Il Farmaco 60 (2005) 163169

http://france.elsevier.com/direct/FARMAC/

Determination of tramadol hydrochloride in ampoule dosage forms by

using UV spectrophotometric and HPLC-DAD methods
in methanol and water media
Aysel Kk a, Ycel Kadoglu b,*
a

Department of Chemistry, Faculty of Science & Arts, University of Ataturk, 25240 Erzurum, Turkey
Department of Analytical Chemistry, Faculty of Pharmacy, University of Ataturk, 25240 Erzurum, Turkey
Received 23 July 2004; received in revised form 14 December 2004; accepted 28 December 2004

Abstract
Two newly developed simple and sensitive methods for determination of tramadol hydrochloride in ampoule dosage forms were described
and validated. Measurements for spectrophotometric method were performed using UV-Vis Spectrophotometer in ranges of 200400 nm. The
solutions of standard and the samples were prepared in methanol and water media and the UV absorption spectrums of tramadol were
monitored with maximum absorptions at 275 and 271 nm for both mediums, respectively. The standard calibration curves of tramadol were
constructed by plotting absorbance vs. concentration in the concentration range with the final dilution of 10100 g ml1. Reversed phase
chromatography for HPLC method was conducted using a Phenomenex Bondclone C18 column with an isocratic mobile phase consisting of
25% acetonitrile in 75% 0.01 M phosphate buffer (pH 3). The effluent was monitored on a DAD detector at 218 nm. Linear response (r > 0.99)
was observed over the range of 0.540 g ml1 for methanol and water and run on six different occasions. The methods were applied successfully to pharmaceutical ampoule forms, but also for comparison in two different solvent media. Besides, it was completely validated and
proven to be rugged.
2005 Elsevier SAS. All rights reserved.
Keywords: Tramadol; Pharmaceutical dosage form (ampoule)

1. Introduction
Tramadol hydrochloride, [()-trans-2-(dimethylaminomethyl)-1-(m-methoxyphenyl)-cyclohexanol hydrochloride,
C16H25O2N.HCl, F.W. = 299.84 g mol1] (Fig. 1), is a centrally acting analgesic agent which has been shown to be a
synthetic analogue of codeine [1]. It is metabolised by the
cytochrome P450 enzyme system in the liver to form 11 metabolites of which M1 (O-desmethyltramadol) predominates
and possesses analgesic properties [2]. It has been used since
1977 for the relief of strong physical pain and has been the
most widely sold opioid analgesic drug in the world [3].
The tramadol was determined by HPLC with UV detection [46,9], fluorescence detection [7,11,12] or electrochemical detection [10]. Capillary gas chromatography was also
* Corresponding author. Tel.: +90 0442 2311536; fax: +90 0442
2360962.
E-mail address: ykadiogli@yahoo.com (Y. Kadoglu).
0014-827X/$ - see front matter 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.12.002

used for determining tramadol [8]. The literature was reported

two spectrophotometric methods differed from our described
work for a kinetic investigated study of the oxidation reaction of the drug [13], influence of substitution and solvent for
a few drugs [14] and a reversed phase high performance liquid chromatographic method for the quantitation of tramadol
in pharmaceutical dosage forms [5].
To our knowledge, there is no UV spectrophotometric
method and HPLC-DAD method for determination of tramadol in pharmaceutical forms for both methanol and water

Fig. 1. Chemical structure of tramadol.

164

A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

media in literature. Therefore, the aim of the present work is

the development of simple and sensitive analytical methods
for the determination of tramadol hydrochloride in pharmaceutical ampoules. The methods have been validated to determine the tramadol injections in methanol and water solvent
media by using UV-Vis Spectrophotometer and HPLC. It has
been suggested that the two methods are applicable for determination of tramadol in ampoule dosage forms. The results
obtained from spectrophotometer and HPLC in two different
solvent media have been statistically compared.

rate was 1 ml min1 and an injection volume of 25 l was

used. The combination of the mobile phase, consisting of
0.01 M phosphate buffer (pH 3)-acetonitrile (75:25, v/v) with
the addition of 0.1% triethylamine was chosen and filtered
through a 0.47-m nylon membrane filter and degassed ultrasonically before use [16].
2.4. Preparation of standard solutions

The UV system consisted of Shimadzu UV-3101 PC

Model, UV-Vis-NIR Scanning Spectrophotometer, in 1.0 cm
quartz cells, was connected to PC Computer and HewlettPackard DeskJet printer. The absorbance value of tramadol
solutions was determined with a fixed slit width of 5 nm
between in wavelength ranges of 200400 nm.
The HPLC system consisted of a Thermoquest Spectra System P 1500 isocratic pump coupled with a Spectra System
UV 6000 LP photodiode array detection system, a Spectra
System AS 3000 autosampler, a SCM 1000 vacuum membrane degasser, a SN 4000 system controller. The detector
was set to scan from 200 to 800 nm and had a discrete channel set at 218 nm, which was the wavelength used for quantification.

Stock solutions of tramadol for UV determination were

monthly prepared at concentrations of 200 g ml1 in methanol and water and kept at 4 C. The working standard solutions were prepared by diluting the stock solutions in the concentration range from 10 to 100 g ml1 for both solvent
mediums. Five different concentrations of tramadol as the
working standard solutions, chosen for the calibration curve
were 10, 35, 65, 85 and 100 g ml1 (n = 6). The standard
solutions were prepared by dilution of different volumes of
the stock solution to a constant volume with methanol and
water. UV spectra were recorded against methanol and water
as reference substances.
Standard stock solutions containing tramadol for HPLC
determination were prepared monthly in methanol and water
at a concentration of 100 g ml1 and kept stored at 4 C. The
eight standard solutions from 0.5 to 40 g ml1 (0.5, 1, 3, 5,
10, 20, 30, and 40 g ml1) in methanol and water were made
by a serial dilution. The calibration graphs were constructed
in the range of 0.540 g ml1 for tramadol (n = 6). No change
in the stability of the stock solutions over 1 month was
observed.

2.2. Materials and reagents

2.5. Procedure

Tramadol standard was a gift from Grnenthal (Aachen,

Germany) and Abdi Ibrahim Ila San. ve Tic. A.S
q. (Istanbul,
Turkey). Contramal ampoules containing 100 mg tramadol
hydrochloride in 2 ml were obtained from Hospital of
Arastrma, University of Ataturk in Erzurum-Turkey. Purity
of the tramadol was tested by checking its melting point
(181 C), UV, 1H-NMR, 13C-NMR spectra and by HPLC
analysis using UV detection and no impurities were found.
Tramadol solutions were prepared for spectrophotometer with
deionised water and methanol of analytical grade (Merck).
HPLC grade methanol, acetonitrile, and triethylamine were
purchased from Merck. All other chemicals were obtained
from commercial sources and were of analytical grade. Buffer
solution (potassium dihydrogen phosphate, KH2PO4) was prepared with deionised water.

For two methods, accurately measured 5 l of ampoule

was transferred into a flask to give the concentration of
25 g ml1 and diluted to volume of 10 ml with methanol by
shaking for 5 min. Then, an aliquot of 10 l (8 l for HPLCDAD method) from the ampoule was diluted to 10 ml with
methanol into a flask (10 ml) for a theoretical final concentration of 50 g ml1 (40 g ml1 for HPLC-DAD method)
and the flask was shaken for 5 min. The same procedures
were made for deionised water in both of the methods.

2. Experimental
2.1. Apparatus

2.3. Chromatographic conditions

The analytical column was a Phenomenex Bondclone
reversed-phase C18 column with particle size of 10 m (300
3.90 mm I.D.). The column temperature was 30 C. The
control of the HPLC system and data collection were by Vestel computer equipped with Chromquest software. The flow-

3. Results and discussion

3.1. Method development
The UV absorption spectrums of tramadol were monitored a single well-defined maximum peak for methanol
medium at 275 nm and for water medium at 271 nm in the
measuring wavelength range of 200400 nm with a fixed slit
width of 5 nm. No difference was observed in the maximum
wavelengths of all spectra (n = 6). UV spectrums of tramadol
standard solutions both methanol and water media are shown
in Figs. 2 and 3, respectively. No interfering absorbances were

A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

165

Table 1
Features of the calibration curves of tramadol in methanol and water by
UV-Vis spectrophotometry (n = 6)
Features
Regression equation
RSD %
Correlation coefficient
Linear range (g ml1)

Fig. 2. UV spectrums of tramadol in methanol. Concentrations of tramadol,

(a) 10; (b) 25; (c) 35; (d) 50; (e) 65; (f) 75; (g) 85 and (h) 100 g ml1.

Fig. 3. UV spectrums of tramadol in water. Concentrations of tramadol, (a)

10; (b) 25; (c) 35; (d) 50; (e) 65; (f) 75; (g) 85 and (h) 100 g ml1.

found due to the ampoule tramadol. And for HPLC method,

no interfering peak and ghost peak were detected at chromatograms obtained at 218 nm by photodiode array detection system. Retention time for tramadol was determined as 5.82 min
for our HPLC-DAD system (see Fig. 6.).
3.2. Linearity of calibration curves
Five-level calibration series with six analyses at each concentration level were measured for UV determination. The
standard calibration curves of tramadol were constructed by
plotting absorbance vs. concentration for both methanol and
water media in six different days. The results were averaged
and analysed by linear simple regression model of y = mx + b
by the least-squares method. For both calibration curves, a
good linearity within the concentration range of 10

Methanol
y = 0.0069x + 0.0321
0.658.46
0.9923
10100

Water
y = 0.0061x + 0.0389
0.359.51
0.9912
10100

100 g ml1 was showed. The mean regression equations

of calibration curves and correlation coefficients were
y = 0.0069x + 0.0321, r = 0.9923 in methanol medium and
y = 0.0061x + 0.0389, r = 0.9912 in water medium. The correlation coefficient and the regression equation obtained for
the methanol line was higher than that of water as shown in
Table 1.
The tramadol contains a weakly absorbing chromophore
in its molecule [13]. This is the reason why UV detection is
not suitable for the determination of low tramadol concentrations [14]. Besides, the low concentrations indicated poor linearity in order that it is emerged the occurrence of scattering
at these tramadol concentrations. Therefore, the concentration range of 10100 g ml1 has been chosen as the most
suitable at all measurements for this method in UV spectrophotometry.
Eight-level calibration series with six analyses at each concentration level were measured for HPLC method on-line statistical processing of the calibration analyses by the least
squares method was performed automatically using the
Chromquest Software. The linearity of calibration graphs was
demonstrated by the good determination coefficients (r2)
obtained for the regression line in six different days. Typical
correlation coefficients were >0.99 same as UV determination. The mean regression equations were y = 8.106x 1.613,
r = 0.9967 in methanol medium and y = 7.106x 1.189,
r = 0.9948 in water medium. The correlation coefficient and
the regression equation obtained for the methanol line was
higher than that of water similar to UV determination as
shown in Table 4.
3.3. Method validation
The precision of the method, expressed as the relative standard deviation (RSD = 100.SD/mean), was assessed. Accuracy was expressed as the mean percentage of analyte recovered in the assay. Both statistical parameters were calculated
in each concentration level for the sensitivity of the method.
The limit of quantification (LOQ) was determined as the lowest concentration on the standard calibration curve that was
measured with a precision of 20% and accuracy of 80% or
120% [11]. The limit of detection (LOD), expressed as the
lowest amount of analyte that was detected but not quantified, was also calculated [15]. The all RSD values were lower
than 10%. Repeatability was given as inter- and intra-day precision and accuracy where evaluated by analysing three different concentration of tramadol on three different day.
At the UV determination, six replicate determinations at
three different concentrations (25, 50 and 75 g ml1 for tra-

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A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

madol in methanol and water) were carried out to test the

precision of these methods. The RSD values from tramadol
for intra-day precision in methanol and water, respectively,
were found to be 0.658.46% and 0.359.51%. The RSD values of the evaluated inter-day precision were showed as 2.36
5.13% and 1.895.55%, respectively. These data indicated
that the developed methods have a good repeatability. There
was difference between the RSD values of two techniques
and it was showed that the RSD values of methanol were
generally smaller than the values of water as given in Table 2.
The all RSD values for both intra- and inter-day precision
were lower than 10%.
For HPLC determination, precision and accuracy were
determined for tramadol standards at eight concentrations with
respect to a calibration graph prepared every day (n = 6). The
precision of the method was evaluated as the intra- and interday RSD of the measured peak areas standards at four differ-

ent concentrations (1, 5, 20, and 40 g ml1 for tramadol in

methanol and water). All samples for these purposes were
freshly prepared including preparing the standard solutions
from the same stock solutions (100 g ml1). Six replicates
from each pool were assayed on each of 3 days so that both
intra- and inter-day precision and accuracy could be determined. The RSD values from tramadol for intra-day precision in methanol and water, respectively, were found to be
0.867.34% and 1.549.31%. The RSD values of the evaluated inter-day precision were showed as 2.425.09% and
3.286.87% in methanol and water, respectively. These data
indicated that the developed methods have a good repeatability. There was difference between the RSD values of two techniques and it was showed that the RSD values of methanol
were usually smaller than the values of water as given in
Table 5. Precision studies showed acceptable the RSD values
were < 10% for both intra- and inter-day (n = 6) studies.

Table 2
Summary of assay the inter- and intra-day precision obtained data from tramadol in methanol and water by UV-Vis spectrophotometry
Intra-day
Sample
Concentration (g ml1)
Tramadol (Methanol) 25
50
75
Tramadol (Water)
25
50
75

n
6
6
6
6
6
6

X
0.2188
0.4030
0.5694
0.2126
0.3250
0.5159

SD
0.00264
0.00262
0.00179
0.00533
0.00466
0.00180

Inter-day
RSD (%)
1.21
0.65
1.77
2.51
1.43
0.35

X
0.1723
0.3440
0.5098
0.1839
0.3076
0.5020

SD
0.00810
0.01760
0.01200
0.00923
0.01710
0.00950

RSD (%)
4.68
5.13
2.36
5.02
5.55
1.89

Table 3
Statistical evaluation of obtained data from two solvent media in pharmaceutical preparation containing tramadol by UV-Vis spectrophotometry
Statistical values
n
X, Recovery (%)
SD (%)
Standard error (%)

Methanol
10
100.93
9.928
3.140

Water
10
89.12
9.083
2.872

t-test
tc: 2.776
tt: 1.734

n: number of determination, X: mean recovery, SD: standard deviation, tc: calculated t-value, tt: tabulated t-value.
Table 4
Features of the calibration curves of tramadol in methanol and water by HPLC (n = 6)
Features
Regression equation
RSD %
Correlation coefficient
Linear range (g ml1)

Methanol
y = 8.106x 1.613
0.867.34
0.9967
0.540

Water
y = 7.106x 1.189
1.549.31
0.9948
0.540

Table 5
Summary of assay the inter- and intra-day precision obtained data from tramadol in methanol and water by HPLC
Intra-day
Sample
Tramadol (methanol)

Tramadol (water)

Concentration (g ml )
1
5
20
40
1
5
20
40

n
6
6
6
6
6
6
6
6

X
269032
1008984
2940972
5372948
247065
969580
2794384
5887715

SD
14899
25920
25413
252450
22997
48588
78715
90737

Inter-day
RSD (%)
5.54
2.57
0.86
4.70
9.31
5.01
2.82
1.54

X
248349
1007647
3051504
5621313
214357
1008963
2824916
5517920

SD
10419
51313
73902
185887
14726
43148
92550
206120

RSD (%)
4.20
5.09
2.42
3.31
6.87
4.28
3.28
3.74

A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

3.4. Sensitivity
For UV determination, the limit of quantitation (LOQ) was
found as 5 g ml1 in methanol and 6 g ml1 in water. The
LOD was 2 g ml1 in methanol (S/N = 3.2) and 4 g ml1 in
water (S/N = 3.3). For HPLC determination, the limit of
quantitation was found as 0.75 g ml1 in methanol and
0.9 g ml1 in water. The LOD was 0.4 g ml1 in methanol
and 0.7 g ml1 in water (S/N = 2).
3.5. Statistical analysis of dosage forms
They were analysed using the same method which commercial ampoules were prepared as described in Procedure
2.5. This method has been applied successfully to commercial ampoule dosage forms. The UV spectrums of tramadol
concentrations (50 g ml1) prepared from commercial
ampoules in methanol and water were shown in Figs. 4 and
5, respectively. Statistical comparison of the results was performed with regard to accuracy and precision using Stu-

167

Table 6
Statistical evaluation of obtained data from two solvent media in pharmaceutical preparation containing tramadol
Statistical values
N
X, Recovery (%)
SD (%)
Standard error (%)

Methanol
12
99.47
4.039
1.166

Water
12
93.29
2.227
0.643

t-test
tc: 4.643
tt: 2.069

n: number of determination, X: mean recovery, SD: standard deviation, tc:

calculated t-value, tt: tabulated t-value.

dents t-test at 95% confidence level. It was also showed the

statistical evaluation of obtained data from two media with
same method in pharmaceutical preparation containing tramadol 100 mg/ml in Table 3. The Students t-test was showed
that there was significant difference between the two media
for same method with regard to accuracy. The mean recovery
was found to be 100.93% for methanol and 89.12% for water.
Same method was applied successfully to commercial
ampoule dosage forms by HPLC experiments as shown in
Fig. 7.a,b. The students t-test values of 95% confidence level
did not exceed the theoretical values, indicating a significant
difference between the accuracy and the precision of two
media as shown Table 6 (p < 0.05). The mean recovery was
found to be 99.47% for methanol and 93.29% for water. The
best results for tramadol determination were obtained in
methanol as shown Tables 3 and 6.
3.6. Influence of solvents

Fig. 4. UV spectrum of sample contained commercial tramadol ampoule

(50 g ml1) in methanol.

Since, two different solvents have been used, the maximum wavelength has been observed at 275 nm for methanol
and at 271 nm for water [12]. When using water, it has been
observed to a shift towards short-wavelength that is the shift
to blue, at 271 nm [14]. So, water is more a polar solvent
(forming the H-bond) than methanol, the detailed structures
are not appeared and produced the larger spectrums. The
polarity of methanol is lower than water and its dipole
dipole and dielectric effects are not present. Because of this,
the refined structures have seen as more detailed. Its absorption maximum is shifted from 275 to 271 nm with a bathochromic shift of 4 nm. That is, the situation has been recognized as a shift toward the red (bathochromic shift) that the
heteroatoms such as O and N are found in unsaturated structure. It is corresponded to the np* electronic transitions in
the UV spectra region. Thus, the np* energy barrier larges
in a polar solvent and the shift is observed at maximumwavelength. Also, its energy increases since its wavelength is
shortened. Moreover, from these findings obtained for the
absorbance measurements of tramadol, it has been concluded that methanol can be more an appropriate solvent than
water and kmax is in accommodation with absorbance wavelength recorded for methanol in literature [12,14].
4. Conclusions

Fig. 5. UV spectrum of sample contained commercial tramadol ampoule

(50 g ml1) in water.

This is the first such reported method for quantitative determination of tramadol in ampoules for both methanol and water

168

A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

Fig. 6. Chromatogram of tramadol standard of 10 g ml1 in HPLC-DAD system.

Fig. 7. Chromatograms of samples contained commercial tramadol ampoule (25 g ml1) in methanol (A) and in water (B).

solvent media by two different analytical methods. Then, these

developed analytical spectrophotometric and HPLC methods
have been validated for two different solvent media. The
applied methods are advantageous in having simple and rapid
for the determination of the concentration of tramadol in
ampoules when compared with other methods in the literature for the routine determination [13,5]. The different methods can be used as an alternative method for the routine deter-

mination of tramadol and for two different solvent media in

the pure form and in this pharmaceutical formulation with
good linearity, sensitivity, reproducible, accuracy, and precision.
The obtained statistical values indicated difference between
the accuracy and the precision of two analytical methods.
Besides, the comparison of statistical results obtained by two
methods indicated that methanol was more an appropriate sol-

A. Kk, Y. Kadoglu / Il Farmaco 60 (2005) 163169

vent than water for both methods and the recovery for methanol solvent medium were better than water solvent medium
for both methods as shown in Tables 3 and 6. A through investigation was conducted in order to choose the optimum solvent medium for spectrophotometric and HPLC-DAD determination of tramadol.

[6]

[7]

[8]

Acknowledgements
We would like to thank Grunenthal GmbH (Germany),
Abdi Ibrahim Ila San. ve Tic. A. S
q. (Turkey) and its manager
Ayse Kse for her invaluable help in providing drug standard
and Dr. Nazm Dogan (Faculty of Medicine, University of
Ataturk) for providing of Contramal ampoules.

[9]

[10]

[11]

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