetic parameters of detomidine and its metabolites from equine plasma samples were determined using a non-compartmental statistical model with the Phoenix WinNonlin^TM Enterprise Program v5.3 (Pharsight Inc., St. Louis, MO, USA). The substance concentration versus time profiles were graphed using GraphPad Prism 6 (San Diego, CA, USA). All presented data represent the mean ± standard deviation (SD; n = 2).

Method optimization

A selective and sensitive LC-MS/MS method was developed to quantify detomidine and metabolites in horse plasma. To ensure the accuracy and precision of the assay, the deuterated compound of the metabolite 3-hydroxy detomidine was utilized as the ISTD in the study. Detection signal optimization was performed in positive ESI mode. The MRM transitions monitored were 187.4→81.2 for detomidine, 203.2→185.0 for 3-hydroxy detomidine, 217.1→199.1 for 3-carboxy detomidine, and 207.2→81.2 for 3-hydroxy detomidine-d4, respectively. All analytes showed good peak shapes and were eluted within 6 min (Fig. 1). The retention times for detomidine, 3-hydroxy detomidine, 3-carboxy detomidine, and 3-hydroxy detomidine-d4 were 5.69, 2.45, 2.41, and 2.45 min, respectively (Table 1).

Figure 1. MRM chromatograms of detomidine and its metabolites. (A) Detomidine at 0.5 ng/mL, (B) 3-hydroxy detomidine at 0.2 ng/mL, (C) 3-carboxy detomidine at 0.2 ng/mL in plasma, and (D) 3-hydroxy detomidine-d4 (ISTD) at 20 ng/mL. MRM, multiple reaction monitoring; ISTD, internal standard.

Method validation

The MRM chromatograms of equine plasma spiked with detomidine, 3-hydroxy detomidine, and 3-carboxy detomidine at LLOQ concentrations are depicted in Fig. 1. LLOQ for detomidine in equine plasma was 0.5 ng/mL, while for 3-hydroxy detomidine and 3-carboxy detomidine, it was 0.2 ng/mL. The linearity was demonstrated for detomidine in the concentration range of 0.5-1,000 ng/mL and for 3-hydroxy detomidine in the range of 0.2-2,000 ng/mL, as well as for 3-carboxy detomidine in the range of 0.2-2,000 ng/mL, all showing good linearity above an r² value of 0.9920. The QC samples were assayed for precision, accuracy, and stability at 50 ng/mL (n = 3). The precisions were within 1.5% and 2.9%, and accuracies were 94.1-99.3% for all substances. In the stability test of the target substances at –4°C for 7 days, stable results were obtained with a range of 97.1-103.6%. The validation results are summarized in Table 2.

Table 2 . Precision, accuracy, and stability for detomidine and its metabolites in equine plasma.

Compound	LLOQ (ng/mL)	Linearity range (ng/mL)	Correlation coefficient, r2	Precision (RSD, %)	Accuracy (%)	Stability (–4°C, 7 days, %)
Detomidine	0.5	0.5-1,000	0.9941	1.5	94.1	100.8 ± 2.8
3-hydroxy detomidine	0.2	0.2-2,000	0.9978	2.9	98.2	101.0 ± 1.4
3-carboxy detomidine	0.2	0.2-2,000	0.9928	1.9	99.3	100.3 ± 3.2

LLOQ, the lower limit of quantification; RSD, relative standard deviation. Precision, accuracy, and stability were measured at a quality control concentration of 50 ng/mL..

Pharmacokinetic profiling in plasma

The metabolic pathway and the plasma concentration of detomidine and metabolites in horses over time, after IV administration at a dose of 30 μg/kg are shown in Fig. 2. The result of calculated pharmacokinetic parameters of detomidine and metabolites in equine plasma are described in Table 3. When examining the plasma concentration of detomidine in horses, detomidine was detected at levels above LLOQ of 0.5 ng/mL within 6 hours after a single IV administration. The C_max in plasma reached 234.5 ± 155.9 ng/mL, with a measured t_1/2 of 0.52 ± 0.1 h. The area under AUC_0-t and CL values were 64.5 ± 13.4 h·ng/mL and 60.0 ± 12.0 mL/h/kg, respectively. The metabolite 3-hydroxy detomidine was monitored at levels above the LLOQ of 0.2 ng/mL for 48 h, with a C_max of 6.3 ± 3.6 ng/mL reached at T_max of 0.8 ± 0.0 h and a half-life of 8.5 ± 8.7 h. The AUC_0-t value was 31.0 ± 12.2 h·ng/mL. Similarly, the metabolite 3-carboxy detomidine was monitored at levels above the LLOQ of 0.2 ng/mL for 48 hours, with a C_max of 4.0 ± 0.8 ng/mL reached at T_max of 3.0 ± 0.0 h and a half-life of 17.4 ± 4.5 h. The AUC_0-t values were 74.7 ± 2.9 h·ng/mL.

Figure 2. Pharmacokinetic profile of detomidine and its metabolites. (A) Chemical structures and metabolic pathways of detomidine and its metabolites, and (B) the mean plasma concentration vs. time profiles of detomidine and its metabolites following intravenous (IV) injection at 30 μg/kg of detomidine (Provet) in the horse (n = 2).

Table 3 . Pharmacokinetic parameters of detomidine and metabolites in equines (n = 2) after intravenous (IV) administration of detomidine (30 μg/kg).

Pharmacokinetic parameters	Detomidine	3-hydroxy detomidine	3-carboxy detomidine
Tmax (h)	-	0.8 ± 0.0	3.0 ± 0.0
Cmax (ng/mL)	234.5 ± 155.9	6.3 ± 3.6	4.0 ± 0.8
AUC0-t (h·ng/mL)	64.5 ± 13.4	31.0 ± 12.2	74.7 ± 2.9
t1/2 (h)	0.52 ± 0.1	8.5 ± 8.7	17.4 ± 4.5
CL (mL/h/kg)	60.0 ± 12.0	-	-

Data are presented as the mean ± standard deviation (n = 2)..

In this study, an LC-MS/MS method was employed for the quantitative measurement of detomidine and its metabolites in equine plasma. The method validation for quantification was confirmed through good linearity (0.5-1,000 ng/mL or 0.2-2,000 ng/mL), accuracy (94.1-99.3%) with precision (1.5-2.9%), and stability (97.1-103.6%). Following IV administration of 30 μg/kg detomidine, the pharmacokinetic profile in equine plasma was successfully assessed.

The investigation of sensitive and stable drug detection methods is crucial in horse racing to ensure fair competition. This principle is equally applicable to other animal competitions, including those involving humans, emphasizing the importance of fairness. Detomidine, while having a low risk of addiction, is often used in combination with other prohibited drugs, psychotropics, or opioids, making it a potential indicator of the use of these substances (Schatzman et al. 2001; Valverde 2010). Moreover, an overdose of detomidine can cause human poisoning, necessitating strict regulation of this drug (Cummins 2005). In this context, this study focuses on the detection and analysis of detomidine metabolites, a commonly used short- acting tranquilizer and sedative in horses.

The lowest screening detection limit of detomidine and 3-hydroxy detomidine in equine plasma through officially published papers is from the method developed by Ho et al., employing solid phase extraction and high-resolution Orbitrap MS analysis (limit of detection, LOD; 0.03 ng/mL, 0.01 ng/mL, respectively) (Ho et al. 2013). While there may be an unofficial laboratory-specific LOD, considering the current recommended ISL of 0.02 ng/mL, the detection of 3-hydroxy detomidine in laboratories is likely to be determined as a positive result. In this study, detomidine exhibited concentrations above the LLOQ for 6 hours, and both metabolites, 3-hydroxy detomidine, and 3-carboxy detomidine, were detected throughout the experimental period (48 hours). Given the calculated LLOQ exhibited a signal-to-noise ratio above 9, it is expected that the detection period based on ISL concentration will extend beyond this. This expectation takes into account that the ISL is based on the LOD, which generally adopts concentrations with a signal-to-noise ratio of 3 or higher. These findings suggest the utility of monitoring 3-hydroxy detomidine and 3-carboxy detomidine for controlling detomidine misuse. However, considering the longer half-life and the greater AUC of 3-carboxy detomidine, it can be a more potentially useful substance for doping tests than 3-hydroxy detomidine.

Despite the successful detection of drugs and metabolites during the drug monitoring period (48 hours), this study still has limitations. In the experiment, the LLOQ for 3-hydroxy detomidine was measured at 0.2 ng/mL, and assuming the LOD is one-third of the LLOQ, it amounts to approximately 0.067 ng/mL, which is higher than the ISL concentration of 0.02 ng/mL. Therefore, the analytical method used in this study may encounter difficulties in detecting 3-hydroxy detomidine at concentrations between the ISL and the LOD. To address this issue, potential solutions include adding the concentration step in the sample preparation process, employing other analytical techniques such as nano LC-MS that offer higher sensitivity and specificity, utilizing the latest version of high-performance analytical instrument.

The authors declare that they have no conflict of interest.

This study was supported by the Horse Industry Research Center of the Korea Racing Authority and the National Research Foundation of Korea (NRF) grant funded by the Korea government (2023K2A9A1A01098682).