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Quantitative aspects of in vitro phase II glucuronidative metabolism of O-desmethyltramadol (O-DSMT or M1), the active metabolite of the analgesic drug tramadol, by feline, canine and common brush-tailed possum hepatic microsomes are described.Whilst previous studies have focused on the phase I conversion of tramadol to M1, this is the first report in which the phase II glucuronidative metabolic pathway of M1 has been isolated by an in vitro comparative species study.Using the substrate depletion method, microsomal phase II glucuronidative in vitro intrinsic clearance (Clint) of M1 was determined.The in vitro Clint (mean ± SD) by pooled common brush-tailed possum microsomes was 9.9 ± 1.7 μL/min/mg microsomal protein whereas the in vitro Clint by pooled canine microsomes was 1.9 ± 0.07 μL/min/mg microsomal protein. The rate of M1 depletion by feline microsomes, as measured solely by high pressure liquid chromatography, was too slow to determine. Liquid chromatography-mass spectrometry identified O-DSMT glucuronide in samples generated from all three species' microsomes, although the amount detected under the feline condition was minimal.This study indicates that M1 likely undergoes in vitro phase II glucuronidation by canine and common brush-tailed possum microsomes and, to a minor extent, by feline microsomes. The rate of depletion of M1 by phase I metabolism was also undertaken.When incubated with phase I co-factors and common brush-tailed possum microsomes or canine microsomes, M1 had an in vitro Clint of 47.6 and 22.8 μL/min/mg microsomal protein, respectively. However, due to a lack of CYP2B-like activity in the feline liver, unsurprisingly, M1 did not deplete when incubated with feline microsomes. Consequently, major M1 elimination pathways, using feline microsomes, were not determined."

Aim: Tramadol is widely used to treat acute, chronic, and neuropathic pain. Its primary active metabolite, O-desmethyltramadol (M1), is mainly responsible for its µ-opioid receptor-related analgesic effect. Tramadol is metabolized to M1 mainly by the cytochrome P450 (CYP) 2D6 enzyme, and to other metabolites by CYP3A4 and CYP2B6. The aim of this study was to develop a population pharmacokinetic (PK) model of tramadol and its metabolite using healthy Korean subjects. Methods: Data on plasma concentrations of tramadol and M1 were obtained from 23 healthy Korean male subjects after a twice-daily oral dose of 100 mg of tramadol, every 12 hrs, for a total of 5 times. Blood samples were collected at 0 (pre-dose), 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 48 and 72 hrs after last administration. Plasma tramadol concentrations were then analyzed using LC/MS. Population PK analysis of tramadol and its metabolite was performed using a nonlinear mixed-effects modeling (NONMEM). Results: A one-compartment model with combined first-order and zero-order absorption was well fitted to the concentration-time curve of tramadol. M1 was well described by the one-compartment model as an extension of the parent drug (tramadol) model. Genetic polymorphisms of CYP2D6 correlated with the clearance of tramadol, and clearance from the central compartment to the metabolite compartment. Conclusion: The parent-metabolite model successfully characterized the PK of tramadol and its metabolite M1 in healthy Korean male subjects. These results could be applied to evaluate plasma tramadol concentrations after various dosing regimens.

1. The aim of this study was to compare the in vitro cytotoxic effect of tramadol and M1 metabolite in HepG2 cell line, the underlying mechanism, and PI3K/AKT/mTOR as potential target.2. Concentrations representing therapeutic level for tramadol (2 µM) and M1 metabolite (0.5 µM) were used. In addition, other increasing concentrations representing higher toxic levels were used (6, 10 µM for tramadol and 1.5, 2.5 µM for M1 metabolites). Cytotoxicity was assessed at 24, 48 and 72 h.3. Both tramadol and M1 metabolites were able to produce cytotoxicity in a dose and time dependent manner. Insignificant difference was detected between cells exposed to tramadol and M1 metabolite at therapeutic concentrations. Tramadol-induced apoptotic and autophagic cell death while M1 metabolite-induced apoptosis only. For PI3K/AKT/mTOR pathway, the therapeutic concentration of tramadol was only able to increase phosphorylation of AKT while higher toxic concentrations were able to increase phosphorylation of whole pathway; Meanwhile, M1 metabolite was able to increase the phosphorylation of the whole pathway significantly in therapeutic and toxic concentrations.4. In conclusion, both tramadol and M1 are equally cytotoxic. Apoptosis and autophagy both mediate hepatic cell death. PI3K/AKT pathway is involved in apoptosis induction while autophagy is regulated through mTOR independent pathway.

Multimodal analgesia is employed in paediatric pain management to maximise analgesia and minimise side effects. Tramadol is dosed at 1–1.5 mg/kg to treat severe pain in children but the assay for tramadol in plasma samples for pharmacokinetic and toxicology studies does not often consider concurrently administered medications. In this study we developed and validated an HPLC–UV method to quantify tramadol and its main metabolite (O-desmethyltramadol) in human plasma in the presence of seven potentially interfering drugs. Sample preparation method was developed by combining liquid–liquid extraction and protein precipitation. Chromatographic separation was achieved on a BDS-Hypersil-C18 column (5 µm, 250 × 4.6 mm) using a double gradient method. The limit of quantification was 6.7 ng/ml for both tramadol and ODT. The precision and accuracy were in compliance with ICH guidelines. This method was successfully employed to analyse the blood samples of 137 paediatric participants in a tramadol pharmacokinetic trial.

Introduction: A range of opioids are commonly prescribed to manage chronic pain, but individual patient responses vary greatly, especially in older populations. One source of that variability are differences in absorption, metabolism and excretion, i.e. pharmacokinetics. Blood, plasma and serum concentrations of opioids allow that variability to be quantified and may be used to optimise opioid dosing. As an aid to that process, there is an unmet need to rapidly quantify several opioids and their metabolites in a single analytical method. Aims: To develop a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of tramadol, oxycodone, fentanyl and their major metabolites in various human matrices. Methods: Sample preparation involved adding three deuterated internal standards followed by protein precipitation with 100 % acetonitrile, evaporation and reconstitution. Separation of analytes via LC was achieved on a reversed phase column via binary gradient elution using 0.005 % formic acid in water and 100 % acetonitrile as mobile phases. Analytes were detected via MS/MS with multiple reaction monitoring (MRM). Results: The method was accurate with the inter-day and intra-day accuracy of quality control samples (QCs) below 15 %. It was also precise with inter-day and intra-day coefficient of variation below 15 %. The lower limit of quantification (LLOQ) was 0.2 ng/mL for all analytes except tramadol and its metabolites, where the LLOQ was 10 ng/mL. Recovery was greater than 88 % for all analytes, except for O-desmethyltramadol (81 %). Analytes were stable over four freeze-thaw cycles, for 24 h on the bench top and for 24 h post-preparation. The inter- and intra-day variability of concentrations determined in blood and plasma were within 84–124%, whereas the inter- and intra-day variability for blood samples prepared using volumetric absorptive micro-sampling (VAMS) compared to those prepared from whole blood ranged between 83–122%. Conclusion: A LC–MS/MS method is described that is able to accurately and precisely quantify a number of commonly prescribed opioids and their major metabolites in plasma and whole blood, including whole blood collected using VAMS. Refereed/Peer-reviewed

There is currently little information available on the pharmacokinetics and pharmacodynamics of the analgesic opioid tramadol when used in the veterinary medicine of domestic species. In this study, we aimed to determine the pharmacokinetics of tramadol and its active metabolite M1 following intravenous administration of 2 (T2) and 4 (T4) mg/kg to Northeast Brazilian donkeys. Tramadol and M1 plasma levels were quantified using a validated liquid chromatography-tandem mass spectrometry method. We found that plasma levels of tramadol and M1 were higher than those reported as clinically meaningful in humans for at least 3 hr. However, the pharmacokinetic parameter calculation corrected by dose analysis identified no proportional increase with dose for the AUC of tramadol (T2: 2,663 ± 1,827 vs. T4: 2,964 ± 1,038 ng*h/ml) and M1 (T2: 378 ± 237 vs. T4: 345 ± 142 ng*h/ml). This finding appears to be attributable to a significant increase in clearance and a reduction in the terminal half-life of tramadol. The frequency of adverse effects observed at the higher dose indicates that 2 mg/kg administered intravenously would be suitable for donkeys. Clinical studies are required to determine the implications of these observations regarding the pharmacodynamic response to tramadol in Northeast Brazilian donkeys.

Tramadol (TR) is a member of the opioid family and is widely used for pain treatment in clinical patient care. The analgesic effect of tramadol is induced primarily by its main metabolite Odesmethyltramadol (ODMT). Due to interindividual differences in the TR metabolism to ODMT, the responses to TR vary highly between patients. Thus, a fast and selective method for simultaneous detection of TR and ODMT would increase the patient safety and pain treatment efficacy. In this study, a tetrahedral amorphous carbon (ta-C) electrode coated with a thin dip-coated recast Nafion membrane was fabricated for selective electrochemical determination of TR and ODMT. With this Nafion/ta-C electrode, simultaneous detection of TR and ODMT was achieved with linear ranges of 1-12.5 mu M and 1-15 mu M, respectively. The limits of detection were 131 nM for TR and 209 nM for ODMT. Both analytes were also measured in the presence of several common interferents, demonstrating the high selectivity of the fabricated electrode. In addition, the effect of pH on the peak potential was studied to observe the electrochemical behavior of the analytes at the electrode. Finally, clinically relevant concentrations of TR and ODMT were simultaneously detected from diluted human plasma to assess the applicability of the electrode in real samples. The fabricated Nafion/ta-C electrode was found successful in the simultaneous electrochemical detection of TR and ODMT in both buffer solution and in human plasma. (C) 2018 Elsevier Ltd. All rights reserved.

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.

Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT.The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays.Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media.SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans.

A rapid and sensitive method involving liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) coupled to an intracerebral microdialysis technique was developed for the determination and pharmacokinetic investigation of tramadol and its major active metabolite O-desmethyltramadol (ODT) in rat brain. The microdialysis samples were separated on a C18 column and eluted with a mobile phase of acetonitrile-water-formic acid (50:50:0.1; v/v/v) at a flow rate of 0.3 mL/min. The ESI-MS/MS spectra were performed in electrospray positive ion mode, and the analytes were detected by multiple reaction monitoring (MRM) of the transitions m/z [M + H]+ 264.3 → 58.2 for tramadol, m/z [M + H]+ 250.3 → 58.3 for ODT, and m/z [M + H]+ 379.4 → 264.0 for ambroxol (internal standard; IS). The total run time was 4.0 min. A lower limit of quantitation (LLOQ) was achieved as 1 ng/mL for tramadol and 0.5 ng/mL for ODT, with excellent linearity over a concentration range of 1 ~ 500 ng/mL (r > 0.99) for tramadol and 0.5 ~ 50 ng/mL for ODT (r > 0.99), respectively. The proposed method was successfully applied to the pharmacokinetic studies of tramadol and ODT in rat brain. Copyright © 2017 John Wiley & Sons, Ltd.

Tramadol is a &mu, opioid receptor agonist and a monoamine reuptake inhibitor. O-desmethyltramadol (M1), the major active metabolite of tramadol, is produced by CYP2D6. A physiologically-based pharmacokinetic model was developed to predict changes in time-concentration profiles for tramadol and M1 according to dosage and CYP2D6 genotypes in the Korean population. Parallel artificial membrane permeation assay was performed to determine tramadol permeability, and the metabolic clearance of M1 was determined using human liver microsomes. Clinical study data were used to develop the model. Other physicochemical and pharmacokinetic parameters were obtained from the literature. Simulations for plasma concentrations of tramadol and M1 (after 100 mg tramadol was administered five times at 12-h intervals) were based on a total of 1000 virtual healthy Koreans using SimCYP&reg, simulator. Geometric mean ratios (90% confidence intervals) (predicted/observed) for maximum plasma concentration at steady-state (Cmax,ss) and area under the curve at steady-state (AUClast,ss) were 0.79 (0.69&ndash, 0.91) and 1.04 (0.85&ndash, 1.28) for tramadol, and 0.63 (0.51&ndash, 0.79) and 0.67 (0.54&ndash, 0.84) for M1, respectively. The predicted time&ndash, concentration profiles of tramadol fitted well to observed profiles and those of M1 showed under-prediction. The developed model could be applied to predict concentration-dependent toxicities according to CYP2D6 genotypes and also, CYP2D6-related drug interactions.

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations.

Tramadol is a synthetic, centrally acting, opiate-like analgesic that is structurally related to codeine and morphine. The objective of this study was to determine the pharmacokinetics of tramadol hydrochloride and its major active metabolite O-desmethyltramadol (M1) in the California sea lion (Zalophus californianus). A single dose of tramadol was administered orally in fish at 2 mg/kg to a total of 15 wild California sea lions admitted for rehabilitation. Twenty-four total blood samples were collected post drug administration at 10, 20, 30, and 45 min and at 1, 3, 5, 6, 8, 12, and 24 hr. Blood plasma was separated and stored at -80°C until analysis with high-performance liquid chromatography was performed to determine levels of tramadol and M1, the major active metabolite. The results indicate that the plasma levels of parent tramadol are low or negligible during the first 30-45 min and then reach the predicted mean maximum plasma concentration of 358 ng/ml at 1.52 hr. The M1 metabolite was not detectable in 21 of 24 plasma samples, below the level of quantification of 5 ng/ml in one sample, and detectable at 11 and 17 ng/ml in two of the samples. This study suggests that a 2 mg/kg dose would need to be administered every 6-8 hr to maintain concentrations of tramadol above the minimum human analgesic level for mild to moderate pain. Based on dosing simulations, a dose of 4 mg/kg q8 hr or q12 hr, on average, may represent an adequate compromise, but further studies are needed using a larger sample size. Pharmacodynamic studies are warranted to determine if tramadol provides analgesic effects in this species. The potential for tramadol toxicosis at any dose also has not been determined in this species.

Summary Reasons for performing study Tramadol is an analgesic agent used in man and a number of veterinary species. The pharmacokinetics and behavioural effects of tramadol and its active metabolite have been described in mature horses, but not in young foals. Objectives To characterise the pharmacokinetics, metabolism and some induced behavioural and physiological responses following i.v. tramadol administration in the same group of foals on 4 different occasions, from a few days after birth to age 43 days. Study design Experimental. Methods Tramadol was administered i.v. (3 mg/kg bwt) to a group of 8 foals on 4 separate occasions at ages 6–8, 13–15, 20–22 and 40–43 days. Blood samples were collected prior to administration and at multiple times until 48 h post administration. Blood samples were analysed for tramadol and metabolite concentrations and pharmacokinetics determined at each age. Behavioural and physiological effects were also assessed. Results The average volume of distribution was 5.10, 4.63, 4.02 and 3.84 l/kg bwt and clearance 3.44, 3.08, 3.14 and 2.69 l/h/kg bwt when foals were aged 6–8, 13–15, 20–22 and 40–43 days, respectively. There was not a significant difference in the elimination half-life between age groups (1.52, 1.73, 1.13 and 1.51 for ages 6–8, 13–15, 20–22 and 40–43 days, respectively). The metabolites produced were the same as in mature horses; however, glucuronidation capability, appeared to increase with increasing age. Tramadol administration was well tolerated at all ages studied with sedation noted in the 3 older age groups. Conclusions Tramadol appears to be consistently well tolerated following i.v. administration of 3 mg/kg bwt to foals ranging in age from 1 to 6 weeks. Although analgesic concentrations in foals have yet to be established, the results of this study support further study of tramadol for clinical use in foals.

Tramadol has been used as an analgesic for several decades. µ-Opioid receptors (µORs) are the major receptors that mediate the analgesic effects of opioids. Although µORs have been thought to be one of the sites of action of tramadol, there has been no report that directly proves whether tramadol is an agonist of μOR or not. In this study, we examined the effects of tramadol and its main active metabolite O-desmethyltramadol (M1), on the function of µORs using Xenopus oocytes expressing cloned human µORs. The effects of tramadol and M1 were evaluated using the Ca(2+)-activated Cl(-) current assay method for G(i/o)-protein-coupled receptors by using a µOR fused to G(qi5) (µOR-G(qi5)) in Xenopus oocytes. DAMGO [(D-Ala(2), N-MePhe(4), Gly(5)-ol)-enkephalin] evoked Cl(-) currents in oocytes expressing µOR-G(qi5) in a concentration-dependent manner. Tramadol and M1 also evoked Cl(-) currents in the oocytes expressing µOR-G(qi5); however, relatively higher concentrations (compared to DMAGO) were necessary to induce such currents. Tramadol and M1 had a direct effect on µORs expressed in Xenopus oocytes. Although the monoamine uptake system and several types of ligand-gated ion channels are thought to be one of the targets for tramadol, tramadol-induced antinociception may be mediated at least in part, by the direct activation of µORs.

Decomposed bone and plasma samples of rats exposed to tramadol (TRAM) under different dosing patterns were analyzed. Wistar rats received TRAM as one acute dose (n=4, 45 mg/kg, i.p.) or three doses (n=4, 15 mg/kg, i.p.), 40 min apart. Perimortem heart blood was collected, rats were euthanized and placed outdoors to decompose to skeleton. Recovered bone was ground and subjected to methanolic extraction. Bone extracts and plasma samples underwent solid phase extraction and were analyzed using gas chromatography-mass spectrometry. Levels of TRAM and the primary metabolite O-desmethyltramadol (ODMT) were expressed as mass normalized response ratios (RR/m). Levels (RR/m) for TRAM and ODMT did not differ significantly between exposure types in any of the bone types examined or for the pooled bone comparisons (Mann-Whitney, p0.05). However, ratios of analyte levels (RRTRAM/RRODMT) differed significantly between exposure patterns for tibial and skull bone as well as for pooled bone comparisons (Mann-Whitney, p0.05). Levels of TRAM and ODMT, as well as ratios of analyte levels (RRTRAM/RRODMT), differed significantly in plasma between exposure patterns. Bone TRAM and ODMT levels were poorly correlated to corresponding plasma levels (TRAM: r=0.33-0.57; ODMT: r=-0.35-0.23).

O-desmethyltramadol is one of the main metabolites of tramadol widely used clinically and has analgesic activity. The FTIR and FT-Raman spectra of O-desmethyl tramadol hydrochloride are recorded in the solid phase in the regions 4000-400 cm(-1) and 4000-100 cm(-1), respectively. The observed fundamentals are assigned to different normal modes of vibration. Theoretical studies have been performed as its hydrochloride salt. The structure of the compound has been optimised with B3LYP method using 6-31G(**) and cc-pVDZ basis sets. The optimised bond length and bond angles are correlated with the X-ray data. The experimental wavenumbers were compared with the scaled vibrational frequencies determined by DFT methods. The IR and Raman intensities are determined with B3LYP method using cc-pVDZ and 6-31G(d,p) basic sets. The total electron density and molecular electrostatic potential surfaces of the molecule are constructed by using B3LYP/cc-pVDZ method to display electrostatic potential (electron+nuclei) distribution. The electronic properties HOMO and LUMO energies were measured. Natural bond orbital analysis of O-desmethyltramadol hydrochloride has been performed to indicate the presence of intramolecular charge transfer. The (1)H and (13)C NMR chemical shifts of the molecule have been anlysed.

A sensitive and accurate method for the extraction and quantification of tramadol (T) and its active metabolite, O-desmethyltramadol (ODT) in human plasma with high-performance liquid chromatography–diode array detection was developed and validated. The analytes were extracted from plasma samples by tert-butylmethyl ether in the presence of ammonium hydroxide as alkaline medium and back extraction with 1.0 M hydrochloric acid. Propranolol was used as internal standard. The extraction efficiencies of T and ODT were 83.51 and 78.72%, respectively. The calibration curves were linear (r 2 > 0.99) in the concentration range of 250–2000 ng/mL for T and ODT. Limits of detection and quantification were 125 and 250 ng/mL for both analytes. Intra- and interassay precision for T and ODT were ranged from 1.89 to 10.91% and 2.16 to 5.85%, respectively. Intra- and interassay accuracy for T and ODT were ranged from 213.07 to 4.99% and 22.03 to 26.98%, respectively. The method was successfully applied to quantify T and ODT from authentic plasma samples received from Hospital Sohag University. The method was completely validated and can be of interest to clinical and forensic laboratories.

Analytical procedures for the determination of tramadol (T), O-desmethyltramadol (ODT), and N-desmethyltramadol (NDT) in human urine have been developed and validated using gas chromatography-mass spectrometry (GC/MS). Sample preparation involved liquid-liquid extraction with methyl-tert-butyl ether (MTBE) and followed by back extraction with 0.1 M hydrochloric acid. Proadifen (SKF525A) was selected as internal standard (IS). Extraction efficiencies of T, ODT and NDT were 102.12, 101.30, and 98.21%, respectively. The calibration curves were linear (r(2)>0.99) in the concentration range 10-1000 ng/mL for all compounds. Limits of quantification (LOQ) were 10, 10 and 20 ng/mL for T, ODT and NDT, respectively. Intra-assay precision was within 1.29-6.48% and inter-assay precision was within 1.28-6.84% for T, ODT and NDT. Intra-assay accuracy was within 91.79-106.89% for all analytes. This method detected urine concentrations of T, ODT and NDT in six healthy volunteers for 7 days after administration of 50 mg oral doses of tramadol.

Over recent years, hair has become the ideal matrix for retrospective investigation of chronic abuse, including for tramadol. However, in order to exclude the possibility of external contamination, it is also important to quantify simultaneously its main metabolite, O-desmethyltramadol (M1), which presence in hair reflects systemic exposure. In the present study a methodology aimed at the simultaneous quantification of tramadol and M1 in human hair was developed and validated for the first time. After decontamination of hair samples (60 mg), tramadol and M1 were extracted with methanol in an ultrasonic bath (~5 h). Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. Subsequently to derivatization, analysis was performed by gas chromatography-electron impact/mass spectrometry (GC-EI/MS). The method proved to be selective. The regression analysis for both analytes was shown to be linear in the range of 0.1-20.0 ng/mg with correlation coefficients of 0.9995 and 0.9997 for tramadol and M1, respectively. The coefficients of variation oscillated between 3.85 and 13.24%. The limits of detection were 0.03 and 0.02 ng/mg, and the lower limits of quantification were 0.08 and 0.06 ng/mg for tramadol and M1, respectively. The proof of applicability was performed in hair samples from six patients undergoing tramadol therapy. All samples were positive for tramadol and M1.

The accurate assessment of racemic pharmaceuticals requires enantioselective analytical methods. This study presents the development and validation of an enantioselective liquid chromatography with a fluorescence detection method for the concomitant quantification of the enantiomers of tramadol and their metabolites, N-desmethyltramadol and O-desmethyltramadol, in wastewater samples. Optimized conditions were achieved using a Lux Cellulose-4 column 150 × 4.6 mm, 3 μm isocratic elution, and 0.1% diethylamine in hexane and ethanol (96:4, v/v) at 0.7 mL min-1. The samples were extracted using 150 mg Oasis® mixed-mode cation exchange (MCX) cartridges. The method was validated using a synthetic effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor. The method demonstrated to be selective, accurate, and linear (r2 > 0.99) over the range of 56 ng L-1 to 392 ng L-1. The detection and the quantification limits of each enantiomer were 8 ng L-1 and 28 ng L-1 for tramadol and N-desmethyltramadol, and 20 ng L-1 and 56 ng L-1 for O-desmethyltramadol. The feasibility of the method was demonstrated in a screening study in influent and effluent samples from a wastewater treatment plant. The results demonstrated the occurrence of tramadol enantiomers up to 325.1 ng L-1 and 357.9 ng L-1, in the effluent and influent samples, respectively. Both metabolites were detected in influents and effluents.

This study describes the enantioselective analysis of unbound and total concentrations of tramadol and its main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human plasma. Sample preparation was preceded by an ultrafiltration step to separate the unbound drug. Both the ultrafiltrate and plasma samples were submitted to liquid/liquid extraction with methyl t-butyl ether. Separation was performed on a Chiralpak(®) AD column and tandem mass spectrometry consisting of an electrospray ionization source, positive ion mode and multiple reaction monitoring was used as the detection system. Linearity was observed in the following ranges: 0.2-600 and 0.5-250 ng/mL for analysis of total and unbound concentrations of the tramadol enantiomers, respectively, and 0.1-300 and 0.25-125 ng/mL for total and unbound concentrations of the M1 and M2 enantiomers, respectively. The lower limits of quantitation were 0.2 and 0.5 ng/mL for analysis of total and unbound concentration of each tramadol enantiomer, respectively, and 0.1 and 0.25 ng/mL for total and unbound concentrations of M1 and M2 enantiomers, respectively. Intra- and interassay reproducibility and inaccuracy did not exceed 15%. Clinical application of the method to patients with neuropathic pain showed plasma accumulation of (+)-tramadol and (+)-M2 after a single oral dose of racemic tramadol. Fractions unbound of tramadol, M1 or M2 were not enantioselective in the patients investigated.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of −70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a μ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating μ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak® AD column using hexane:ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n=6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+))/((-)) ratio=4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics.

A new LC procedure for the determination of tramadol, an analgesic, and its metabolites has been developed and validated. Following a liquid–liquid extraction using ethyl acetate and hexane, samples were separated by RP-LC on a Symmetry C18 column and quantified using fluorescence detection at an excitation of 202 nm and an emission of 296 nm. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (0.01 M), 0.1% triethylamine (pH 2.9) and acetonitrile, with a flow-rate of 1.1 mL min−1. The standard curve ranged from 5 to 5,000 ng mL−1. Intra-and inter-assay variability for all the compounds were less that 10% and the average recovery was greater than 90%. This assay is suitable for use in pharmacokinetic studies.

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O-Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the micro-receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high-performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA-ESI-MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O-desmethyltramadol (M5). LC/PDA-ESI-MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N-desmethyltramadol (M2), and N,N-didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI-MS/MS techniques in a search for tramadol metabolites in horse plasma samples.

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of tramadol and its active metabolite O-desmethyltramadol in human plasma was validated. The tramadol and its metabolite were separated on a reversed phase column (Zorbax SB-C18, 100 mm x 3.0 mm I.D., 3.5 microm) under isocratic conditions using a mobile phase of a 10:90 (v/v) mixture of acetonitrile and 0.2% (v/v) trifluoroacetic acid in water. The flow rate was 1 ml/min at the column temperature 45 degrees C. In these chromatographic conditions, the retention times were 2.3 min for O-desmethyltramadol and 3.5 min for tramadol, respectively. The detection of both analytes was in SIM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were m/z 264 for tramadol and m/z 250 for its metabolite. The sample preparation was very simple and rapid and consisted in plasma protein precipitation from 0.2 ml plasma using 0.2 ml solution of perchloric acid 7%. Calibration curves were generated over the range of 2-300 ng/ml for both analytes with values for coefficient of correlation greater than 0.998 and by using a weighted (1/y) quadratic regression. The values of precision and accuracy for tramadol at quantification limit were less than 10.9% and 5.1, respectively, both for within- and between-run. For O-desmethyltramadol, precision and accuracy at quantification limit were 10.1% and -9.9% for within-run determinations and 6.7% and 10.4% for between-run determinations, respectively. The mean recovery for both analytes was 96%. Both tramadol and its metabolite demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. This is the first reported method for analysis of tramadol and O-desmethyltramadol in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of both analytes. The validated LC/MS method has been applied to a pharmacokinetic study of 50 mg tramadol tablets on healthy volunteers.

Tramadol (T) is a centrally acting analgesic structurally related to codeine and morphine. This drug displays a weak affinity for the μ and δ-opioid receptors, and weaker affinity for the κ-subtype; it also interferes with the neuronal release and reuptake of serotonin and noradrenaline in the descending inhibitory pathways. The metabolism of this drug has been investigated in different animals (rats, mice, Syrian hamsters, guinea pigs, rabbits, and dogs) and humans; similar metabolites are produced but in different amounts. The major metabolic pathways involved in phase I metabolite production (M1–M5) are O-demethylation, N-demethylation, and N,N-demethylation. The aim of the current study is to evaluate the pharmacokinetic profile of T in the horse, and its M1, M2, and M5 metabolites after single-dose administration (5 mg/kg body weight [BW]) by intravenous, sustained-release tablets and immediate-release capsules. We also will investigate the potential effects of fasting and feeding on bioavailability of immediate-release capsules. The study design was divided into four randomized phases. Twenty-four gelding Italian trotter race horses were divided into four groups (6 animals each) and administered T intravenously, with T immediate-release capsules in a fasting status, T immediate-release capsules in a feeding status, and T sustained-release in fasting status. Blood samples were collected at different times and analyzed by high-pressure liquid chromatography (HPLC) with fluorimetric detection. The limit of quantification was 5 ng/ml for T, M1, and M2, and 10 ng/ml for M5. A one-compartment model best fit the plasma concentrations of T and M2 after all treatments. Unfortunately, for M1 and M5, it was not always possible to fit plasma curves because of very low and variable concentrations. M2 was the main metabolite produced in the four different treatments and its concentration was higher than the concentration of T after sustained-release administration. Conversely, M1, the main metabolite in humans, and M5 seemed to be only marginally produced in the horse. When T was administered in both fasted and fed states, variations in some pharmacokinetic parameters were not considered clinically significant. We concluded that T could be administered in either a fasted or a fed condition.

Tramadol, an analgesic agent, and its main metabolites O -desmethyltramadol (M1), N -desmethyltramadol (M2) and O,N -didesmethyltramadol (M5) were determined simultaneously in human plasma, saliva and urine by a rapid and specific HPLC method. The sample preparation was a simple, one-step, extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using a mixture of methanol:water (19:81, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection ( λ ex 200 nm/ λ em 301 nm) was used. The calibration curves were linear ( r 2 > 0.996) in the concentration ranges in plasma, saliva and urine. The lower limit of quantification was 2.5 ng/ml for all compounds. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were acceptable in all analyzed body fluids The developed procedure was applied to assess the pharmacokinetics of tramadol and its main metabolites following administration of 100 mg single oral dose of tramadol to healthy volunteers.

The influence of CYP2D6 genotype and CYP2D6 inhibitors on enantiomeric plasma levels of tramadol and O-desmethyltramadol as well as response to tramadol was investigated. One hundred and seventy-four patients received one hundred intravenous tramadol 3 mg/kg for postoperative analgesia. Blood samples drawn 30, 90, and 180 min after administration were analyzed for plasma concentrations of the enantiomers (+)-, (-)tramadol and (+)-, (-)O-desmethyltramadol by liquid chromatography-tandem mass spectrometry. Different CYP2D6 genotypes displaying zero (poor metabolizer (PM)), one (heterozygous individual (HZ)/intermediate metabolizer (IM)), two extensive metabolizer (EM), and three (ultra rapid metabolizer (UM)) active genes were compared. Concentrations of O-desmethyltramadol differed in the four genotype groups. Median (1/3 quartile) area under the concentration-time curves for (+)O-desmethyltramadol were 0 (0/11.4), 38.6 (15.9/75.3), 66.5 (17.1/118.4), and 149.7 (35.4/235.4) ng x h/ml for PMs, HZ/IMs, EMs, and UMs (P

The objective of this study was to determine the pharmacokinetics of two orally administered doses of tramadol (5 and 10 mg/kg) and its major metabolite (O-desmethyltramadol) (M1) in loggerhead sea turtles (Caretta caretta). After oral administration, the half-life of tramadol administered at 5 and 10 mg/kg was 20.35 and 22.67 hr, whereas the half-life of M1 was 10.23 and 11.26 hr, respectively. The maximum concentration (Cmax) for tramadol after oral administration at 5 mg/kg and 10 mg/kg was 373 and 719 ng/ml, whereas that of M1 was 655 and 1,376 ng/ml, respectively. Tramadol administered orally to loggerhead sea turtles at both dosages provided measurable plasma concentrations of tramadol and O-desmethyltramadol for several days with no adverse effects. Plasma concentrations of tramadol and O-desmethyltramadol remained ≥100 ng/ml for at least 48 and 72 hr when tramadol was administered at 10 mg/kg.

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization is presented for quantification (selected-ion mode) of tramadol (T) and O-desmethyltramadol (ODT) in blood plasma after liquid-liquid extraction. The enantiomeric separation was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The validation data were within acceptable limits. The assay was successfully applied to authentic plasma samples allowing confirmation of diagnosis of overdose or intoxication as well as monitoring of patients' compliance. In postoperative patients receiving T, mean therapeutic plasma concentrations were determined as follows: 422.8 and 440.8 ng/mL for (+)-T and (-)-T, respectively, and 23.7 and 40.7 ng/mL for (+)-ODT and (-)-ODT, respectively. Quantitative results demonstrated a great interindividual variability in postoperative plasma analgesic drug concentrations and, therefore, a wide therapeutic concentration range. The 7.7% of the samples that revealed negative results for ODT are likely explained by genetic polymorphisms resulting in absent enzyme activity of CYP2D6.

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.

A sensitive, enantioselective high-performance liquid chromatographic method has been developed for the separation and individual quantitative determination of (+)- and (-)-tramadol and (+)- and (-)-O-desmethyltramadol (M1) in plasma and urine. Extraction from plasma and urine was performed by solid-phase extraction (SPE) on disposable butyl silica (100 mg) extraction cartridges. Separation of the enantiomers of tramadol and M1 was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine, 97:2.8:0.1 (v/v). Quinidine was used as internal standard. The analytes were detected by use of fluorescence detection. The limit of quantification for tramadol and M1 was 5 nM in plasma and 25 nM in urine. Recoveries were approximately 90% for tramadol and M1 in both plasma and urine. Linearity was observed for both enantiomers of tramadol and M1 in both plasma (r2 > 0.999) and urine (r2 > 0.997). The intra and inter-day precision (CV) did not exceed 6.0%. The applicability of the method was demonstrated by means of two clinical studies - a pharmacokinetic study in which a healthy volunteer received 150 mg tramadol hydrochloride as a single oral dose and a study in which poor and extensive CYP2D6 metabolizers received 50 mg tramadol hydrochloride as a single oral dose.

This case discusses the synergistic pharmacodynamic interaction between tramadol and bupropion resulting in new-onset seizures. A possible pharmacokinetic interaction is also discussed.

Although sheep are widely used as an experimental model for various surgical procedures there is a paucity of data on the pharmacokinetics and efficacy of analgesic drugs in this species. The aims of this study were to investigate the pharmacokinetics of intravenously (IV) administered tramadol and its active metabolite O-desmethyltramadol (M1) and to assess the mechanical antinociceptive effects in sheep. In a prospective, randomized, blinded study, six healthy adult sheep were given 4 and 6 mg/kg tramadol and saline IV in a cross-over design with a 2-week wash-out period. At predetermined time points blood samples were collected and physiological parameters and mechanical nociceptive threshold (MNT) values were recorded. The analytical determination of tramadol and M1 was performed using high performance liquid chromatography. Pharmacokinetic parameters fitted a two- and a non-compartmental model for tramadol and M1, respectively. Normally distributed data were analysed by a repeated mixed linear model. Plasma concentration vs. time profiles of tramadol and M1 were similar after the two doses. Tramadol and M1 plasma levels decreased rapidly in the systemic circulation, with both undetectable after 6 h following drug administration. Physiological parameters did not differ between groups; MNT values were not statistically significant between groups at any time point. It was concluded that although tramadol and M1 concentrations in plasma were above the human minimum analgesic concentration after both treatments, no mechanical antinociceptive effects of tramadol were reported. Further studies are warranted to assess the analgesic efficacy of tramadol in sheep.

An HPLC system using a simple liquid–liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the 11 above-mentioned tramadol metabolite and N ,N -dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 25034 mm chromatographic column with LiChrospher 60 RP-selectB 5-mm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v / v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v / v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection ( l 202 nm /l 296 nm for tramadol and its ex em 11 metabolite, l 264 nm /l 344 nm for N ,N -dimethylsulfanilamide) was used. The validated analytical method was ex em applied to pharmacokinetic studies of tramadol in human volunteers. © 2002 Elsevier Science B.V. All rights reserved.

Objective Tramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-10. Study design In vitro pharmacodynamic study. Animals Six healthy dogs. Methods Blood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. Results No differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. Conclusions Tramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. Clinical relevance These data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

1. Metabolism of the analgesic agent, tramadol hydrochloride, was investigated after a single oral administration of 14C-tramadol to four rats (50)mgkg(-1) and two dogs (20)mg kg(-1). 2. Recovery of total radioactivity in rat and dog urine samples over 24 h was 73 and 65% of the radioactive dose, respectively. 3. Unchanged tramadol and a total of 24 metabolites, consisting of 16 Phase I metabolites and eight conjugates (seven glucuromides, one sulphate), were isolated and tentatively identified, which accounted for > 52% of the dose in urine of both species. 4. Of the metabolites, five (M1-5) were previously identified. 5. The metabolites were formed via the following six metabolic pathways: O-demethylation, N-demethylation, cyclohexyl oxidation, oxidative N-dealkylation, dehydration and conjugation. 6. Pathways 1-3 appear to be major steps, forming seven O-desmethyl/N-desmethyl and hydroxy-cyclohexyl metabolites in major quantities. 7. Pathways 1-3 in conjunction with pathway 6 produced four glucuronides along with four minor conjugates. 8. In addition, the in vitro metabolism of tramadol was conducted using rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged tramadol (30% of the sample) plus nine metabolites, M1-7, tramadol-N-oxide (M31) and OH-cyclohexyl-M1 (M32), were profiled and tentatively identified based on MS and MS/MS data.

Pharmacokinetic studies require sensitive analytical methods to allow the determination of low concentrations of drugs and metabolites. When drugs present an asymmetric center, the enantiomeric determination of the compounds of interest should be performed. The method developed is based on on-line LC-MS-MS using atmospheric pressure chemical ionization as an interface determination of enantiomers of tramadol (T) and its active metabolite O-desmethyltramadol (ODT) in human plasma. This determination is preceded by an off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs), performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and water. The washing step was performed with water and the analytes were finally eluted by dispensing methanol. The collected eluate was then evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The enantiomeric separation of tramadol and ODT was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine (97:3:0.1, v/v). The LC system was then coupled to a tandem mass spectrometry system with an APCI interface in the positive ion mode. The chromatographed analytes were detected in the selected reaction monitoring mode. The MS-MS ion transitions monitored were 264-->58 for tramadol, 250-->58 for ODT, and 278-->58 for ethyltramadol, used as internal standard. The method was validated. The recoveries were around 90% for both T and ODT. The method was found to be linear for each enantiomer of both compounds (r2>0.999). The mean RSD values for repeatability and intermediate precision were 3.5 and 6.4% for T enantiomers and 5.0 and 5.6% for ODT enantiomers, respectively. Moreover, the method was found to be selective towards other metabolites, N-desmethyltramadol and N,O-desmethyltramadol (NODT). The method developed was successfully used to investigate plasma concentration of enantiomers of T and ODT in a pharmacokinetic study.

Efficient enzymatic resolutions of the analgesic δ-hydroxytramadol rac - 3 and e-hydroxytramadol rac - 4 have been achieved through pig liver esterase- and Candida rugosa lipase-catalyzed hydrolyses of the corresponding butyrates. The Candida rugosa lipase-catalyzed hydrolysis of O -desmethyltramadol butyrate rac - 8a , having a remote aromatic acyloxy group as the only functional group amendable to a hydrolase-catalyzed reaction, proceeded with a good selectivity.

Aims Tramadol, a centrally acting analgesic, is used as a racemate containing 50% of a (+)- and 50% of a (−)-enantiomer. This paper presents the pharmacokinetic results of postoperative patient-controlled analgesia using (+)-tramadol, (−)-tramadol or the racemate. Methods Ninety-eight patients recovering from major gynaecological surgery were treated in a randomised, double-blind study with (+)-tramadol, (−)-tramadol or the racemate. Following an i.v. bolus up to a maximum of 200 mg, patient-controlled analgesia with demand doses of 20 mg was made available for 24 h. Prior to each demand, the serum concentrations of the enantiomers of tramadol and its metabolite M1 were measured in 92 patients. Results The mean concentrations of tramadol during the postsurgery phase were 470±323 ng ml−1, 590±410 ng ml−1 and 771±451 ng ml−1 in the (+)-, racemate- and (−)-group, respectively ((+) vs (−), P

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O -demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10–1000 ng/ml for ODMT and 10–10 000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 μl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.

A sensitive and stereoselective high-performance liquid chromatographic assay for the quantitative determination of the analgesic tramadol and O-demethyltramadol, an active metabolite, is described in this work. Ketamine was used as internal standard. The assay involved a single tert-butymethylether extraction and liquid chromatography analysis with fluorescence detection. Chromatography was performed at 20 degrees C on a Chiracel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, preceded by an achiral end-capped C18 column. The mobile phase was a mixture of phosphate buffer (containing sodium perchlorate (0.2 M) and triethylamine (0.09 M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol and its active metabolite by this method was 0.5 ng/mL; only 0.5 mL of the plasma sample was required for the determination. The calibration curve was linear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 ng/mL for O-demethyltramadol enantiomers. Intra and interday precision [coefficient of variation (CV)] did not exceed 10%. Mean recoveries of 95.95 and 97.87% for (+)R,R- and (-)S,S-tramadol and 97.70 and 98.79% for (+)R,R- and (-)S,S-O-demethyltramadol with CVs < 2.15% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received 100 mg of tramadol by the intravenous route.

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4. A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO4 concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5-150 ng/ml concentration range [r2=0.999 for (+)- and (-)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (-)-tramadol, respectively.

Heroin is one of the most dangerous abused drugs in the world. Tramadol is an additive recently found at high concentration levels in street heroin seizures in Egypt. This substance could affect the usual analytical method for the detection of heroin and metabolites, as well as the pharmacokinetic and disposition of single analytes. One shortfall regarding this issue is present in the literature. This study describes a validated, simple, sensitive and selective method to determine tramadol, O-desmethyltramadol, 6-acetylmorphine and free morphine in the blood, brain, liver and kidney of Wistar rats, intraperitoneally treated with a combination of heroin and tramadol (10 and 70 mg/kg, respectively) using liquid-liquid extraction and gas chromatography-mass spectrometry detection. The calibration curves of tramadol, O-desmethyltramadol and 6-acetylmorphine in blood were linear in the concentration range from 25-5,000 ng/mL and morphine was found in the concentration range 50-5,000 ng/mL. The analytes were detected in all tested matrices, except 6-acetylmorphine, which was not detected in liver. The highest concentrations of tramadol and O-desmethyltramadol were observed in kidney (22,9381 and 28,498 ng/g), while 6-acetylmorphine and morphine were found at the highest levels in brain (3,280 and 3,899 ng/g, respectively). The present method is simple, rapid and sensitive and can be used to study the pharmacokinetics, disposition and interaction of these drugs in several animal models.