Structural elucidation of tramadol, its derivatives, and metabolites using chemical derivatization and liquid chromatography-high-resolution tandem mass spectrometry.

RATIONALE: Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information-limited mass spectra with one prominent peak representing the amine-containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI-MS) and ESI-MS/MS spectra could be misleading. METHODS: A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N-desmethyltramadol and O-desmethyltramadol) was developed using chemical modification and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) with Orbitrap technology. RESULTS: After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N-desmethyltramadol metabolite in urine samples at low ng/mL levels. CONCLUSIONS: An efficient derivatization-aided strategy was developed for rapidly elucidating the structure of tramadol-like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories.

Nickel oxide nanoparticles modified with dimethylglyoxime grafted on a cellulose surface as an efficient adsorbent for thin film microextraction of tramadol in biological fluids followed by its determination using HPLC.

In the current study, nickel oxide nanoparticles (NiO NPs) modified with dimethylglyoxime (DMG) were deposited onto the cellulose surface (Ni(DMG)2-NiO-Cell) and used as an efficient adsorbent for thin film microextraction (TFME) of tramadol (TRA). The extracted TRA was determined using a high-performance liquid chromatography-ultraviolet detector (HPLC-UV). NiO NPs were synthesized by co-precipitation method on the surface of the cellulose substrate; afterward, its surface was modified by DMG to increase the extraction capability of the thin film toward TRA. The synthesized NiO-Cell and Ni(DMG)2-NiO-Cell thin films were characterized using various techniques. The effect of modification of the NiO thin film with DMG reagent on the extraction efficiency was investigated. The crucial parameters influencing the extraction efficiency, including extraction time, desorption time, desorption solvent, pH and salt content, were investigated via a one-at-a-time approach. The figures of merit for the developed method were evaluated in urine, plasma, and deionized water under the optimized extraction and desorption condition. The limits of detection and limits of quantification were in the range of 0.1 to 1 ng mL-1 and 0.3 to 3 ng mL-1, respectively, for the studied samples. The linear dynamic ranges of the developed TFME-HPLC-UV method were 0.3-1000, 1-2500, and 3-5000 ng mL-1 for the deionized water, urine, and plasma samples, respectively. The reproducibility and repeatability of the developed method was assayed in terms of intra-day, inter-day, and inter-thin film precisions by conducting six-replicate experiments at the concentration level of 0.1 and 1 µg mL-1, which were in the range of 5.9% to 8.3%. The sufficiency and applicability of the developed TFME-HPLC-UV method was investigated by determining TRA in urine and plasma samples, and the resulting relative recoveries (RR%) were 85.9% and 91.7%, respectively.

Comprehensive overview of analytical and bioanalytical methodologies for the opioid analgesics - Tramadol and combinations.

Synthetic opioids like Tramadol are used to treat mild to moderate pain. Its ability to relieve pain is about a tenth that of morphine. Furthermore, Tramadol shares similar effects on serotonin and norepinephrine to several antidepressants known as serotonin-norepinephrine reuptake inhibitors (SNRIs), such as venlafaxine and duloxetine. The present review paper discusses the recent developments in analytical methods for identifying drugs in pharmaceutical preparations and toxicological materials, such as blood, saliva, urine, and hair. In recent years, a wide variety of analytical instruments, including capillary electrophoresis, NMR, UV-visible spectroscopy, HPTLC, HPLC, LC-MS, GC, GC-MS, and electrochemical sensors, have been used for drug identification in pharmaceutical preparations and toxicological samples. The primary quantification techniques currently employed for its quantification in various matrices are highlighted in this research.

Ultra-fast retroactive processing by MetAlign of liquid chromatography/high-resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program.

RATIONALE: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. METHODS: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). RESULTS: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. CONCLUSIONS: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.

Ecologically evaluated and FDA-validated HPTLC method for assay of pregabalin and tramadol in human biological fluids.

The introduced research presents a novel in vivo quantitative method for assay of mixtures of pregabalin and tramadol as a common combinations approved for treatment of neuropathic pain. Green analytical chemistry is a recently emerging science concerned with control of the use of chemicals harmful to the environment in various analytical methods. Consequently, a green high-performance thin layer chromatography (HPTLC) method was achieved for determination of the mixture in human plasma and urine satisfying both analytical and environmental standards. The separation was achieved on HPTLC sheets using a separating mixture of ethanol-ethyl acetate-acetone-ammonia solution (8:2:1:0.05, by volume) as a mobile phase. The sheets were dried in air then scanned at two wavelengths. For tramadol, 220 nm was chosen; however, pregabalin is an unconjugated drug, so its determination was a challenge. Hence for pregabalin, the plates were sprayed with ethanolic solution of ninhydrin (3%, w/v), to obtain a conjugated complex, which could be assessed at 550 nm. Furthermore, the developed method fulfilled the US Food and Drug Administration validation guidelines, and proved to be useful in therapeutic drug monitoring of this combination. The Eco-scale assessment protocol was implemented to determine the greenness profile of the applied method.

A hierarchical 3D camellia-like molybdenum tungsten disulfide architectures for the determination of morphine and tramadol.

A practical technique was applied to fabricate MoWS2 nanocomposite through a one-pot hydrothermal method for use as the electrocatalyst. The characterization of MoWS2 nanocomposite was investigated by several techniques to identify the size, crystal structure, and elemental composition. MoWS2 nanocomposite exhibited a unique and well-defined hierarchical structure with neatly and densely piled nanopetals acting as the active sites in the electrocatalytic reactions. A carbon screen-printed electrode (CSPE) modified with interesting MoWS2 nanopetals (MoWS2/CSPE) was constructed. Subsequently, the electrochemical oxidation of morphine on fabricated MoWS2/CSPE was studied. Experimental results confirm that under optimized conditions, the maximum oxidation current of morphine occurs at 275 mV in the case of MoWS2/CSPE that is around 100 mV more negative than that observed in the case of the unmodified CSPE and about 2.6 times increase was observed for the oxidation peak current. The analytical approach was obtained by differential pulse voltammetry in accordance with the relationship between the oxidation peak current and the morphine concentration. The oxidation peak currents for morphine were found to vary linearly with its concentrations in the range of 4.8 × 10-8-5.05 × 10-4 M with the detection limit of 1.44 × 10-8 M. Two completely separated signals occured at the potentials of 275 mV and 920 mV for oxidation of morphine and tramadol at the surface of MoWS2/CSPE which are sufficient for determination of morphine in the presence of tramadol. The presence of morphine was also detected in real samples using the introduced approach. Graphical abstract Schematic representation of fabrication of the MoWS2 nanocomposite through a one-pot hydrothermal method for use as the electrocatalyst. A carbon screen-printed electrode was modified with MoWS2 nanocomposite. Subsequently, the electrochemical oxidation of morphine on the fabricated electrode was studied.

A screen-printed electrochemical sensing platform surface modified with nanostructured ytterbium oxide nanoplates facilitating the electroanalytical sensing of the analgesic drugs acetaminophen and tramadol.

An electrochemical sensing platform based upon screen-printing electrodes (SPEs) modified with nanostructured lanthanide metal oxides facilitate the detection of the widely misused drugs acetaminophen (ACP) and tramadol (TRA). Among the metal oxides examined, Yb2O3 nanoplates (NPs) were found to give rise to an optimal electrochemical response. The electroanalysis of ACP and TRA individually, and within mixtures, was performed using cyclic and differential pulse voltammetry. The ACP and TRA exhibited non-overlapping voltammetric signals at voltages of +0.30 and + 0.67 V (vs. Ag/AgCl; pH 9) using Yb2O3-SPEs. Pharmaceutical dosage forms and spiked human fluids were analyzed in wide linear concentration ranges of 0.25-654 and 0.50-115 µmol.L-1 with limits of detection (LOD) of 55 and 87 nmol.L-1 for ACP and TRA, respectively. The Yb2O3-SPEs offer a sensitive and chemically stable enzyme-free electrochemical platform for ACP and TRA assay. Graphical abstractSchematic presentation of one-shot electrochemical analysis of misused drugs, tramadol (TRA) and acetaminophen (ACP) by utilizing ytterbium oxide nanoplates modified screen-printed electrodes (Yb2O3-SPEs). The Yb2O3-SPEs showed interesting responses for ACP and TRA within pharmaceutical formulations and human fluids.

Tramadol Does Not Improve Performance or Impair Motor Function in Trained Cyclists.

PURPOSE: To investigate the hypothesis that a therapeutic oral dose of Tramadol improves cycling time trial performance and compromises motor-cognitive performance in highly trained cyclists. METHODS: Following two familiarization trials, 16 highly trained cyclists completed a preloaded time trial (1 h at 60% of peak power followed by a 15-km time trial) after ingestion of 100 mg Tramadol or placebo in a double-blind placebo-controlled counterbalanced crossover design separated by at least 4 d washout. Visuomotor tracking and math tasks were completed during the preload (n = 10) to evaluate effects on cognition and fine motor performance. RESULTS: Time trial mean power output (298 ± 42 W vs 294 ± 44 W) and performance (1474 ± 77 s vs 1483 ± 85 s) were similar with Tramadol and placebo treatment, respectively. In addition, there were no differences in perceived exertion, reported pain, blood pH, lactate, or bicarbonate concentrations across trials. Heart rate was higher (P < 0.001) during the Tramadol time trial (171 ± 8 bpm) compared with placebo (167 ± 9 bpm). None of the combined motor-cognitive tasks were impaired by Tramadol ingestion, in fact fine motor performance was slightly improved (P < 0.05) in the Tramadol trial compared with placebo. CONCLUSIONS: In highly trained cyclists, ingestion of 100 mg Tramadol does not improve performance in a 15-km cycling time trial that was completed after a 1-h preload at 60% peak power. Additionally, a therapeutic dose of Tramadol does not compromise complex motor-cognitive or simple fine motor performances.

Oral Coadministration of Fluconazole with Tramadol Markedly Increases Plasma and Urine Concentrations of Tramadol and the O-Desmethyltramadol Metabolite in Healthy Dogs.

Tramadol is used frequently in the management of mild to moderate pain conditions in dogs. This use is controversial because multiple reports in treated dogs demonstrate very low plasma concentrations of O-desmethyltramadol (M1), the active metabolite. The objective of this study was to identify a drug that could be coadministered with tramadol to increase plasma M1 concentrations, thereby enhancing analgesic efficacy. In vitro studies were initially conducted to identify a compound that inhibited tramadol metabolism to N-desmethyltramadol (M2) and M1 metabolism to N,O-didesmethyltramadol (M5) without reducing tramadol metabolism to M1. A randomized crossover drug-drug interaction study was then conducted by administering this inhibitor or placebo with tramadol to 12 dogs. Blood and urine samples were collected to measure tramadol, tramadol metabolites, and inhibitor concentrations. After screening 86 compounds, fluconazole was the only drug found to inhibit M2 and M5 formation potently without reducing M1 formation. Four hours after tramadol administration to fluconazole-treated dogs, there were marked statistically significant (P < 0.001; Wilcoxon signed-rank test) increases in plasma tramadol (31-fold higher) and M1 (39-fold higher) concentrations when compared with placebo-treated dogs. Conversely, plasma M2 and M5 concentrations were significantly lower (11-fold and 3-fold, respectively; P < 0.01) in fluconazole-treated dogs. Metabolite concentrations in urine followed a similar pattern. This is the first study to demonstrate a potentially beneficial drug-drug interaction in dogs through enhancing plasma tramadol and M1 concentrations. Future studies are needed to determine whether adding fluconazole can enhance the analgesic efficacy of tramadol in healthy dogs and clinical patients experiencing pain.

Mixed hemimicelle magnetic dispersive solid-phase extraction using carbon-coated magnetic nanoparticles for the determination of tramadol in urine samples.

Ionic liquid carbon-coated magnetic nanoparticles were successfully applied as an adsorbent in a mixed hemimicelle magnetic dispersive solid-phase extraction method for the determination of tramadol from urine samples coupled with high-performance liquid chromatography with UV-vis detection. The significant parameters affect the extraction efficiency including type and amount of adsorbent, sample volume, pH, ionic strength, type and amount of elution solvent, time of extraction and desorption, time of ionic liquid loading on the adsorbent and stirring rate were studied and optimized. The proposed method provided a fast, straightforward, environmentally friendly and adsorbent recyclable approach for tramadol analysis. The linear range for the tramadol determination was from 100 to 1500 ng/mL. Precisions and accuracies were within 6%. The applicability of the proposed method in clinical trial was tried successfully on determination of tramadol in addicted subjects under tramadol therapy. The mean percent recovery of the patient samples was 94%. The results proved that the proposed method could be applied in clinical and forensic laboratories for determination of tramadol from biological urine samples.

Negligible impact of birth on renal function and drug metabolism.

BACKGROUND: Transition from the intrauterine to the extrauterine environment in neonates is associated with major changes in blood flow and oxygenation with consequent increases in metabolic functions. The additional impact of birth on renal function and drug metabolism above that predicted by postmenstrual age and allometry is uncertain. Increased clearance at birth could reduce analgesic effect attributable to a lowering of plasma concentration. These elimination processes can be described using the clearance concept. METHODS: Data from four publications that investigated the time course of glomerular filtration rate and clearance of paracetamol, morphine and tramadol were reanalyzed. The effect of birth, based on postnatal age, was used in conjunction with a theory-based allometric size scaling and maturation based on postmenstrual age. RESULTS: Postnatal age had a short-term effect on the time course of clearance distinguishable from the well-known slower maturation based on postmenstrual age. While elimination might be relatively reduced by 15%-45% at birth, there is a rapid increase in elimination for 1-3 weeks after birth to be 15% greater than that predicted by postmenstrual age alone. CONCLUSION: Birth is associated with a small increase in clearance in addition to that described by postmenstrual age for common analgesic drugs cleared by glucuronide conjugation (morphine, paracetamol) or by the P450 cytochrome oxidase (tramadol) and renal systems. While the increase is of biological interest, it would not be expected to have any clinically relevant impact on renal function or drug dosing. The processes of maturation described by these models are potentially applicable to any drug elimination process.

Simultaneous chiral separation of tramadol and methadone in tablets, human urine, and plasma by capillary electrophoresis using maltodextrin as the chiral selector.

The stereoselective analysis and separation of racemic drugs play an important role in pharmaceutical industry to eliminate the unwanted isomer and find the right therapeutic control for the patient. Present study suggests a maltodextrin-modified capillary electrophoresis method for a single-run chiral separation of two closely similar opiate pain relief drugs: tramadol (TRA) and methadone (MET). The best separation method possible for the both enantiomers was achieved on an uncoated fused-silica capillary at 25°C using 100 mM phosphate buffer (pH 8.0) containing 20% (w v-1 ) maltodextrin with dextrose equivalent of 4-7 and an applied voltage of 16 kV. Under optimal conditions, the baseline resolution of TRA and MET enantiomers was obtained in less than 12 minutes. The relative standard deviations (n = 3) of 20 µg mL-1 TRA and MET were 2.28% and 3.77%, respectively. The detection limits were found to be 2 µg mL-1 for TRA and 1.5 µg mL-1 for MET. This method was successfully applied to the measurement of drugs concentration in their tablets, urine, and plasma samples.

RETRACTED: Anisotropic (spherical/hexagon/cube) silver nanoparticle embedded magnetic carbon nanosphere as platform for designing of tramadol imprinted polymer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief following concerns raised by a reader. The particles shown in Fig. 3F appear to be copies of each other as they share the identical arrangement of the characteristic speckles inside the particles. In addition, the extraordinary similarities observed between the data presented in Fig. 4C and in Fig. 3C in ACS Biomater. Sci. Eng., 2017, 3 (9), pp 2120­2135, 10.1021/acsbiomaterials.7b00089, Fig. 4A in Colloids and Surfaces B: Biointerfaces, Volume 142, 1 June 2016, Pages 248-258 10.1016/j.colsurfb.2016.02.053 and Fig. 1D in Biosensors and Bioelectronics, Volume 73, 15 November 2015, Pages 234-244, 10.1016/j.bios.2015.06.005 are highly unlikely. The problems with the data presented cast doubt on all the data, and accordingly also the conclusions based on that data, in this publication. As such this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Metabolic Patterns of Fentanyl, Meperidine, Methylphenidate, Tapentadol and Tramadol Observed in Urine, Serum or Plasma.

Drug testing is a useful tool to identify drug use or monitor adherence to prescription drugs. The interpretation of drug results can be complicated based on the pattern and proportional concentrations of drugs and/or drug metabolite(s). The purpose of this retrospective study was to detect the positivity rates and metabolic patterns of five prescription drugs, including fentanyl, meperidine, methylphenidate, tapentadol and tramadol. Retrospective data were retrieved from the laboratory information system in a national reference laboratory. Drug testing was performed using four mass spectrometry methods that were validated for clinical use. For urine specimens, the positivity rate was the highest for methylphenidate (62.3%, n = 2,489), followed by tramadol (43.7%, n = 3,483), fentanyl (41.9%, n = 4,657), tapentadol (37.9%, n = 736) and meperidine (8.3%, n = 138). Among positive samples, both parent drug and metabolite(s) was detectable in 94.9% of meperidine samples, 94.5% of tramadol samples, 93.8% of fentanyl samples, 89.9% of methylphenidate and 86.6% of tapentadol samples. For serum or plasma specimens, the positivity rate was the highest for tapentadol (75.0%, n = 39), followed by methylphenidate (74.2%, n = 569), fentanyl (53.6%, n = 113), meperidine (41.9%, n = 18) and tramadol (28.9%, n = 213). Similar metabolic patterns were found in serum or plasma. Of positive results, both parent drug and metabolite(s) were found in 94.7% of fentanyl samples, 83.3% of meperidine samples, 79.6% of methylphenidate samples, 53.8% of tapentadol samples and 44.1% of tramadol samples. Our data demonstrates the metabolic patterns of five drugs from a random urine or serum/plasma collection in patients that have been prescribed these medications. The data presented can be used to guide clinicians in determining drug adherence by assessing the positivity rates of the parent drug and corresponding metabolite(s).

Solid-phase microextraction of ultra-trace amounts of tramadol from human urine by using a carbon nanotube/flower-shaped zinc oxide hollow fiber.

A new method is successfully developed for the separation and determination of a very low amount of tramadol in urine using functionalized multiwalled carbon nanotubes/flower-shaped zinc oxide before solid-phase microextraction combined with gas chromatography. Under ultrasonic agitation, a sol of multiwalled carbon nanotubes and flower-shaped zinc oxide were forced into and trapped within the pore structure of the polypropylene and the sol solution immobilized into the hollow fiber. Flower-shaped zinc oxide was synthesized and characterized by Fourier transform infrared spectroscopy. The morphology of the fabricated solid-phase microextraction surface was investigated by scanning electron microscopy and X-ray diffraction. The parameters affecting the extraction efficiencies were investigated and optimized. Under the optimized conditions, the method shows linearity in a wide range of 0.12-7680 ng/mL, and a low detection limit (S/N = 3) of 0.03 ng/mL. The precision of the method was determined and a relative standard deviation of 3.87% was obtained. This method was successfully applied for the separation and determination of tramadol in urine samples. The relative recovery percentage obtained for the spiked urine sample at 1000 ng/mL was 94.2%.

Detection and quantification of the opioid tramadol in urine using surface enhanced Raman scattering.

There is an on going requirement for the detection and quantification of illicit substances. This is in particular the case for law enforcement where portable screening methods are needed and there has been recent interest in breath tests for a range of narcotics. In this study we first developed surface enhanced Raman scattering (SERS) for the detection of tramadol in water and establish robust and reproducible methods based on silver hydroxylamine colloid. We used 0.5 M NaCl as the aggregating agent, with the pH ∼ 7.0 and SERS data were collected immediately (i.e., the analyte association and colloid aggregation times were zero). The limit of detection was rather high and calculated to be 5 × 10(-4) M which would not be practical in the field. Undeterred we continued with spiking tramadol in artificial urine and found that no aggregating agent or modification of pH was necessary. Indeed aggregation occurred spontaneously due to the complexity of the medium which is rich in multiple salts, which are commonly used for SERS. We estimated the limit of detection in artificial urine to be 2.5 × 10(-6) M which is equivalent to 657.5 ng mL(-1) and very close to the levels typically found in individuals who use tramadol for pain relief. We believe this opens up opportunities for testing SERS in real world samples and this will be an area of future study.

Simultaneous Determination of Tramadol and Its Metabolite in Human Urine by the Gas Chromatography-Mass Spectrometry Method.

A sensitive and efficient method was developed for determination of tramadol and its metabolite (O-desmethyltramadol) in human urine by gas chromatography-mass spectrometry. Tramadol, O-desmethyltramadol and medazepam (internal standard) were extracted from human urine with a mixture of ethylacetate and diethylether mixture (1 : 1, v/v) at basic pH with liquid-liquid extraction. The calibration curves were linear (r = 0.99) over tramadol and O-desmethyltramadol concentrations ranging from 10 to 200 ng/mL and 7.5 to 300 ng/mL, respectively. The method had an accuracy of >95% and intra- and interday precision (relative standard deviation %) of ≤4.93 and ≤4.62% for tramadol and O-desmethyltramadol, respectively. The extraction recoveries were found to be 94.1 ± 2.91 and 96.3 ± 3.46% for tramadol and O-desmethyltramadol, respectively. The limit of quantification using 0.5 mL human urine was 10 ng/mL for tramadol and 7.5 ng/mL for O-desmethyltramadol. After oral administration of 100 mg of tramadol hydrochloride to a patient, the urinary excretion was monitored during 24 h. About 15% of the dose was excreted as unchanged tramadol.

Clinical pharmacokinetics of tramadol and main metabolites in horses undergoing orchiectomy.

BACKGROUND: Tramadol is a synthetic codeine analogue used as an analgesic in human and veterinary medicine. It is not approved for use in horses, but could represent a valid tool for pain treatment in this species. OBJECTIVES: The serum pharmacokinetic profile and urinary excretion of tramadol and its metabolites (O-desmethyltramadol [M1], N-desmethyltramadol [M2] and N,O-desmethyltramadol [M5]) was investigated in a multidrug anaesthetic and analgesic approach for orchiectomy in horses. The evaluation of the degree of cardiovascular stability, the intraoperative effect and postoperative analgesia obtained by the visual analogue scale are also reported. Animal and methods: Tramadol (4 mg/kg BW) was administered intravenously to eight male yearlings as a bolus over 60 seconds, 5 min after intubation and 15 min prior to surgery. Drug quantification was performed in serum and urine for tramadol, M1, M2 and M5 by high-performance liquid chromatography with fluorimetric detection. RESULTS: Mean tramadol concentration was 14.87 ± 11.14 µg/mL at 0.08 h, and 0.05 ± 0.06 µg/mL at 10 h. Serum concentrations of M1 and M2 metabolites were quite limited. For M1 and M2, median maximum concentration (Cmax) and time to achieve maximum concentration (Tmax) were 0.05 µg/mL and 0.75 h, and 0.08 µg/mL and 2 h, respectively; M5 was never detected. In urine, tramadol was the most recovered compound, followed by M1, M2 and M5. CONCLUSIONS AND CLINICAL RELEVANCE: Showing no adverse events and based on the kinetic behaviour, pre-operative tramadol IV at a dose of 4 mg/kg BW might be useful and safe as analgesic in horses undergoing surgery.