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The Veterinary Journal 178 (2008) 272–277

TheVeterinary Journal

The pharmacokinetics, metabolism and urinary detection timeof tramadol in camels

M. Elghazali a,*, I.M. Barezaik a, A.A. Abdel Hadi a, F.M. Eltayeb a,J. Al Masri b, I.A. Wasfi a

a Camel Racing Forensic Laboratory, Forensic Science Laboratory, P.O. Box 253, Abu Dhabi, United Arab Emiratesb Veterinary Research Center, P.O. Box 72437 Al Ain, United Arab Emirates

Accepted 5 July 2007

Abstract

The pharmacokinetics of tramadol in camels (Camelus dromedarius) were studied following a single intravenous (IV) and a singleintramuscular (IM) dose of 2.33 mg kg�1 bodyweight. The drug’s metabolism and urinary detection time were also investigated. Follow-ing both IV and IM administration, tramadol was extracted from plasma using an automated solid phase extraction method and theconcentration measured by gas chromatography–mass spectrometry (GC/MS). The plasma drug concentrations after IV administrationwere best fitted by an open two-compartment model. However a three-compartment open model best fitted the IM data.

The results (means ± SEM) were as follows: after IV drug administration, the distribution half-life (t1/2a) was 0.22 ± 0.05 h, the elim-ination half-life (t1/2b) 1.33 ± 0.18 h, the total body clearance (ClT) 1.94 ± 0.18 L h kg�1, the volume of distribution at steady state (Vdss)2.58 ± 0.44 L kg�1, and the area under the concentration vs. time curve (AUC0–1) 1.25 ± 0.13 mg h L�1. Following IM administration,the maximal plasma tramadol concentration (Cmax) reached was 0.44 ± 0.07 lg mL�1 at time (Tmax) 0.57 ± 0.11 h; the absorption half-life (t1/2ka) was 0.17 ± 0.03 h, the (t1/2b) was 3.24 ± 0.55 h, the (AUC0–1) was 1.27 ± 0.12 mg h L�1, the (Vdarea) was 8.94 ± 1.41 L kg�1,and the mean systemic bioavailability (F) was 101.62%.

Three main tramadol metabolites were detected in urine. These were O-desmethyltramadol, N,O-desmethyltramadol and/or N-bis-desmethyltramadol, and hydroxy-tramadol. O-Desmethyltramadol was found to be the main metabolite. The urinary detection timesfor tramadol and O-desmethyltramadol were 24 and 48 h, respectively. The pharmacokinetics of tramadol in camels was characterisedby a fast clearance, large volume of distribution and brief half-life, which resulted in a short detection time. O-Desmethyltramadol detec-tion in positive cases would increase the reliability of reporting tramadol abuse.� 2007 Published by Elsevier Ltd.

Keywords: Tramadol; Pharmacokinetics; Metabolism

Introduction

The use of analgesic agents in veterinary medicine hasincreased substantially in recent years. Despite advancesin the development and availability of non-steroidal anti-inflammatory drugs (NSAIDs), their application can belimited due to their harmful side effects. Tramadol is a lreceptor agonist antitussive compound (Nosal’ova et al.,1991), but despite demonstrating an opioid mechanism of

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doi:10.1016/j.tvjl.2007.07.008

* Corresponding author. Tel.: +971 50 5817 969; fax: +971 2 4463 470.E-mail address: mezali@yahoo.com (M. Elghazali).

action, clinical practice has shown it to be unique amongother centrally acting opioid analgesics.

The therapeutic use of tramadol has not been associatedwith significant adverse effects, such as respiratory depres-sion, constipation or sedation, and the drug has been usedsuccessfully in humans to produce analgesia in post-opera-tive patients following orthopaedic and major gynaecolog-ical surgeries (Lehmann et al., 1990; Tuncer et al., 2003).Tramadol produced superior analgesia when used incombination with morphine compared to morphine alone,thereby decreasing morphine requirements (Webb et al.,2002). Tramadol is available as a racemic mixture

M. Elghazali et al. / The Veterinary Journal 178 (2008) 272–277 273

composed of (+) and (�) enantiomers in equal propor-tions. Despite its long-term use, the understanding and pre-diction of the time course of its pharmacological effects arestill hampered by the presence of active metabolites andcoexistence of opioid and non-opioid mechanisms.

Tramadol metabolism has been investigated in mice,hamsters, rats, guinea-pigs, rabbits, dogs and humans(Lintz et al., 1981). The principle metabolic pathways, O-and N-demethylation, involve cytochrome P-450 isoen-zymes 2D6, 2B6 and 3A4 (Subrahmanyam et al., 2001).O- and N-desmethylated compounds undergo further sul-phation or glucuronidation in phase II reactions. Onlyone of these metabolites, O-desmethyltramadol (M1metabolite), is pharmacologically active (Lee et al., 1993;Lintz et al., 1981; Subrahmanyam et al., 2001). A numberof new metabolites of tramadol, products of oxidative N-dealkylation and 1,6-dehydration, have recently been dis-covered in the urine of humans, rats, and dogs (Wuet al., 2001). In post-operative human patients, the mini-mum effective plasma concentrations for tramadol and itsmain metabolite M1 have been reported to be between298 ± 171 and 590 ± 410 and 39.6 ± 29.5 and 84 ± 34 ngmL�1, respectively (Lehmann et al., 1990; Grond et al.,1999).

Data concerning tramadol disposition, its metabolismand minimum effective plasma concentration in camelsare not available. Therefore, the purpose of this workwas: (1) to develop an assay to quantify tramadol in camelsplasma, (2) to determine the pharmacokinetics of tramadolfollowing intravenous (IV) and intramuscular (IM) singledose administrations, (3) to investigate the drug’s metabo-lism, and (4) to determine the urinary detection time oftramadol and its metabolites.

Materials and methods

Animals

Six camels (Camelus dromedarius), three males and three females, 5–7years of age and weighing approximately 300 kg were used in this study.The camels were deemed to be healthy, as confirmed by physical exami-nation and previous serum biochemical analysis. They were housed inshaded, ventilated stalls and received hay and water ad libitum. Theinvestigation was conducted under the supervision of the Ministry ofAgriculture, Veterinary Department.

Treatment

The study was carried out in a 2-period cross-over design, with animalsrandomly divided into two groups of three camels. In the first period, threecamels received tramadol (as tramadol hydrochloride solution, Grunen-thal) as a single IV dose of 2.33 mg kg�1 bodyweight, and three animalsreceived the same single dose of tramadol IM. The first period was fol-lowed by washout period of 15 days. After the washout time, the secondpart of the study was undertaken in which the treatments were reversed.

Sample collection

Blood samples (15 mL) were collected into heparinised Vacutainersfrom the opposite jugular vein prior to tramadol administration (0 time),

and 5, 10, 15, 30, and 45 min and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and24 h after IV drug administration. Blood samples were also collected fromthe jugular vein before drug administration (pre-injection, time 0), and 10,20, 30, and 45 min, and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24 h after IMdrug injection. The blood samples were immediately placed on ice, cen-trifuged at 1000 g at room temperature for 10 min, the separated plasmawas then stored at �80 �C until analysis to determine the tramadolconcentration.

Drug assay procedure

The concentration of tramadol in the plasma was measured by gaschromatography–mass spectrometry (GC/MS). Automated solid phaseextraction was carried out using Rapid trace workstations (Zymark) andASPEC XL (Gilson). To 1 mL plasma was added 100 lL internal stan-dard (diphenhydramine [1 lg mL�1]), and 3.5 mL phosphate buffer(100 mM pH 6.0). Extraction cartridges (Clean Screen, Worldwide Mon-itoring) were conditioned sequentially with methanol (5 mL) and 0.1 Mphosphate buffer (pH 6.3, 3 mL). The samples were passed through thecartridges and were followed by 0.1 M phosphate buffer (3 mL). Thecartridges were then washed with 1 M acetic acid (2 mL and then driedwith air at 45 psi for 5 min and washed with hexane (2 mL). Acid andneutral fractions were washed with dichloromethane (5 mL) and tramadolwas eluted with ethyl acetate: isopropyl alcohol (80:20 with 2% ammonia;3 mL). The organic solvent was transferred to clean test tubes and thesolvent was evaporated under nitrogen at 40 �C. The dried sample wasreconstituted in 50 lL of ethyl acetate and 2 lL were injected into the GC/MS system.

GC/MS analysis

GC/MS was carried out using an Agilent Technologies 5973 NetworkMass selective Detector interfaced to a 6890 N network GC system with7683B series auto-injector and sample tray. Injection was made in thesplitless mode onto a 30 m · 0.25 mm HP-5MS column (Hewlett-Pack-ard). The initial column temperature was 100 �C and it was programmedto rise to 310 �C at 20 �C min�1. Helium was used as the carrier gas. Datawere acquired in the selected ion monitoring (SIM) mode examining theions’ mass to charge ratios (m/z) which were 165 and 263 for diphenhy-dramine (ISTD) and tramadol, respectively. The linearity of the methodwas from 10 to 2000 ng mL�1 of tramadol in spiked plasma (r2 > 0.995).The inter assay coefficient of variation for 200 and 500 ng mL�1 (n = 6)determined on three consecutive days were 8.3% and 10.6%, respectively.The intra assay coefficient of variation for 10 ng mL�1 (n = 6) and 200 ngmL�1 (n = 10) were 10.9% and 4.03%, respectively. The recovery of spikedcamel plasma at concentrations of 200 and 2000 ng mL�1 was >91.0 %.The limit of quantification based on an S:N ratio P3 was 10 ng mL�1.

Determination of detection time

One of the objectives of this study was to determine the withdrawaltime for tramadol and its major metabolites in racing camels. Urinesamples from four camels were collected and pooled at the pre-injectionsampling (0-time), and at 24, 48, 72, 96, 120, 144, 168 and 192 h after drugadministration. Determination of the drug was carried out in the urinesamples using an extraction and derivatisation method for screening basicdrugs in post-race urine samples (Wasfi et al., 1998a).

Identification of tramadol metabolites

The metabolites of tramadol in the urine of camels (n = 4) wereinvestigated using a 5 mL aliquot of samples collected at 0, 5, 24, and 48 hafter IV drug administration. Metabolites were extracted using our routinesolid phase extraction method for acidic and basic drug screening in post-race camel urine (Wasfi et al., 1998a). Tramadol metabolites were theninvestigated from the chromatographic peaks obtained with the urinesamples taken after administration of tramadol compared with the urine

Table 1Mean ± SEM values for tramadol pharmacokinetic variables after intra-venous and intramuscular administration of 2.33 mg kg�1 in camels (IV,

274 M. Elghazali et al. / The Veterinary Journal 178 (2008) 272–277

samples taken before the drug administration (0 time) and confirmed bymatching against a commercial available spectral data-bases (Associationof Official Racing Chemists).

n = 5; IM, n = 6)

Variable IV IM

A (lg mL�1) 1.55 ± 0.37 1.40 ± 0.63B (lg mL�1) 0.51 ± 0.20 0.30 ± 0.11K12 (h�1) 1.38 ± 0.44 0.75 ± 0.44K21 (h�1) 1.50 ± 0.55 0.91 ± 0.28K(elim) (h�1) 1.61 ± 0.24 0.61 ± 0.05a (h�1) 3.92 ± 0.95 2.00 ± 0.61b (h�1) 0.56 ± 0.10 0.27 ± 0.05Tmax (h) – 0.57 ± 0.11Cmax (lg mL�1) – 0.44 ± 0.07t1/2a (h) 0.22 ± 0.05 0.42 ± 0.12t1/2b (h) 1.33 ± 0.18 3.24 ± 0.55AUC0–1 (mg h L �1) 1.25 ± 0.13 1.27 ± 0.12Vdarea (L kg�1) 3.98 ± 0.50 8.94 ± 1.41ClT (L h kg �1) 1.94 ± 0.18 1.91 ± 0.17Vdss (L kg�1) 2.58 ± 0.44 6.71 ± 1.12F (%) 101.62 ± 9.38Ka (h�1) 4.98 ± 1.01t1/2ka (h) 0.17 ± 0.03

Data analysis

Using a computerised pharmacokinetics and drug disposition program(PCModfit Ver 1.70 1999), pharmacokinetic parameters were calculatedfrom the best fitting relationship between plasma concentrations versustime. The best fitting model was chosen by Akaike’s minimum informationcriterion estimation (Yamaoka et al., 1978). They were also computedusing a non-compartmental analysis based on the statistical momentstheory (Gabaldi and Perrier, 1982; Martinez, 1998). The area under theplasma concentration-time curve (AUC) was calculated by the trapezoidalmethod with extrapolation to infinite time. Following IM administration,the absolute bioavailability (F) was calculated from the followingequation:

F ð%Þ ¼ ðAUCIMÞ=ðAUCIVÞ � 100

where AUCIM and AUCIV are the AUC of the drug from (0–1) after IVand IM administration. The volume of distribution (area method) was cal-culated by use of the following equation:

Vdarea ¼ Dose=ðAUC� bÞ:

A and B, zero time plasma concentration intercepts of biphasic IV and IMdisposition curves; a, hybrid rate constant of the slope of distribution; b,hybrid rate constant of the slope of elimination; Ka, hybrid rate constantof the slope of absorption; AUC0–1, the area under the concentration-time curves from zero to infinity; Vdarea, volume of distribution based onarea under the curve to infinity; t1/2abs, the absorption half-life; t1/2a, thedistribution half-life; t1/2b, the elimination half-life; K12, and K21. first-order transfer rate constants for drug distribution from the centralcompartment to the peripheral compartment and from the peripheralcompartment to the central compartment; K(elim), first order rate constantfor drug elimination; ClT, total body clearance; Vdss, volume of distri-bution at steady state; F%, percentage bioavailability.

Results

The plasma profile and pharmacokinetics of tramadolfollowing IV and IM administration is presented in Fig. 1and Table 1. A bi-exponential model best fitted the plasmaconcentration after IV administration in the six camels,while a 3-exponential model best fitted the data obtainedafter IM administration (n = 6). The curve (Fig. 1) of themean plasma concentration after IV administration indi-cated an initial rapid phase, corresponding to the distribu-tion of the drug with a distribution half-life (t1/2a) of0.22 ± 0.05 h. The distribution phase was followed by aslower elimination phase with an elimination half-life(t1/2b) of 1.33 ± 0.18 h.

Following IM administration, the peak concentration oftramadol (Cmax) in plasma was 0.44 ± 0.07 lg mL�1 at

0 2 4 6 8 10 120.01

0.10

1.00

10.00

Time (h)

TR

AM

Con

cent

ratio

n μg

/mL

Fig. 1. (Mean ± SEM) plasma concentration vs. time of tramadolfollowing IV (s) and IM (d) administration of 2.33 mg kg�1 bodyweight(n = 6).

0.57 ± 0.11 h (Tmax). The absorption half-life t1/2ka was0.17 ± 0.03 h. The mean elimination half-life (t1/2b) was3.24 ± 0.55 h following IM drug administration, whichwas longer and not significantly different (P = 0.58) fromthat obtained after IV administration (1.33 ± 0.18 h). Thesystemic bioavailability after IM administration was101.62% ± 9.38.

The basic fraction in urine samples collected 4.5–6 hafter tramadol administration (IV and IM), before andafter enzyme hydrolysis, showed several metabolites oftramadol. GC/MS analysis of the unhydrolysed basic frac-tion confirmed the presence of free tramadol and the freemajor metabolites O-desmethyltramadol, O-N-desmethyl-tramadol or N-desmethyltramadol, and hydroxy-tramadol.Tramadol and these metabolites were excreted in free andconjugated forms (glucuronide and/or sulphate).

The intensity of the GC peaks of tramadol and the threemajor metabolites obtained from non-hydrolyzed urinesamples was very low compared with enzymatically hydro-lyzed urine samples (Fig. 2). The main metabolite, O-des-methyltramadol (M1; Fig. 2), had a relative retentiontime (retention time of the metabolite/retention time tram-adol) of 1.04 and a molecular ion fragment m/z of 249, andremained detectable in urine for 48 h. The other metabolite(M5; Fig. 2) had a relative retention time of 1.05, a

Fig. 2. Reconstructed total ion chromatogram (TIC) of extracted camel urine after an IV dose of 2.33 mg kg�1 tramadol. A, pre-injection (time 0); B, 5 hpost-administration sample; C, 5 h post-administration enzyme hydrolyzed sample: tramadol (T), O-desmethyl tramadol (M1), O-N-desmethyl tramadolor N-bisdesmethyl tramadol (M5), and hydroxy tramadol (OH-T).

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molecular ion fragment m/z of 235 and could be detected inurine samples for 24 h. Two hydroxy-tramadol metaboliteswith molecular ion fragment m/z of 179 and with relativeretention times of 1.08 and 1.10 were identified as

hydroxy-tramadol, suggesting a hydroxy group in the phe-nyl and cyclohexane rings, respectively.

Metabolites due to the dehydration of tramadol andsimultaneous hydroxylation and dehydration were detected

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but their identification was not confirmed due to theirunavailability in our MS data-bases and lack of referencematerials. There was no evidence of metabolites in acidand neutral fractions. The amount of O-desmethyltram-adol excreted in urine was not quantified as total urine col-lection was not performed.

Using our routine methods for screening post-race urinesamples, we were able to detect tramadol, its main metab-olite (O-desmethyltramadol), and (O-N- or N-N-bides-methyltramadol) for 24, 48, and 24 h, respectively.

Discussion

Tramadol is a synthetic analgesic of the aminocyclohex-anol group with a complex interaction between opiate,adrenergic and serotonin receptors. Following IV adminis-tration of a single dose of tramadol to dromedary camelsthe disposition of the drug was described by a two-com-partment open model, while following IM administrationof a single dose, first-order absorption preceded a biphasicdisposition which was described by a two-compartmentmodel (Fig. 1).

Intravenous tramadol administration to dogs, humaninfants and other neonates (but not rabbits) have led tothe same pattern of fit (Kukanich and Papich, 2004; Allega-ert et al., 2005; Kucuk et al., 2005). In dogs, following asingle IV dose, tramadol had a t1/2b of 1.8 h and a Vdss

of 3.0 L kg�1 (Kukanich and Papich, 2004). In comparisonwith the results reported in the present study, the t1/2b of1.33 h and Vdss of 2.57 L kg�1 indicate that tramadol dis-position in dogs and camels is similar but unlike that foundin humans and rabbits. After a single IV injection of tram-adol in human children and adults, the mean t1/2b was6.4 h, with a Vd of 3.1 L kg�1 and total plasma clearanceof 6.1 mL min kg�1 (Murthy et al., 2000). In rabbits, phar-macokinetic studies following IV tramadol revealed thatthe Cmax and AUC were 14.3 lg mL�1 and 42.2 lg h mL�1,respectively (Kucuk et al., 2005). In the present study, theratio of K12 to K21 was 0.92, which suggested that the drugwas not retained in tissues, and the volume of the centralcompartment ranged from 0.714 to 2.170 L kg�1.

After IM administration to camels, the maximal plasmaconcentration and time to maximal plasma concentrationwere variable (Cmax range: 0.71–0.25 ng mL�1 and Tmax

ranged from 0.33 to 1.00 h). Although all camels were ofsimilar size and were treated in the same way prior to drugadministration in order to minimise variability caused byfood and management, the differences in bioavailabilityranged from 72.30% to 139.87%. Rate of absorption canaffect maximal plasma concentration; in humans, the peakplasma concentration was reached in 0.17–1.5 h and thebioavailability was 100% after IM administration (Chaoet al., 2000). In comparison, the results reported here afterIM tramadol administration to camels showed that peakplasma concentration was reached in 0.33–1.00 h, t1/2b

was 3.24 h and the drug was completely absorbed with amean systemic bioavailability of 101.63%.

The pharmacokinetics of tramadol in humans and ratsindicates gender-related differences (Liu et al., 2003; Hui-Chen et al., 2004). In contrast, the results reported in thisstudy suggest no therapeutically relevant gender differencesin the camel. We suggest future work be conducted to lookin greater detail at the pharmacokinetics of the differenttramadol enantiomers and to examine these for any gen-der-related effects. It will also be interesting to investigatethe effects of different routes of administration ofenantiomers.

The metabolic pathways of tramadol in the camel weresimilar to those reported in humans, mice, hamsters, rats,guinea-pigs, rabbits, and dogs (Lintz et al., 1981; Paaret al., 1996; Rudaz et al., 1999, 2000). The principal meta-bolic pathway is O- and N-desmethylation, and thisappears to be mediated by cytochrome P450 isoenzyme2D6 (pivotal), 2B6 and 3A4 (Subrahmanyam et al.,2001). We detected many metabolites in 4.5-6 h post-administration urine samples, but four major metaboliteswere identified (Fig. 2). The metabolism of tramadol incamels is extensive, producing basic metabolites. The majorroute of tramadol elimination, however, was as conjugatedtramadol and O-desmethyltramadol in the basic fraction;this could be a glucuronide and/or sulphate conjugatebecause of the nature of the enzyme we used. O-desmethyl-tramadol was found to have a much longer half-life thanunchanged tramadol or other tramadol metabolites.

It would appear from the results obtained from 4.5 to6 h post-administration urine samples (three camels IV,one camel IM) that the route of administration doesnot affect tramadol’s metabolic pathway. However, inhumans the metabolic pathway appears to be ratherslower and is affected by the route of administration(Lintz et al., 1981). Similar to humans, rats, and dogs(Wu et al., 2001), camels eliminate tramadol in hydroxyl-ated forms. Aromatic hydroxylation appears to be acommon phase 1 metabolic pathway in this species.For example, we have previously demonstrated hydro-xyl–flunixin, hydroxyl-tolfenamic acid, hydroxyl-tripelen-namine, and hydroxyl-diclofenac metabolites (Wasfiet al., 1998a,1998b,2000,2003).

One of the objectives of the present study was to deter-mine how long tramadol and its metabolites remain detect-able in camels. We were able to detect tramadol and itsmain metabolite, O-desmethyltramadoldol, in enzymati-cally hydrolyzed urine samples for 24 and 48 h, respec-tively, following IV and IM administration. Thesedetection times, despite tramadol’s short half-life of1.33 h and fast clearance of 1.93 L kg h�1, might reflectthe alkaline nature of camel urine (Wasfi et al., 1998a)where the drug will be in unionised form, and so reab-sorbed in the renal tubules. Also, glucuronide conjugatesare unstable in alkaline media, so the hydrolysis of trama-dol and O-desmethyltramadol glucuronides would beexpected and would enhance the recycling of tramadoland its main metabolite so extending its detection time incamels. This is likely as the camel has a small urine volume

M. Elghazali et al. / The Veterinary Journal 178 (2008) 272–277 277

(about 1.0 L day�1; Wasfi et al., 1998a) and a low glomer-ular filtration rate of 0.55–0.65 mL kg�1 min�1 (Wilson,1984), allowing ample time for in vivo hydrolysis and reab-sorption. We routinely perform enzyme or alkaline hydro-lysis in post-race urine samples to free conjugated drugsand we screen by very sensitive GC/MS methods (Wasfiet al., 1998a). Therefore, for precautionary measures, werecommend withholding tramadol administration to cam-els before racing for a minimum period of 3 days.

Acknowledgments

The authors thank Brigadier Dr. Ahmed Al Awadhi,Director of the Forensic Science Laboratory for his supportand advice. Thanks are also extended to Dr. Basal, S. Wajed,A. Alatoum, and N. Al Braiki for technical assistance.Special thanks go to all staff of the doping laboratory.

References

Allegaert, K., Anderson, B.J., Verbesselt, R., Debeer, A., de Hoon, J.,Devlieger, H., Van Den Anker, J.N., Tibboel, D., 2005. Tramadoldisposition in the very young: an attempt to assess in vivo cytochromeP450 2D6 activity. British Journal of Anaesthesia 95, 231–239.

Chao, C.K., Yu, L.L., Su, L.L., Liu, C.M., Yang, T.H., Chen, C.M., 2000.Bioequivalence study of tramadol by intramuscular administration inhealthy volunteers. Arzneimittelforshung 50, 636–640.

Gabaldi, M., Perrier, D., 1982. Pharmacokinetics, second ed. MarcelDekker Inc., New York, pp. 1–494.

Grond, S., Meuser, T., Uragg, H., Stahlberg, H.J., Lehmann, K.A., 1999.concentrations of tramadol enantiomers during patient-controlledanalgesia. British Journal of Clinical Pharmacology 48, 254–257.

Hui-Chen, L., Yang, Y., Na, W., Ming, D., Jian-Fang, L., Hong-Yuan,X., 2004. Pharmacokinetics of the enantiomers of trans-tramadol andits active metabolite, trans-O-demethyltramadol, in healthy male andfemale Chinese volunteers. Chirality 16, 112–118.

Kucuk, A., Kadiolu, Y., Celebi, F., 2005. Investigation of the pharma-cokinetics and determination of tramadol in rabbits plasma by a high-performance liquid chromatography-diode array detector methodusing liquid–liquid extraction. Journal of Chromatography B 816,203–208.

Kukanich, B., Papich, M.G., 2004. Pharmacokinetics of tramadol and themetabolite O-desmethyltramadol in dogs. Journal of VeterinaryPharmacology and Therapeutics 27, 239–246.

Lee, C.R., McTavish, D., Sorkin, E.M., 1993. tramadol. A preliminaryreview of its pharmacodynamic and pharmacokinetic properties, andtherapeutic potential in acute and chronic pain states. Drugs 46, 313–340.

Lehmann, K.A., Kratzenberg, U., Schroeder-Bark, B., Horrichs-Haer-meyer, G., 1990. Postoperative patient-controlled analgesia withtramadol: analgesic efficacy and minimum effective concentrations.Clinical Journal of Pain 6, 212–220.

Lintz, W., Erlacin, S., Frankus, E., Uragg, H., 1981. Biotransformation oftramadol in man and animal. Arzneimittelforschung 31, 1932–1943.

Liu, H., Jin, S., Wang, Y., 2003. Gender-related differences in pharma-cokinetics of enantiomers of trans-tramadol and its active metabolite,trans-O-demethyltramadol, in rats. Acta Pharmacologica Sinica 24,1265–1269.

Martinez, M.N., 1998. Non compartmental methods of drug character-ization: statistical moment theory. Journal of the American VeterinaryMedicine Association 213, 974–980.

Murthy, B.V., Pandya, K.S., Booker, P.D., Murray, A., Lintz, W.,Terlinden, R., 2000. Pharmacokinetics of tramadol in children after i.v.or caudal epidural administration. British Journal of Anesthesia 84,346–349.

Nosal’ova, G., Strapkova, A., Korpas, J., 1991. Relationship between theantitussic and analgesic activity of substances. Acta PhysiologicaHungary 77, 173–178.

Paar, W.D., Frankus, P., Dengler, H.J., 1996. High-performance liquidchromatographic assay for the simultaneous determination of tram-adol and its metabolites in microsomal fractions of human liver.Journal of Chromatography B 286, 221–227.

Rudaz, S., Cherkaoui, S., Dayer, P., Fanali, S., Veuthey, J.-L., 2000.Simultaneous stereoselective analysis of tramadol and its main phase 1metabolite by on-line capillary zone electrophoresis–electrosprayionization mass spectrometery. Journal of Chromatography A 868,295–303.

Rudaz, S., Veuthey, J.-L., Desiderio, C., Fanali, S., 1999. Simultaneoussteroselective analysis by capillary electrophoracesis of tramadolenantiomers and their main phase (metabolites in urine. Journal ofChromatography A 846, 227–237.

Subrahmanyam, V., Renwick, A.B., Walters, D.G., Young, P.G.,Price, R.J., Tonelli, A.P., Lake, B.G., 2001. Identification ofcytochrome P-450 isoformsresponsible for cis-tramadol metabolismin human liver microsomes. Drug Metabolism and Disposition 29,1146–1155.

Tuncer, S., Pirbudak, L., Balat, O., Capar, M., 2003. Adding ketoprofento intravenous patient-controlled analgesia with tramadol after majorgynecological cancer surgery: a double-blinded, randomised, placebo-controlled clinical trial. European Journal of Gynaecology andOncology 24, 181–184.

Wasfi, I.A., Abdel Hadi, A.A., Elghazali, M., Boni, N.S., Alkatheeri,N.A., Barezaiq, I.M., Hamid, A.M., 2000. Comparative disposition oftripelennamine in horses and camels after intravenous administration.Journal of Veterinary Pharmacology and Therapeutic 23, 145–152.

Wasfi, I.A., Boni, N.S., Abdel Hadi, A.A., El Ghazali, M., Zorob, O.,Alkatheeri, N.A., Barezaiq, I.M., 1998a. Pharmacokinetics, metabo-lism and urinary detection time of flunixin after intravenous admin-istration in camels. Journal Veterinary Pharmacology andTherapeutics 21, 203–208.

Wasfi, I.A., ElGhazali, M., Hadi, A.A., Zorob, O., Boni, N.S., Alkatheeri,N.A., Barezaiq, I.M., 1998b. Pharmacokinetics of tolfenamic acid andits detection time in urine after intravenous administration of the drugin camels (Camelus dromedarius). American Journal of VeterinaryResearch 59, 1451–1458.

Wasfi, I.A., Hussain, M.M., Elghazali, M., Alkatheeri, N.A., Hadi, A.A.,2003. The disposition of diclofenac in camels after intravenousadministration. The Veterinary Journal 166, 277–283.

Webb, A.R., Leong, S., Myles, P.S., Burn, S.J., 2002. The addition oftramadol infusion to morphine patient-controlled analgesia afterabdominal surgery: a double-blind placebo-controlled randomisedtrial. Anesthesia and Analgesia 95, 1713–1718.

Wilson, R.T., 1984. The Camel. The Print House Pte., Singapore, pp. 74.Wu, W.N., McKown, L.A., Gauthier, A.D., Jones, W.J., Raffa, R.B.,

2001. Metabolism of the analgesic drug, tramadol hydrochloride, in ratand dog. Xenobiotica 31, 423–441.

Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’sinformation criterion (AIC) in the evaluation of linear Pharmacoki-netic equations. Journal of Pharmacokinetics and Biopharmaceutics 6,165–175.