Bioequivalence Study of Tramadol by

Intramuscular Administration in Healthy

Volunteers
Chih-Kai Chao a, Li-Li Yua, Li-Li Sua, Chu-Min Liua. Tsung-Hsien Yang a, and Chi-Ming Chena, b

Department of Pharmaceutical Technology, Mithra Bioindustry Co.a, Taipei Hsien (Taiwan),

and School of Pharmacy, Taipei Medical Collegeb, Taipei (Taiwan)

Summary

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Tramadol hydrochloride (CAS 36282-47-0) is a centrally acting analgesic agent binding to µ
opiate receptors. The bioavailability of a new tramadol hydrochloride injection (Limadol)
was compared with a commercially available reference product by intramuscular administra-
tion in twelve healthy Chinese male volunteers by a standard two-way cross-over trial. Each
volunteer received a single 100 mg injection of tramadol HCl in each phase. The bioavailability
was compared using the area under the plasma concentration-time curve from time 0 to 30 h
(AUC0−30), the area under the plasma concentration-time curve from time 0 to infinity
(AUC0−⬁), peak plasma concentration (Cmax), and time to reach peak plasma concentration
(Tmax). No statistically significant difference was observed between the Tmax, Cmax, AUC0−30
and AUC0−⬁ of the two preparations. It is concluded that test and reference formulations of
tramadol hydrochloride are bioequivalent for both the extent and rate of absorption after a
single intramuscular injection.

Zusammenfassung

Untersuchung der Bioäquivalenz von Tramadol nach intramuskulärer Injektion

bei gesunden Probanden
Tramadol-Hydrochlorid (CAS 36282-47-0) ist ein zentral wirkendes Analgetikum, das an µ-
Opiat-Rezeptoren bindet. Die Bioverfügbarkeit von Tramadol-Hydrochlorid aus einer neuen
Zubereitung zur Injektion (Limadol) wurde mit der eines handelsüblichen Produkts nach
intramuskulärer Injektion bei zwölf gesunden männlichen chinesischen Probanden im standar-
disierten Zweifach-cross-over-Versuch untersucht. Jeder Proband erhielt in jeder Phase eine
einmalige Injektion mit 100 mg Tramadol-HCl. Die Bioverfügbarkeit wurde an Hand der
Fläche unter der Plasmakonzentration-Zeit-Kurve vom Zeitpunkt 0 bis 30 h (AUC0−30) bzw.
bis unendlich (AUC0−⬁), der Spitzenkonzentration im Plasma (Cmax) und der Zeit bis zum
Erreichen der Spitzenkonzentration im Plasma (Tmax) bestimmt. Zwischen den beiden Präpa-
raten wurden keine signifikanten Unterschiede bezüglich Tmax, Cmax, AUC0−30 und AUC0−⬁
gefunden. Daraus wird geschlossen, daß Test- und Referenzformulierung von Tramadol-HCl
nach einmaliger intramuskulärer Injektion sowohl im Hinblick auf Ausmaß und Geschwindig-
keit der Absorption bioäquivalent sind.

Key words Analgesics · CAS 36282-47-0 · Limadol, bioavailability, bioequivalence · Trama-

dol hydrochloride, intramuscular administration

Arzneim.-Forsch./Drug Res. 50 (II), 636−640 (2000)

Arzneim.-Forsch./Drug Res. 50 (II), Nr. 7 (2000)

636 Chao et al. − Tramadol
 2.2. Chromatographic conditions
HPLC was performed using an Intelligent LC-10AT
HPLC pump (Shimadzu, Tokyo, Japan) and WISP 712
autosampler (Waters, Milford, MA, USA) with RJ-10A
fluorescence detector (Shimadzu, Tokyo, Japan) at an
excitation wavelength of 272 nm and an emission wave-
length of 299 nm. A HP-3396A integrator (Hewlett-
Packard, Boise, IH, USA) as used to measure the peak
areas. HPLC separation was carried out on a silica col-
umn (Ultrasphere, 25 cm × 4.6 mm, I.D. 5 µm)
(Beckman, Fullerton, CA, USA) with acetonitrile and
20 mol/l (NH4)2HPO4 (adjusted to pH 7.0) in the ratio
40 : 60 (v/v) at a flow rate of 1 ml/min.

2.3. Protocol design

Scheme 1: Chemical structure of tramadol HCl (I) and N-
acetylprocainamide (II) internal standard.
The protocols of this study were approved by the De-
partment of Health, Republic of China (Taiwan). Twelve
healthy Chinese male volunteers (21 to 24 years) were
enrolled in the study after providing written informed
consent. Health status was determined before and after
the study based on medical history, physical examina-
tion, and laboratory findings. Exclusion criteria were re-
cent drug use, history of drug abuse or drug intolerance,

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1. Introduction and any clinically significant deviation from normality
in either the physical examination and laboratory test
Tramadol hydrochloride (trans-(±)-2-[(dimethyl- results. No drugs were allowed for 2 weeks before and
amino)methyl]-1-(3- methoxyphenyl)cyclohexanol during the study. Alcohol ingestion or smoking was not
hydrochloride, CAS 36282-47-0) (Scheme 1) is a permitted for 48 h before the study. Subjects were fasted
centrally acting analgesic which possesses opioid for 10 h before the study. Reference and test preparations
agonist properties. It inhibits pain by binding to µ of tramadol hydrochloride were given randomly to the
subjects as shown in Table 1, and one week wash-out
opiate receptors and inhibiting norepinephrine and time was allowed.
serotonin reuptake [1−3]. Unlike typical opioid an-
algesics, tramadol HCl has not been associated
with clinically significant side effects such as respir- 2.4. Drug administration and blood sampling
atory depression, constipation, sedation or cardio- On the first day of the study an indwelling venous cath-
vascular effects [4−7]. The euphorogenic [8] and de- eter was placed into the antecubital vein, and 20 ml
pendence [9, 10] effects of tramadol HCl are also blood was taken as the zero-hour sample. Each subject
then received a tramadol HCl injection (100 mg/2 ml) in
relatively slight. the upper outer quadrant of the buttocks. After dosing,
Tramadol may be administered orally, rectally, or 10 ml of blood was collected at 0.167, 0.333, 0.667, 1,
parenterally. After a single oral administration of 1.5, 2, 4, 6, 8, 12, 24 and 30 h. Blood samples were cen-
tramadol (100 mg) capsules or tablets to young trifuged at 3000 rpm for 10 min within half an hour after
healthy volunteers, a Cmax of 280 to 308 ng/ml was collection and the plasma was frozen at −20 °C.
achieved after 1.6 to 2 h (Tmax) [11, 12]. The elim-
ination half-life (T1/2) of tramadol HCl (100 mg)
following a single intravenous or oral dose was 5.1
to 5.9 h [11, 12]. Following oral administration of
tramadol (100 mg) to German subjects, the AUC
was 2488 ± 774 h · ng/ml [12]. The mean oral Table 1: Data of subjects and scheme of randomized admini-
bioavailability, the volume of distribution and the stration of tramadol HCl.
total clearance of tramadol were 68 %, 306 ± 52 l
and 710 ± 174 ml/min, respectively [12]. In this Volunteer Sex
Age Weight Height
Order of
admin-
study, the comparative bioavailability of two tram- (years) (kg) (cm)
istrationa)
adol injections after intramuscular administration
was investigated. 1 M 22 64 160 A B
2 M 22 60 171 B A
3 M 21 68 172 A B
4 M 22 60 175 B A
5 M 22 65 172 A B
6 M 24 73 170 B A
2. Materials and methods 7 M 21 64 173 A B
8 M 21 63 175 B A
2.1. Materials and reagents 9 M 23 58 170 A B
Limadol as test formulation was supplied by Nang Ku- 10 M 23 60 171 B A
ang Pharmaceutical Co. Ltd., Tainan (Taiwan). The ref- 11 M 22 63 174 A B
erence product was obtained by the respective manufac- 12 M 21 70 176 B A
turer. N-Acetylprocainamide was from Sigma (St. Louis, a)
A: reference preparation (100 mg/2 ml); B: test preparation
MO, USA), and both acetonitrile and ammonium phos- (100 mg/2 ml).
phate were from E. Merck, Darmstadt (Germany).
Arzneim.-Forsch./Drug Res. 50 (II), Nr. 7 (2000)
Chao et al. − Tramadol 637
2.5. Determination of tramadol concentration in
plasma
Plasma samples containing N-acetylprocainamide in-
ternal standard (Scheme 1) were quantified by HPLC
with fluorescence detection according to the method de-
scribed by Novilis et al. [13] with some modifications.
Tramadol in plasma (1.0 ml) was alkalinized with 8 N
NaOH solution (500 µl) and then extracted with diethyl
ether (7.0 ml). Following centrifugation at 3000 rpm for
10 min, the organic solvent was evaporated under a N2
stream and reconstituted with 200 µl of morbile phase.
25 µl of sample solution was injected into the HPLC
column. Quantitation analysis of tramadol in plasma
was performed by computing the ratio of the area under
the peak of tramadol and the internal standard versus
the concentrations. Concentrations of tramadol were Fig. 1: Plasma concentration-time profiles of tramadol (100 mg)
found to be linearly related to the peak ratio between after intramuscular administration of two formulation to twelve
600 ng/ml and 10 ng/ml. Intra-batch and inter-batch Chinese subjects. Each point represents the mean ± SD.
accuracy, calculated as the relative error of the measured
versus the known concentrations, were less than 7 %. In-
tra-batch and inter-batch precision, determined as the
coefficient of variation, i.e. the ratio between the mean
of the found concentrations and its S.D., was less than 3160 ± 527 h · ng/ml as 3120 ± 622 and 3250
6 %. The lower limit of quantitation was 10 ng/ml. The ± 674 h · ng/ml, respectively. Table 3 shows the

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average recovery of tramadol from plasma by organic ANOVA test results of the logarithmically trans-
solvent extraction was 81.0 % formed values of Cmax, AUC0−30 and AUC0−⬁ for
reference and test drugs. No significant differences
2.6. Pharmacokinetic and statistical analysis were observed between the values of ln(Cmax),
Following intramuscular administration of tramadol ln(AUC0−30) and ln(AUC0−⬁) for reference and test
HCl (100 mg) to volunteers, the major pharmacokinetic products. The power values of ln(Cmax), ln(AUC0−
parameters were estimated from the plasma concentra- 30) and ln(AUC0−⬁) for reference and test drugs
tions versus time data. The total area to infinity after were also higher than 0.8. By two one-sided test
the last dose (AUC0−⬁) was calculated by the summation procedures, the 90 % confidence interval of
of AUC0−30 and C30/Kel. The AUC0−30 was calculated
using the trapezoidal rule from time 0 to 30 h. C30 was ln(Cmax), ln(AUC0−30) and ln(AUC0−⬁) ratios were
the drug plasma concentration at 30 h. The elimination also within the 80−125 % interval (Table 4). Tmax
rate constant (Kel) was derived from the terminal slope is a primary indication of the rate of drug absorp-
of the plasma drug concentration-time profile. Cmax, tion and is critical for the analgesic effect of trama-
peak drug concentration and Tmax, time to peak drug dol hydrochloride. Because longer sampling inter-
concentration were obtained directly from drug concen- vals would give larger variance, non-parametric
tration versus time curve. Bioavailability of the two tra- tests were evaluated. The results show that the val-
madol formulations was assessed by comparing AUC, ues of Tmax were not significantly different between
Tmax and Cmax using two-way cross-over analysis of
ANOVA with two one-sided tests.
Table 2: Pharmacokinetic parameters of tramadol after intra-
2.7. Onset and duration of action muscular administration (100 mg) of reference and test formu-
The onset and duration of tramadol were defined as the lation.
time when the plasma concentration reached and main-
Volun- Cmax Tmax AUC0−30 AUC0−⬁
tained 100 ng/ml [14, 15]. The onset and duration values (ng/ml) (h) (h · ng/ml) (h · ng/ml)
were obtained by linear interpolation in the ascending teer
and descending sections of the plasma concentration- no. A B A B A B A B
time curves near 100 ng/ml.
1 391 476 0.670 0.170 2660 2590 2760 2680
2 371 353 0.170 1.000 2630 2720 2710 2800
3 206 281 4.000 1.500 2820 3220 2950 3360
4 370 335 1.500 1.500 2900 2960 3020 3070
3. Results 5 381 316 0.330 0.330 2420 3110 2560 3200
6 267 389 1.000 1.500 3130 3310 3240 3450
Fig. 1 illustrates the plasma concentration-time 7 327 378 1.500 1.000 3320 3340 3470 3490
profiles of tramadol over a 30-h period in twelve 8 423 438 0.670 0.670 2780 2710 2880 2810
subjects after intramuscular injection of the refer- 9 474 471 0.330 0.670 4370 4930 4550 5210
ence and test formulations. Table 2 shows the Cmax, 10 312 436 0.330 0.170 3090 3050 3260 3200
Tmax, AUC0−30 and AUC0−⬁ values calculated for 11 250 232 0.670 4.000 3400 2850 3520 2940
12 485 320 0.170 1.000 2920 2700 3030 2820
the two preparations. These results show that both Mean 355 369 0.945 1.13 3040 3120 3160 3250
formulations were rapidly absorbed into the blood SD 86.2 76.7 1.070 1.03 508 622 527 647
stream. Maximum drug concentrations in plasma A: reference preparation (100 mg/2 ml); B: test preparation (100
of 355 ± 86.2 and 369 ± 76.7 ng/ml were reached mg/2 ml); Cmax: maximum plasma concentration; Tmax: peak
for reference and test formulation at the average time of maximum plasma concentration; AUC0−30: area under
times of 0.945 ± 1.07 and 1.13 ± 1.03 h, respec- plasma concentration-time curve from time 0 to time 30 h;
tively. The mean AUC0−30 and AUC0−⬁ for refer- AUC0-⬁: area under plasma conentration-time curve from time
0 time infinity.
ence and test formulations were 3040 ± 508 and
Arzneim.-Forsch./Drug Res. 50 (II), Nr. 7 (2000)
638 Chao et al. − Tramadol
Table 3: Results of cross-over analysis of pharmacokinetic para- drug and 5.14 ± 2.29 min for test drug. The mean
meter variance of tramadol injections. duration for reference and test formulations were
DF MS F-Value P-Value Power 11.2 ± 2.23 h and 11.2 ± 2.45 h, respectively (Table
5). The onset and duration for both tramadol in-
ln(Cmax) Subj(Seq) 10 0.0906 2.99 0.0496 jections were almost identical.
Seq 1 0.0557 0.614 0.451
Period 1 0.00172 0.0567 0.817
Treatment 1 0.0133 0.438 0.523 0.807
Error 10 0.0303
ln(AUC0−30) Subj(Seq) 10 0.0464 7.55 0.00183 4. Discussion
Seq 1 0.0521 1.12 0.314
Period 1 0.00410 0.668 0.433 The bioequivalence of tramadol formulations was
Treatment 1 0.00375 0.611 0.453 1.00 evaluated after intramuscular administration to
Error 10 0.00614 healthy volunteers. The curves of the concentra-
ln(AUC0-⬁) Subj(Seq) 10 0.0485 8.34 0.00122 tions of tramadol in plasma showed the similarities
Seq 1 0.0547 1.13 0.313 of the two formulations. None of the pharmacoki-
Period 1 0.00346 0.595 0.458 netic parameters observed or calculated from the
Treatment 1 0.00331 0.570 0.468 1.00
Error 10 0.00581 concentration-time curve were significantly differ-
ent. Intramuscular administration of tramadol
DF: degree of freedom; MS: mean square; Cmax: maximum showed higher Cmax, greater AUC and longer elim-
plasma concentration; AUC0−30: area under plasma concentra-
tion-time curve from 0 to 30 h; AUC0-⬁: area under plasma
ination half-life than those found after administra-
concentration-time curve from time 0 to time infinity. tion of the same oral dose to healthy subjects [16].
The onset of the analgesic effect by oral tramadol
(100 mg) was reached within 30−40 min [16] and

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the duration was 8−9 h [16, 11]. From the present
Table 4: Results of cross-over analysis of two one-sided tests of
pharmacokinetic parameters. study, it appears that intramuscular injections of
tramadol hydrochloride provided a more rapid on-
90 % Confidence set and longer duration than that of the oral route.
Sample size intervals
In conclusion, the bioavailability of the two trama-
ln(Cmax) 12 (96.8−108 %) dol products was equivalent and these two injec-
ln(AUC0−30) 12 (96.8−109 %) tions can be used in place of each other.
lnAUC0-⬁) 12 (92.1−119 %)
Cmax: maximum plasma concentration; AUC0−30: area under
plasma concentration-time curve from 0 to 30 h; AUC0-⬁: area
under plasma concentration-time curve from time 0 to time infi- 5. References
nity.
[1] Lee, C. R., McTavish, D., Sorkin, E. M., Tramadol,
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and Q3 of 0.219, 0.386 and 0.653, respectively. [2] Raffia, R. B., Fridrichs, E., Reimann, W. et al.,
Opioid and nonopioid components independently con-
Based on a threshold concentration of 100 ng/ml, tribute to the mechanism of action of tramadol, an
the onset and duration of the analgesic effect of “atypical” opioid, J. Pharmacol. Exp. Ther. 260, 275
tramadol was estimated. After intramuscular ad- (1992)
ministration of tramadol hydrochloride (100 mg), [3] Dayer, P., Desmeules, J., Collart, L., Pharmacology
the mean onset was 5.65 ± 3.56 min for reference of tramadol, Drugs 3 (Suppl. 2), 18 (1997)
[4] Vogel, W., Burchardi, H., Sihler, K. et al., The effect
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Correspondence: Prof. Dr. Chi-Ming Chen,
School of Pharmacy, Taipei Medical College,
250 Wu-Hsing Street, Taipei (Taiwan)
E-mail: pcmc@tmc.edu.tw

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ArzneimForsch
DrugRes
Antiemetika · Magen-Darm-Therapeutika · Urologika

Antiemetics · Gastrointestinal Drugs · Urologic Drugs

Determination of Trimebutine and Desmethyl-

trimebutine in Human Plasma by HPLC
Michel Lavit a, b, Sylvie Saivina, b, Hamid Boudraa, b, Frank Michela, Arnaud Martin c, Gérard Cahiez c,
Jean Pierre Labauned, Jean Marie Chomarde, and Georges Houina, b

Laboratoire de Pharmacocinétique et de Toxicologie Clinique, Centre Hospitalier Universitairea,

Toulouse (France), Laboratoire de Cinétique des Xénobiotiques, Faculté des Sciences Pharmaceutiques b,
Toulouse (France), Centre de Recherche, Ecole supérieure de Chimie Organique et Minérale (ESCOM)c,
Cergy-Pontoise (France), Rivopharm S.A.d, Manno, Lugano (Switzerland), and Laboratoires Fournier,
Direction EuroGalenae, Chenôve (France)

Summary

A simple and sensitive HPLC method has been developed to measure trimebutine (CAS
39133-31-8, maleate: CAS 34140-59-5) and its main metabolite desmethyl-trimebutine in hu-
man plasma. The method was validated according to the Washington Consensus Conference
on the Validation of Analytical Methods. It involved extraction of the plasma with n-hexane
containing 2-pentanol, followed by reversed-phase HPLC using a Partisil ODS2 10 µm col-
umn and UV detection at 265 nm. The retention times of the internal standard (procaine),
desmethyl-trimebutine and trimebutine were 2.4, 4.3 and 6.5 min, respectively. The standard
Arzneim.-Forsch./Drug Res. 50 (II), Nr. 7 (2000)
640 Lavit et al. − Trimebutine and desmethyl-trimebutine