American Journal of Anal yt ical Chemistry, 2011, 2, 768-775
doi:10.4236/ajac.2011.27088 Published Online November 2011 (
Copyright © 2011 SciRes. AJAC
Flow Injection Determination of Tramadol Based on Its
Sensitizing Effect on the Chemiluminescent Reaction of
Xun Yao, Jingkai Zhang, Jianguo Li*
College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, China
E-mail: *
Received July 14, 2011; revised August 15, 2011; accepted August 27, 2011
In this paper, a novel chemiluminescence (CL) method for the determination of tramadol has been developed
by combining the flow injection technique and its sensitizing effect on the weak CL reaction between sulfite
and acidic KMnO4. A mechanism for the CL reaction has been proposed on the basis of fluorescent and CL
spectra. Under the optimized conditions, the proposed method allows the measurement of tramadol hydro-
chloride over the range of 0.04 - 4 g/mLwith a correlation coefficient of 0.9995 (n = 8) and a detection limit
of 0.01 g/mL (3σ), and the relative standard deviation for 2.0 g/mL tramadol (n = 11) is 2.1%. The utility
of this method was demonstrated by determining tramadol hydrochloride in tablets and injections.
Keywords: Chemiluminescence, Tramadol hydrochloride, KMnO4, Sodium Sulfite, Flow-Injection Analysis
1. Introduction
y-phenyl)-cyclohexanol] (Figure 1) hydrochloride is a
centrally acting analgesic agent which possesses an an-
algetic action with a potency ranging between weak
opioids and morphine [1,2]. It is mainly metabolized by
liver and essentially excreted by the kidney [2,3]. Clini-
cal studies have shown that respiratory depression, pro-
nounced opioid side-effect profile and analgetic toler-
ance hardly happen after repeated administration are ob-
served with tramadol [4]. Therefore, it is successfully
used since 1977 for the relief of short-term and long-term
pains which may be induced by the different illnesses or
traumas in the world [5].
Various methods have been reported for the determi-
nation of tramadol hydrochloride in biological fluids and
in pharmaceutical preparations, including HPLC [6-11],
HPTLC [12], GC [13,14], capillary electrophoresis(CE)
[15,16], spectrophotometry [17-19], voltammetry [20]
and potentiometry [21]. Each of the above methods had
its own merits and disadvantages, for example , some of
them were accurate and selective, but involved expensive
instrumentation and were time consuming, needed
preconcentration before determination because of lacking
high sensitive detector; some were inherent simple but
have low sensitivity . Therefore, it is very important to
develop a rapid, simple, sensitive and accurate method
for the determination of tramadol in order to obtain op-
timal therapeutic concentrations for quality assurance in
pharmaceutical preparations.
Flow-injection chemilluminescence(CL) methods are
powerful analytical techniques that have been frequently
used for the analysis of pharmaceuticals in recent years
and have excellent sensitivity, wide linear dynamic range,
a high degree of reproducibility, which requires rela-
tively simple and inexpensive instrumentation [22-25].
To the best of our knowledge, up to the present time,
nothing has been published concerning the determination
of tramadol with a flow injection CL method.
Figure 1. Chemical structures of tramadol.
In order to develop a simple, rapid, and sensitive
method for the determination of tramadol hydrochloride
in both pure form and its pharmaceutical preparations,
this work studied the sensitizing effect of tramadol on the
CL intensity emitted from the reaction of sulfite with
acidic KMnO4. A method for tramadol detection is pro-
posed on the basis of this sensitizing effect. This method
is simple and less expensive than the above-mentioned
techniques and at the same time offers good accuracy
and precision. Combining with flow injection technique,
this effect provides a sensitive and convenient method
for the determination of tramadol in pharmaceutical
preparations. It has been used to determine tramadol hy-
drochloride in tablets and injections successfully. The
possible CL reaction mechanism has also been discussed
on the basis of CL spectra.
2. Experimental
2.1. Apparatus
The CL emission was recorded with a set of flow-injec-
tion CL analyzer (IFFL-E, Xi’an Ruike Electronic equip-
ment Corporate, Xi’an, China). The schematic diagram
of the flow-injection CL analytical system was shown in
Figure 2. Two peristaltic pumps were used to deliver
flow streams. PTFE tubing (0.8 mm i.d.) was used to
connect all components in the flow system. A flow cell
that positioned in front of the detection window of the
photomultiplier tube (PMT) (CR-105, Hamamatsu, Bei-
jing, China) was a 25 cm length of spiral glass tubing
(1.0 mm i.d.) and the distance between injection valve
and flow cell was about 10 cm. Sample solution of
tramadol hydrochloride was injected into Na2SO3 carrier
stream using a six-way injection valve equipped with a
80 µL sample loop, merged with KMnO4 solution stream,
and then generated CL emission in the flow cell. The CL
signal was detected by the PMT with no wavelength dis-
crimination and recorded with computer employing an
IFFL-E flow-injection CL analysis system software. The
fluorescence and absorption spectra were monitored us-
ing a F-2500 fluorescence spectrometer (Hitachi, Tokyo,
Japan) and a Shimadzu UV-2450 UVvisible recording
spectrophotometer (Shimadzu, Kyoto, Japan), respec-
tively. The CL spectrum was obtained with a series in-
terference filters by the static method. The filters were
inserted between the sample cuvette and the photomulti-
plier tube (PMT).
2.2. Reagents and Chemicals
Pure powders of tramadol hydrochloride was obtained
from Nanjing Institute for Drug Control (Nanjing, China).
KMnO4 and Na2SO3 were purchased from Shanghai
Chemical Reagent Limited Company (Shanghai, China).
All other reagents and chemicals were commercially
available and of analytical reagent grade. All solutions
were prepared with sub-boiling distilled deionized water.
The standard solution of tramadol hydrochloride
(1.000 mg/ml) was prepared by dissolving 0.0500 g
tramadol hydrochloride with water and diluting to 50mL
with water, stored in the refrigerator (4˚C) and diluted as
required daily. The Na2SO3 solution (1.0 × 102 mol/L) was
prepared by dissolving 0.1260 g Na2SO3 with 100 mL
water daily and kept in a dark place. Potassium perman-
ganate solution (1.0 × 102 mol/L) was prepared by dis-
solving 1.58 g of KMnO4 in 1 L of boiled water and fil-
tering through glass wool.
2.3. Procedure
As shown in Figure 2, flow lines (a-d) were connected
with tramadol hydrochloride solution, Na2SO3 solution,
water and KMnO4 with H2SO4 solution, respectively.
Flow rate was set at 4.2 mL/min for the KMnO4 solution
and water, respectively; sample and Na2SO3 solution at
2.5 mL/min. Pumps were started to wash the whole sys-
tem until a stable blank signal was recorded. A 80µL
mixture of tramadol and Na2SO3 was injected into the
carrier stream (H2O), which was merged with acidic
KMnO4 solution for producing CL signal. The concen-
tration of tramadol hydrochloride was quantified by the
CL intensity (peak height).
Figure 2. Schematic diagram of CL flow system. a, tramadol;
b, sodium sulfite solution; c, carrier(H2O); d, KMnO4; P1
and P2, peristaltic pump; V, six-way injection valve; F, CL
flow cell; PMT, photomultiplier tube; HV, negative high
voltage supply; AMP, amplitude; R, recorder; W, waste
Copyright © 2011 SciRes. AJAC
3. Results and Discussion
3.1. The Kinetic Characteristics of the CL
The kinetic characteristics of the CL reaction were stud-
ied by a stop-flow injection method after the baseline
was steadily recorded. Then 3.0 × 103 mol/L Na2SO3
was injected into 0.08 mol/L sulphuric acid solution
containing 1.0 × 104 mol/L KMnO4 and the CL kinetic
curve was simultaneously recorded by IFFM-E lumi-
nometer (see Figure 3, dashed line). Solid line in Figure
3 was the CL kinetic curve obtained when the mixture
solution of 2.0 g/mL tramadol and 3.0 × 103 mol/L
Na2SO3 were injected into 0.6 mol/L sulphuric acid solu-
tion containing 1.0 × 104 mol/L KMnO4. It was found
that the rate of the reaction was so fast that the CL inten-
sity reached the peak maximum only 0.7 s from reagent
mixing, and it took about 0.8 s for the signal decline to
the base line. The results indicate that the CL signal was
remarkably increased in the presence of tramadol.
3.2. Optimization of the Experimental
To establish the optimum conditions for the determina-
tion of tramadol hydrochloride, various parameters were
investigated using a series of univariate approachs which
were performed on reagent concentration, conditions of
reaction medium, reagent flow rate and injection sample
The kinds and concentration of acids in the reaction
system influence the CL intensity. Therefore, six differ-
ent acids including HCl, HNO3, CH3COOH, H3PO4,
H6P4O13 or H2SO4 of different concentration in the range
of 0.02 - 0.2 mol/L were tested, respectively. The results
showed the best signal was obtained in sulphuric acid, so
sulphuric acid was selected as the optimum medium.
With the increasing concentration of H2SO4, the CL in-
tensity increased and reached a maximum value at 0.08
mol/L (Figure 4). Thus, 0.08 mol/L H2SO4 was selected
as the acidic medium for the KMnO4 solution.
The effect of KMnO4 concentration on CL intensity
was examined in the range of 0.2 × 104 to 1.6 × 104
mol/L (Figure 5). With the increasing concentration of
KMnO4, the CL intensity increased and reached a flat value
at 1.0 × 104 mol/L. Thus, 1.0 × 104 mol/L KMnO4
concentration was chosen for further experiments.
The effect of Na2SO3 concentration upon the CL in-
tensity was examined within the range of 1.0 × 103 to
4.0 × 103 mol/L (Figure 6). With the increase of
Na2SO3 concentration, the CL intensity increased and
reached a maximum value at 3.0 × 103 mol/L. Therefore,
3.0 × 103 mol/L was used as the optimum concentration
of Na2SO3.
Figure 3. CL intensity-time profiles of KMnO4-Na2SO3 re-
action (dashed line) and KMnO4-Na2SO3-tramadol hydro-
chloride reaction(solid line).
Figure 4. Effect of H2SO4 concentration on CL intensity of
1.0 × 104 mol/L KMnO4 + 1.0 g/mL tramadol hydrochlo-
ride + 3.0 × 103 mol/L Na2SO3.
Figure 5. Effect of KMnO4 concentration in 0.08 mol/L
H2SO4 on CL intensity of 1.0 g/mL tramadol hydrochlo-
ride + 3.0 × 103 mol/L Na2SO3.
Copyright © 2011 SciRes. AJAC
Figure 6. Effect of Na2SO3 concentration on CL intensity of
1.0 g/mL tramadol hydrochloride + 1.0 × 104 mol/L
KMnO4 concentration in 0.08 mol/L H2SO4.
Because the proposed CL reaction was very fast, the
distance between Y-shaped mixing element and the flow
cell was made to be as short as possible, and the flow
rate of pump P2 was studied in the range of 1.0 to 6.0
mL/min in order to determine the maximum of the CL
signal. When the flow rate was 4.2 mL/min, the relative
CL intensity, reproducibility of signal, peak shape, and
ratio of signal to noise was the best. Therefore, the flow
rate of 4.2 mL/min was employed throughout the ex-
At the flow rate of 4.2 mL/min, the determination of
tramadol hydrochloride, including sampling and washing,
could be performed in 20s, giving a sample measurement
frequency of about 180 samples per hour. Thus, it was
decided to supply the KMnO4 solution and carrier stream
at 4.2 mL/min, respectively. Considering the reagent
consumption, 2.5 mL/min was chosen as the flow rate of
the sample and Na2SO3 solution.
In flow injection analysis, it is necessary to optimize
the injection volume to achieve the desired sensitivity.
The influence of the sample injection volume on the CL
intensity was tested at 40, 60, 80, 100 and 120 L of 2.0
g/mL tramadol hydrochloride. The biggest relative CL
intensity and the best ratio of signal to noise were ob-
tained when it was fixed between 80 and 100 L. Thus, a
80 L sample solution was injected into the carrier
3.3. Analytical Characteristics of Tramadol
Under the optimum conditions mentioned above, the
relative CL intensity was linearly related to the concen-
tration of tramadol hydrochloride from 0.04 to 4 g/mL.
Figure 7 shows the flow injection chemiluminescence
signals for tramadol. The maximum peak height increased
linearly with the increasing of tramadol concentration,
with a linear regression equation of ΔI (relative units) =
754.1 c (g/mL) + 121.6 (r = 0.9995, n = 8). The detec-
tion limit was 0.01 g/ml, which was calculated accord-
ing to IUPAC regulation that is three times of standard
deviation of blank value (3σ). The relative standard de-
viation for 11 parallel determinations of 1.0 g/mL
tramadol was 2.1%, showing a good reproducibility.
3.4. Interferences Experiments
The influence of some common inorganic ion and related
organic compounds was studied by the determination of
1.0 g/ml tramadol hydrochloride solution. The tolerance
limit was taken as the amount which caused a relative
error ±5% in the peak height. The results were shown in
Table 1 that some ions and the studied excipients in the
tablets did not interfere with the determination of tramadol
hydrochloride in this system. Therefor, this method can
be used for the determination of tramadol hydrochloride
in pharmaceutical preparations.
Figure 7. Typical recorder response for the determination
of tramadol (a-h).
Table1. Tolerance to different substances in the determina-
tion of 1.0 g/mL tramadol hydrochloride.
Species added Maximum tolerable ratio
Na+, K+, Mg2+, Zn2+, Al3+,
NO 500
Ca2+, Cu2+ 100
starch, Fe3+, Pb2+ 50
glucose, sucrose, CH3COO 20
Citric acid, Ni2+, Cr3+ 10
ascorbic acid, tartaric acid 5
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
3.5. Determination of Tramadol Hydrochloride
in Pharmaceutical Preparations
ing action of these non-fluorescent compounds are dif-
ferent from each other, the sensitizing effect of CAPS is
attribute to the presence of the cyclohexyl ring [31,34].
On the one hand the molecule of tramadol hydrochlo-
ride (Figure 1) contains a cyclohexyl ring, on the other
hand the CL spectrum of KMnO4-Na2SO3-H2SO 4 system
in the presence and absence of tramadol hydrochloride
showed similar emission profile extending from 450 to
600 nm (Figure 8), which was measured by means of a
series of interference filters. According to the suggested
reports, the emitter is the excited sulfur dioxide. There-
fore, it may be concluded that tramadol hydrochloride
played a role of an enhancer in the reaction.
The proposed method was successfully applied to the
determination of tramadol in a commercial pharmaceuti-
cal formulation. The tramadol hydrochloride tablets and
injections from different manufacturers were bought
from the local market. The average content of tablets was
calculated from the contents of 25 tablets, they were then
finely ground, homogenized and a portion of the powder
which was equivalent to 20 mg was weighed accurately
and diluted with 50 mL water. The mixture was soni-
cated for 10 min and then filtrated. The filtrate was di-
luted further with water. On the contrary, the injection of
tramadol hydrochloride needed not any pre-treatment.
The sample solution was prepared by directly diluting
the injection and the filtrate with water in order that the
concentration of tramadol was in the working range of its
determination. According to the proposed method,
tramadol hydrochloride was determined and the results
were shown in Table 2. The t-test indicated that there
were no significant differences between the results ob-
tained by the proposed method and those obtained by the
Chinese Pharmacopoeia method at confidence level of
The fluorescence spectra of tramadol solution were
scanned in the range 250 - 700 nm, using a F-2500 fluo-
rescence spectrometer (Hitachi, Tokyo, Japan). Since the
fluorescence spectrum of tramadol (λex = 200 nm, λem =
300 nm) was not identical with the CL spectrum obtained,
and the reaction product of KMnO4 with tramadol was a
non-fluorescent compound, an energy-transfer mecha-
nism was excluded.
The absorbance spectra of KMnO4-H2SO4 solution and
KMnO4-H2SO4 -tramadol solution were scanned (Figure
9). The absorbance peaks of KMnO4 at 524 nm and 545
nm decreased in the presence of tramadol and the de-
creased quantity was related to the concentration of
tramadol. According to the suggestion reported in [32],
the mechanism of tramadol-KMnO4-Na2SO3-H2SO4 sys-
tem could be expressed as follows:
3.6Possible CL Mechanism
The reaction of sulphite with strong oxidants such as
KMnO4 in acid media is accompanied by a weak CL that
has been reported extensively [26,27], it is proposed that
the excited is the emitter and the emission spec-
trum is between 450 and 600 nm [28,29]. This weak CL
reaction can be sensitized by not only several fluorescent,
which is the energy-transfer mechanism[22,30] but also
non-fluorescent compounds such as 3-cyclohexylamino-
propane sulfonic acid (CAPS) [31], ibuprofen[32] and
iproniazid [33] etc. Although the causes of the sensitiz-
43 34
264 2
KMnOtramadol products + energy
SO energy SO
Table 2. Results of determinationof tramadol hydrochloride in injections and tablets.
Samples Nominal
(%, n = 5)
(%, n = 5)
Injection 1(H20023786) 50 mg/2mL 49.7 ± 0.4
(mg/2mL) 2.0 49.8 ± 0.2 1.6
Injection 2
(J20020086) 100 mg/2mL 99.9 ± 0.5
(mg/2mL) 1.8 99.7 ± 0.4 1.5
Tablet 1
(H20033330) 100 mg/tablet 99.7 ± 0.6
(mg/2mL) 1.9 99.6 ± 0.3 1.4
Tablet 2 (H10960043) 50 mg/tablet 49.6 ± 0.7
(mg/2mL) 2.1 49.7 ± 0.5 1.5
Figure 8. CL spectra of 1.0 × 104 mol/LKMnO4 + 3.0 × 103
mol/LNa2SO3 (dashed line) and 20.0 g/mL tramadol hy-
drochloride + 1.0 × 104 mol/LKMnO4 + 3.0 × 103 mol/L
Na2SO3 (solid line).
Figure 9. Absorbance spectra of: (a) 1.0 × 104 mol/L
KMnO4 + 0.08 mol/L H2SO4; (b) 1.0 × 104 mol/L KMnO4 +
0.08 mol/L H2SO4 + 10.0 g/mL tramadol; and (c) 1.0 × 104
mol/L KMnO4 + 0.08 mol/L H2SO4 + 20.0 g/mL tramadol.
The CL of KMnO4-Na2SO3 reaction was very weak.
The CL signal was remarkably increased in the presence
of tramadol. Therefore, it can be concluded that tramadol
played a role of an enhancer in the reaction.
4. Conclusions
A new flow injection CL method was proposed for the
determination of tramadol hydrochloride based on the
KMnO4-sodium sulfite system. The experimental condi-
tions affecting the CL reaction were optimized and the
analytical characteristics for the determination of tramadol
hydrochloride are presented here. The possible CL reac-
tion mechanism has also been discussed. The proposed
method has a simple, rapid, inexpensive and high sensi-
tivity for the determination of tramadol hydrochloride in
tablets. This method is practical and valuable in clinical
and biochemical laboratories for the determination of
5. Acknowledgements
This work was supported by the Science Fund from the
National Natural Science Foundation of China (No.
21075087), the Provision Science Fund from Soochow
University (No. Q3109955), the Open Project Program
of State Key Laboratory of Food Science and Technol-
ogy, Jiangnan University (No. SKLF-KF-200908), Edu-
cation bureau of Jiangsu Province (No. 08KJB150014).
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