Original Article

Development and validation of RP-HPLC method for

estimation of Tapentadol hydrochloride in bulk
and tablet dosage forms
Y. Indira Muzib
a,
*, J. Ravi Kumar Reddy
b,c
, K.P.R. Chowdary
d
, E. Swathi
c
a
Department of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, India
b
Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, India
c
Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, India
d
Department of Pharmaceutics, Andhra University, Vishakhapatnam 530003, Andhra Pradesh, India
a r t i c l e i n f o
Article history:
Received 6 March 2013
Accepted 27 May 2013
Available online 6 August 2013
Keywords:
Licrosphere column
Methanol
Isocratic mode
Quantitative analysis
Tapentadol hydrochloride
a b s t r a c t
Objective: To develop a simple, novel, sensitive, precise and specic RP-HPLC method for the
determination of Tapentadol hydrochloride in bulk and its tablet dosage forms.
Methods: The chromatographic separation was achieved on C18 Licrosphere column
(150 mm 4.6 mm inner diameter, 5 mm particle size) as a stationary phase using Meth-
anol: 0.1 mM Dipotassium Phosphate buffer (pH 4, adjusted with ortho phosphoric acid) as
mobile phase at detection wavelength 280 nm in isocratic mode at a ow rate of 1 ml/min.
Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 mg/ml.
The correlation coefcient (r
2
) value was found to be 0.9994. Precision study showed % CV
value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro-
chloride are in the range of 99.96e100.01%. The limit of detection and limit of quantica-
tion for Tapentadol hydrochloride were found to be 0.25 mg/ml and 0.75 mg/ml respectively.
Conclusion: The developed method has good sensitivity, reproducibility and specicity for
the determination of Tapentadol hydrochloride in bulk and its tablet dosage forms. This
method was simple, fast, accurate, and precise. Hence this method was validated and
found to be suitable for determining the purity of Tapentadol hydrochloride in bulk drugs
and pharmaceutical formulations. The proposed validated method was successfully used
for the quantitative analysis of commercially available dosage form.
Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.
1. Introduction
Tapentadol, 3-[(1R,2R)-3-(dimethylamino)-1-ethyl-2-methyl]-
propylphenol hydrochloride (TAP), differs distinctly from
previously characterized centrally acting analgesics in that a
peculiar dual mechanism of action that has demonstrated
efcacy in clinical application.
1
Tapentadol is a centrally
acting synthetic analgesic, received initial U.S. approval in
* Corresponding author. Tel.: 91 9441593292, 91 7702229333.
E-mail address: yindira2002@rediffmail.com (Y.I. Muzib).
Available online at www.sciencedirect.com
j ournal homepage: www. el sevi er. com/ l ocat e/ i j cas
i nt e r na t i o na l j our na l of c he mi c a l a nd a na l yt i c a l s c i e nc e 4 ( 2 0 1 3 ) 6 7 e7 2
0976-1209/$ e see front matter Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijcas.2013.07.001
2008
2
and was then placed into the schedule II category of
the Controlled Substances Act in May, 2009.
3
It is suggested
that the broad analgesic prole of Tapentadol and its rela-
tive resistance to tolerance development may be due to a
dual mode of action consisting of both MOR activation and
NE reuptake inhibition
4
(Fig. 1).
To date, only two LCeMS methods to detect Tapentadol in
biological matrices (urine and urine and oral uid)
5
have been
reported in the literature; however there have been no studies
on HPLC method for detection of Tapentadol in pharmaceu-
tical formulations. To address this shortfall, the aim of the
present paper was to develop and validate a new simpler
methodology to quantify Tapentadol in tablet formulation
using HPLC with diode array detection (HPLCeDAD).
2. Materials and methods
2.1. Chemicals and reagents
Tapentadol hydrochloride working standard powder was
gifted by MSN Laboratories, Hyderabad and was used
without further purication. Tapentadol hydrochloride
tablets containing 100 mg were purchased from local
pharmacy, Tirupathi. HPLC grade Methanol and Dipotas-
sium Phosphate buffer was purchased from S.D. Fine Chem.
(Mumbai, India). All solutions were ltered through 0.45
micron membrane lters purchased from Pall Pharmalab
Filtration Pvt. Ltd. (Mumbai, India). All chemicals were of
analytical grade unless stated otherwise and used as
received. Puried HPLC grade water was obtained by reverse
osmosis and ltration through a milli-Q system and was
used to prepare all solutions.
2.2. Instrumentation
The HPLC analysis was carried out by using system(Shimadzu
Co., Kyoto, Japan) consisted of a Shimadzu model LC-10 ADVP,
SPD 10 A VP variable wavelength detector (possessing deute-
rium lamp with a sensitivity of 0.005 AUFs and adjusted to an
absorbency of 280 nm), a Shimadzu model C-R5A chromato-
graph integrator module (chart speed at 10 mm/min), a Shi-
madzu model SIL-6A auto injector, and a Shimadzu module
SCL-6A system controller.
2.3. Chromatographic conditions
Chromatographic separation was achieved on Isocratic
elution of the mobile phase Methanol: 0.1 mM Dipotassium
Phosphate buffer (pH 4, adjusted with ortho phosphoric acid)
with the ow rate of 1 ml/min. Separation was performed on
C18 Licrosphere column (150 mm 4.6 mm inner diameter,
5 mm particle size). The ow rate was 1.0 ml/min and detector
wavelength was kept at 280 nmfor monitoring the separation.
The column back pressure was maintained at 110e115 kg/cm.
Integration of the detector output was performed using the
Shimadzu Empower software to determine the peak area. The
contents of the mobile phase were ltered through a 0.45-mm
membrane lter and degassed by sonication before use. In-
jection volume was 20 mL and total run time was 10 min, and
column temperature was maintained at ambient. The eluent
was detected at 280 nm.
2.4. Preparation of standard stock solution
The stock solution of Tapentadol hydrochloride was prepared
by dissolving accurately weighed 10 mg in 10 mL of methanol
to obtain a nal concentration of 1.0 mg/mL. The prepared
stock solution was stored at specied temperatures in amber
glass scintillation vial. The diluted solutions were ltered
through 0.45 mm membrane lter. From this stock solution
Tapentadol hydrochloride calibration standards were freshly
prepared prior to analysis prepared at concentrations of
75e600 mg/mL from a standard solution of 100 mg/mL by
appropriate dilution with mobile phase.
2.5. Preparation of sample solution
Twenty tablets of Tapentadol hydrochloride were weighed,
crushed and mixed in a mortar and pestle to ne powder. A
portion of powder equivalent to the weight of one tablet was
accurately weighed into each of six 25 ml volumetric asks
and 10 ml of mobile phase was added to each ask. The
volumetric asks were sonicated for 20 min to effect com-
plete dissolution of the Tapentadol hydrochloride and the
solutions were then made up to the volume with mobile
phase. Suitable aliquots of solution were ltered through a
0.45 mm nylon lter. One microlitre of the ltered solution
was transferred to a volumetric ask and made up to the
volume with mobile phase to yield concentration of Tapen-
tadol hydrochloride in the range of linearity previously
described.
2.6. Assay
A mass of not less than 20 tablets was prepared by grinding
them to a ne, uniform particle size powder using a mortar
and pestle. After calculating the average tablet weight, a
composite equivalent to the 10 mg was accurately weighed
and quantitatively transferred into a 100 ml volumetric ask.
Approximately, 10-ml milli-Q water was added, the solution
was sonicated for 10 min, 70 ml diluent was added to it, and
mechanically shaken for 10 more minutes. The ask was
equilibrated to room temperature, carefully lled to volume
with the diluent, and mixed well. Aportion of the solution was
ltered through a 0.45 mm membrane lter, discarding the
rst 2e3 ml of the ltrate. A portion of the ltered sample
(5.0 ml) was diluted into a 50 ml volumetric ask with the
mobile phase and mixed well. Fig. 1 e Structure of Tapentadol.
i nt e r na t i o na l j o ur na l o f c he mi c a l a nd a na l y t i c a l s c i e nc e 4 ( 2 0 1 3 ) 6 7 e7 2 68
3. Method validation
The developed method was validated for assay of Tapentadol
hydrochloride in accordance with ICH guidelines.
6
3.1. Detection and quantitation limits (sensitivity)
Limits of detection (LOD) and limit of quantitation (LOQ) were
estimated from the signal-to-noise ratio.
7,8
LOD is dened as
the lowest concentration resulting in a peak area of three
times the baseline noise. LOQ is dened as the lowest con-
centration that provides a signal-to-noise ratio higher than 10,
with precision (%CV) and accuracy (% bias) within their
acceptable range (10%).
3.2. Linearity (calibration curve)
Thecalibrationcurveswereconstructedwithsixconcentrations
of Tapentadol hydrochloride ranging from 75 to 450 mg/mL.
Calibration curves were constructed by plotting the ratio of the
mean peak area of either Tapentadol hydrochloride versus the
concentration. The linearity was assessed by linear regression
analysis, which was calculated by the least square method.
3.3. Accuracy and precision
Precision of the assay was determined by repeatability (intra-
day) and intermediate precision (inter-day) for 3 consecutive
days.
8e10
Three different concentrations of Tapentadol hy-
drochloride were analyzed in six independent series in the
same day (intra-day precision) and 3 consecutive days (inter-
day precision). Every sample was injected in triplicate. The
accuracy of the method, which is dened as the nearness of
the true value and found value, was evaluated as % bias for
Tapentadol hydrochloride according to the following equation:
%Accuracy observedconcentration=nominal concentration
100:
3.4. System suitability
The system suitability was evaluated by six replicate analyses
of a Tapentadol hydrochloride at a concentration of 60 mg/
mL.
9,10
The acceptance limit was 2% for the percent coef-
cient of variation (% CV) of the peak area and the retention
time of Tapentadol hydrochloride.
3.5. Recovery
The absolute recovery was calculated from the peak area of
Tapentadol hydrochloride methanolic standard solutions to
those containing Tapentadol hydrochloride at three different
concentrations.
3.6. Statistical analysis
Data collected in this study were analyzed using JMP statistical
software package by one-way analysis of variance (ANOVA).
Table 1 e Optimized HPLC conditions for the estimation
of Tapentadol HCl.
S. no Parameter Description/value
1 Stationary phase C18 Licrosphere column
(150 mm 4.6 mm inner
diameter, 5 mm particle size)
2 Mobile phase Methanol: 0.1 mM Dipotassium
Phosphate buffer (pH 4, adjusted
with ortho phosphoric acid)
3 Flow rate 1 ml/min
4 Detection wavelength 280 nm
5 Detector UV (diode array detector)
6 Elution Isocratic
7 Injection volume 20 ml
8 Column temperature 25

C
9 Run time 6 min
10 Diluent Mobile phase
Fig. 2 e Typical chromatogram of Tapentadol HCL.
i nt e r na t i o na l j our na l of c he mi c a l a nd a na l yt i c a l s c i e nc e 4 ( 2 0 1 3 ) 6 7 e7 2 69
Univariate linear regression analysis using least square
method was applied to test the tted model. Correlation co-
efcient was calculated and the results of the statistical anal-
ysis were considered signicant if their corresponding p-values
were less than 0.05.
4. Results and discussion
4.1. Method development and optimization
The chromatographic conditions were optimized for the
determination of Tapentadol hydrochloride within a short
analysis time (<6 min). To accomplish these objectives, the
chromatographic column was rst chosen based on peak
shapes and resolution. C18 Licrosphere column
(150 mm 4.6 mm inner diameter, 5 mm particle size), main-
tained at ambient temperature (25

C) was used for the sepa-
ration and the method validated for the determination of
Tapentadol hydrochloride in pharmaceutical dosage forms.
The stressed samples were initially analyzed using a mobile
phase consisting of Methanol: 0.1 mMDipotassiumPhosphate
buffer (pH 4, adjusted with ortho phosphoric acid) at a ow
rate of 1 ml per min and UV detection at 280 nm. The Rt of
Tapentadol was found to be 3.1 min. Optimized HPLC
conditions for the estimation of Tapentadol HCl given in
Table 1 and typical chromatogram of Tapentadol HCl shown
in Fig. 2.
4.2. Validation
The method was validated with respect to parameters
including linearity, limit of quantitation (LOQ), and limit of
detection (LOD), suitability, precision and accuracy.
4.3. Accuracy
The accuracy of the proposed analytical method was deter-
mined by recovery experiments. The recovery studies were
carried out at three different concentration levels in triplicate
(80, 100, and 120%). The analyzed samples yielded high re-
covery values from the developed method. The % recovery
results of the method are given in Table 2.
4.4. Linearity
The linearity of the calibration curve for Tapentadol hydro-
chloride was calculated and constructed by plotting the mean
peak area versus concentration. The correlation coefcient of
regression r
2
0.9999 over a concentration range (75e450 mg/
ml), the representative linear regression equation for Tapen-
tadol hydrochloride Y 21349x 32996 as shown in Fig. 3, and
the corresponding results given in Table 3.
4.5. Precession (reproducibility)
In order to demonstrate the reproducibility of the method for
the assay of a tablet pharmaceutical preparation, ve tablet
extracts were injected in to the capillary in duplicate. The
resultant RSDs for migration time and peak are were 0.25%
and 0.65%, respectively for Tapentadol hydrochloride. The
results are shown in Table 4. Fig. 3 e Linearity of Tapentadol.
Table 2 e Accuracy (recovery) of Tapentadol hydrochloride.
Sample
no.
Spiked
level
Sample
weight (mg)
Sample
area
mg/ml added mg/ml found % recovery % mean
recovery
% mean
recovery
1 50% 135.45 4288421 199.9926 195.7401 98 100 100
2 50% 135.45 4408154 199.9926 201.2051 101
3 50% 135.45 4309312 199.9926 196.6936 98
4 50% 135.45 4440198 199.9926 202.6677 101
5 50% 135.45 4362190 199.9926 199.1072 100
6 50% 135.45 4420919 199.9926 201.7878 101
1 100% 270.91 8857334 400.0000 404.2829 101 100
2 100% 270.91 8725612 400.0000 398.2706 100
3 100% 270.91 8660061 400.0000 395.2786 99
1 150% 406.36 12766075 599.9926 582.6929 97.12 99
2 150% 406.36 12889590 599.9926 588.3306 98.06
3 150% 406.36 13052655 599.9926 595.7735 99.30
4 150% 406.36 13112499 599.9926 598.5050 99.75
5 150% 406.36 13047794 599.9926 595.5516 99.26
6 150% 406.36 13143562 599.9926 599.9228 99.99
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4.6. Determination of the main drug in bulk and tablet
dosage form (assay)
Six solutions of Tapentadol hydrochloride prepared from the
bulk drug and tablet dosage form were prepared and analyzed
with the same experimental conditions and found to be drug
content within the specied limits. The results were shown in
Table 5.
4.7. Ruggedness and robustness
Preliminary experiments revealed that amongst the many
operating parameters involved. The buffer pH is the most
inuential parameter on the repeatability of the method,
when suitable precautions have been taken with regard to
instrumental aspects of injection and capillary condition-
ing. The method was employed for two different in-
struments and with two different operators. In these
experiments, ve standard solutions of Tapentadol hydro-
chloride (at 0.05 mg ml
1
) were assessed on each of two
occasions and the results showed no signicant statistical
differences between operators or between instruments. The
RSD values for migration time and peak area for the initial
start time were 0.30% and 0.83% (n 5) respectively, and for
measurements at two months the RSDs were 0.26% and
1.05%, respectively.
4.8. Limits of detection and quantication
The LOD for Tapentadol hydrochloride, on the basis of a
signal-to-noise ratio of 3, was determined to be 0.001 mg/ml
(the sample injection time was 4 s). In addition, the LOQ, based
on a signal-to-noise ratio of ten was found to be 0.003 mg/ml.
The results were shown in Table 6.
5. Conclusion
A rapid, precise, user friendly and reproducible HPLC method
for estimation of Tapentadol hydrochloride in bulk and its
tablet pharmaceutical dosage forms was developed and vali-
dated as per ICH Guidelines. The LOD and LOQ measurements
were also established for the further scope of utilizing this
method. Because of its wide range of linearity, use of readily
available mobile phase and RSD values for all parameters
were found to be less than 2, which indicates the validity of
method and results obtained by this method fairly reliable.
This method can be used by the industries and academic in-
stitutions for the estimation of hydrochloride.
Conict of interest
All authors have none to declare.
Acknowledgements
The authors are expressing sincere thanks and appreciation to
JPR solutions for funding of this research work to publish in the
journal. The authors extend thanks to the Management of Anna
macharya college of Pharmacy and School of Pharmaceutical
Sciences, JNTU-K, Kakinada for their cooperation in the present
research work. The corresponding author expresses deep appre
ciation to B. Mohammed Ishaq for his help during this work.
r e f e r e n c e s
1. Giorgi M, Meizler A, Mills PC. Quantication of tapentadol in
canine plasma by HPLC with spectrouorimetric detection:
Table 3 e Linearity data.
Linearity level Concentration (mg/ml) Peak area
25% 75 555890
50% 150 1116635
75% 225 1673640
100% 300 2228914
125% 375 2782306
150% 450 3335847
Table 4 e Precision of Tapentadol hydrochloride.
S. no Sample
name
Inj.
(20 ml)
Name RT Area
1 Precision1 1 Tapentadol 3.155 2156575
2 Precision2 1 Tapentadol 3.143 2150804
3 Precision3 1 Tapentadol 3.136 2159065
4 Precision4 1 Tapentadol 3.127 2154633
5 Precision5 1 Tapentadol 3.120 2150334
6 Precision6 1 Tapentadol 3.114 2150547
Mean 2153660
Std. dev. 3676
% RSD 0.2
Table 5 e Assay of formulation.
Sample no. Sample
weight (mg)
Sample
area e 1
% Assay e 1
1 271.0 8735989 99.10
2 271.0 8809425 99.93
3 271.0 8675493 98.41
4 271.0 8898726 100.94
5 271.0 8852017 100.41
6 271.0 8903936 101.00
Average Assay: 100
STD 1.04
% RSD 1.04
Table 6 e System suitability parameters.
Parameters Value
Calibration range 75e450 mg/ml
Theoretical plates 5021
Rt 3.126
Tailing Factor 0.58
LOD 0.001 mg/ml
LOQ 0.003 mg/ml
i nt e r na t i o na l j our na l of c he mi c a l a nd a na l yt i c a l s c i e nc e 4 ( 2 0 1 3 ) 6 7 e7 2 71
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