Chinnalalaiah Runja et al.

/ Journal of Pharmacy Research 2012,5(12),5489-5492

Research Article Available online through
ISSN: 0974-6943 http://jprsolutions.info
New RP-HPLC Method Development and Validation for Estimation of Tapentadol
hydrochloride in Bulk and Pharmaceutical Dosage form
Chinnalalaiah Runja and Ravikumar Pigili*
*
Department of Pharmaceutical Chemistry, Joginpally B R Pharmacy College, Moinabad,R.R Dist., Andhra Pradesh, India.
Received on:14-07-2012; Revised on: 19-08-2012; Accepted on:17-09-2012

ABSTRACT
A simple, rapid, selective, precise and accurate new RP-HPLC method was developed and validated for assay of Tapentadol Hydrochloride in bulk and tablet
dosage form. The method was developed by using the solvent system, Phosphate Buffer: Methanol: Acetonitrile in the ratio of 50:20:30 (v/v/v) and Hibar
C-18 (250 x 4.6 mm, 5µ) as stationary phase. The mobile phase was pumped at a flow rate of 1ml/min and the eluent was detected at 272 nm by PDA
detector. The retention time was 3.31 min. Linearity curve was plotted and the linearity range was found to be 50-150 µg/ml with correlation coefficient of
0.999. The accuracy of the proposed method was determined and mean percentage was found to be 100.63 %. The method was successfully validated
according to the ICH guidelines. The proposed method can be successfully applied for the estimation of Tapentadol hydrochloride in pharmaceutical dosage
form and it was concluded that the developed method was accurate, sensitive, precise, robust and useful for the rapid determination of Tapentadol in
pharmaceutical dosage forms.

Keywords: Tapentadol HCl, Assay, Validation, RP-HPLC

INTRODUCTION
Tapentadol 1, a novel centrally acting synthetic opioid analgesic agent, used
in the treatment of acute pain management. The mechanism of action is not
known however the analgesic activity may be due to acting on µ-opioid
receptor. It was also found that tapentadol showed antidepressant like activ-
ity 2 and noradrenalin reuptake inhibition 3 properties. Recently, FDA has
approved Tapentadol extended-release tablets 4 for treatment of chronic
osteoarthritis, low back pain, and pain associated with diabetic peripheral
neuropathy. Chemically tapentadol 5 is, 3-[(1R, 2R)-3-(3-dimethylamino) -
1- ethyl -2- methyl propyl] phenol. The structure of tapentadol is shown in
figure 1.
and marketed product Nucynta 100 mg was purchased from local market.
Acetonitrile, Water, were obtained from Merck. Mumbai and Potassium
dihydrogen ortho phosphate, Ortho Phosphoric Acid obtained from
RANKEM Mumbai. All solvents used in this work are HPLC grade.
Standard solutions and Chromatographic conditions
Preparation of Buffer pH 5
Transfer 6.8gm of Potassium dihydrogen ortho phosphate in to a 1000ml
beaker add about 900ml of milli-Q water and sonicate and make up to the
final volume, then adjust the solution to pH 5 with Ortho Phosphoric Acid
solution.

Preparation of Tapentadol standard stock solution

Figure 1: Molecular Structure of Tapentadol HCl
Literature survey revealed that, there are very few analytical techniques were
reported for estimation of tapentadol by HPLC, UPLC 6, Spectrofluorimetric
method 7 and LC/MS 8. But most of the above methods are highly expensive
and are not easily available in quality control laboratories. So authors objec-
tive is to develop a simple, accurate, precise RP-HPLC method for estima-
tion of tapentadol HCl in bulk and Pharmaceutical dosage form. After devel-
oping the method it was validated for assay determination which includes
accuracy, precision, linearity range, selectivity and robustness.
10 mg of Tapentadol HCl working standard was weighed and transferred into
a 10 ml clean dry volumetric flask, and 7 ml of diluent was added, sonicated
for 5 minutes and make up to the final volume with diluent (standard stock).
Sample preparation of Tapentadol in tablet dosage form
5 tablets were weighed and crushed into powder, and transferred into a 50 mL
volumetric flask, 30mL of diluent added and sonicated for 25 min, further the
volume made up with diluent and filtered. From the filtered solution 1.0ml
was pipeted out into a 100 ml volumetric flask and made upto 100ml with
diluent.
HPLC INSTRUMENTATION AND CONDITIONS

MATERIAL AND METHODS Instrument

Chromatographic analysis was performed with Waters 2695 seperation module
equipped with waters 2996 Photodiode Array Detector. The column used
Reagents and Chemicals was Hibar C-18 (250 x 4.6 mm, 5µ). The Empower 2 software was em-
Tapentadol was obtained as gift sample from MSN laboratory in Hyderabad ployed for LC peak integration along with data acquisition and data process-
ing. Ultrasonic bath was used for sonication and degassing of mobile phase.
*Corresponding author. Waters PDA detector was used to obtain the overlay spectra of the drug to
Dr. P. Ravi Kumar determine the analytical wavelength.
Department of Pharmaceutical Chemistry,
Joginpally B R Pharmacy College, Chromatographic conditions
Moinabad,R.R Dist., Andhra Pradesh, India. Mobile phase consisting of Phosphate Buffer: Methanol: Acetonitrile in the

Journal of Pharmacy Research Vol.5 Issue 12.Decemeber 2012 5489-5492

 Chinnalalaiah Runja et al. / Journal of Pharmacy Research 2012,5(12),5489-5492
ration of 50:20:30 (v/v/v) at 1 ml/min flow rate was used. It was filtered LODσ = 3.3*SD / Slope of response SD- Standard Deviation
through 0.45µm nylon filter and sonicated for 5 min in ultrasonic bath. LOQ= 10 /Slope of response σ - Standard deviation
Samples were analyzed at 272 nm at an injection volume of 20 µL and
separation was carried by using Hibar C-18 (250 x 4.6 mm, 5µ). The opti- RESULTS AND DISCUSSIONS
mized chromatographic conditions are given in table 1
Optimization of the chromatographic conditions
Table 1: Optimized chromatographic conditions Different chromatographic conditions were employed for the analysis of the
Tapentadol in bulk and pharmaceutical dosage form. The optimized mobile
S.No Parameter Optimized condition
phase used in this method is Phosphate Buffer: Methanol: Acetonitrile in the
1 Instrument Waters 2695 Separation module RP-HPLC ratio of 50:20:30 (v/v/v) at a flow rate 1 ml/min. Sample was detected by
2 Column µ
Hibar C-18 (250 x 4.6 mm, 5 ). PDA detector at 272 nm. The retention time for Tapentadol was found to be
3 Mobile phase Phosphate Buffer: Methanol: Acetonitrile in 3.31 min (Figure 2).
the ratio of 50:20:30 (v/v/v)
4 Flow Rate 1 ml/ min
5 Detection PDA Detector at 272 nm
6 Injection volume 20µl
7 Temperature Ambient
8 Retention Time 3.31 min

METHOD DEVELOPMENT AND VALIDATION

Preliminary trials were made by changing various chromatographic param-
eters to develop a new method for analyzing the Tapentadol HCl. Finally a
suitable method was developed with a mobile phase Phosphate Buffer:
Methanol: Acetonitrile in the ratio of 50:20:30 (v/v/v) at a flow rate of 1.0 ml/
min. After method development, validation of the current test method for
Tapentadol HCl was performed for assay determination of active ingredients
in bulk and finished pharmaceutical products which include accuracy, preci-
sion, selectivity, robustness, limit of quantification limit of detection, linear-
ity and range.

Accuracy (% Recovery studies)

The accuracy of the method was determined by calculating recoveries of
Tapentadol Hydrochloride by the standard addition method. For this known Figure 2: RP-HPLC Chromatogram of Tapentadol HCl
amount of standard solutions of Tapentadol Hydrochloride 50%, 100%, and
Assay of Tapentadol in Tablet
150% was prepared. The detailed results were given in Table 2.
Assay of marketed product was carried out by using the developed method.
Sample solutions were prepared and injected into HPLC system. The sample
Precision
solution was scanned at 272 nm. The assay limits were found to be 96 -
Precision of an analytical method is usually expressed as the standard devia-
106%. A single peak was observed with retention time 3.25. (Figure 3)
tion. The % Relative Standard Deviation was determined by injecting five
freshly prepared Tapentadol standard samples in the same day under the
Formulation Dose Concentration Amount % Drug
similar conditions. The results were given in Table 3.
(µg/ml) found Estimated

Linearity and range NUCYNTA Tablets 100 mg 100 99.86 99.86

Linearity was evaluated by calculating the correlation coefficient. Five differ-
ent concentrations of Tapentadol were prepared and a calibration curve was
constructed by plotting the peak area vs concentrations. From this, correla-
tion coefficient was calculated and linearity range was found to 50-150µg/ml.
The data was given in table 4.

Robustness
To evaluate the robustness, the sample was analyzed by changing flow rate
and temperature. The flow rate of mobile phase was changed by ± 20% and
temperature was changed to ±5 0C. This deliberate change in the method has
no affect on the peak tailing, peak area and theoretical plates and finally the
method was found to be robust. The results are placed in Table 5.

LOD and LOQ

LOD and LOQ were determined based on standard deviation of the re-
sponse and slope. The lowest amount of the sample is determined quantita-
tively and its limits were expressed as concentration of analyte (parts per
million). The following two formulas were used for LOD and LOQ detec-
tion. Figure 3: Tapentadol HCl chromatogram in Nucynta Tablet

Journal of Pharmacy Research Vol.5 Issue 12.Decemeber 2012 5489-5492

 Chinnalalaiah Runja et al. / Journal of Pharmacy Research 2012,5(12),5489-5492
Method Validation Table 4: Linearity data
Accuracy S.No Concentration (µg/ml) Area
A known amount of standard drug solution at three concentration levels
1 50 433932
(50%, 100%, and 150 %) was added to the pre analyzed sample solution.
2 70 593563
The assay was repeated over three consecutive days to obtain intermediate 3 100 828172
precision data. Good recovery of the spiked drug was obtained at each added 4 120 1001201
concentration, and the mean percentage recovery of Tapentadol was found to 5 150 1264202
be 100 % which suggest that method development is very accurate. The 8 Correlation coefficient 0.999
acceptance limit of % recovery should be between 98.0-102.0%
Robustness
Table 2: % Recovery data of Tapentadol The flow rate as per the method is 1ml.This was changed to 0.8ml and 1.2ml.
The effect of column temperature was studied at 35 and 25°C instead of
S.N Concentration % Amount Amount Mean %RSD 30°C. System suitability results were within the limit. It was found that
Recovery added found %Recovery ± S.D
(µg/ml) (µg/ml) (n=3)
there was no drastic changes occur on chromatogram when flow rate and
column temperature changes.
1 50% 100.41 50 50.20 100.646±0.205 0.203
100.75 50 50.37 Table 5: Robustness Data
100.78 50 50.39
2 100% 99.86 100 99.86 100.20±0.298 0.297 Standard Flow Rate Temperature ( 30 ±5 oC)
100.42 100 100.42 Solution 0.8 ml 1.2 ml 25 0 C 35 0 C
100.32 100 100.32 Area Rt Area Rt Area Rt Area Rt
3 150% 101.15 150 151.72 100.636±0.661 0.656
100.87 150 151.30 1 978284 3.28 963156 3.28 905014 3.29 949543 3.24
2 969417 3.29 956393 3.31 910250 3.23 950238 3.23
99.89 150 149.83 3 970632 3.28 962639 3.26 920693 3.26 930621 3.24
AVG 972777.7 3.28 960729.3 3.28 911985.7 3.26 943467.3 3.24
Precision SD 4807.16 0.0057 3764.26 0.0152 7982.30 0.03 11130.68 0.0057
A known standard solution of drug substance was injected five times and % RSD 0.494 0.173 0.391 0.463 0.875 0.920 1.179 0.175
corresponding peak areas were recorded. The % relative standard deviation
SD- Standard Deviation, RT- Retention Time % RSD- Relative Standard Deviation
of peak area was determined. Based on the results obtained from the data it
was suggested that developed method is precise. System Suitability
System suitability was performed for Tapentadol standard solution by in-
Table 3: Precision method of proposed RP-HPLC method jecting five replicates. A standard solution was prepared and was injected
Injection Area Retention Time (Rt) five times in to the HPLC. The % RSD of the retention time and area of the
peak was calculated from the chromatograms obtained. The % RSD of area
1 909543 3.24
2 905979 3.28 was found to be within the limits. It was found that the number of theoretical
3 905014 3.29 plates for Tapentadol is more than 10000 and tailing factor is below 2.0. The
4 918021 3.27 results of system suitability and system suitable parameters were given in
5 903680 3.27 Table 6 & 7.
6 900756 3.28
Average 907165.5 3.27 Table 6: System suitability for RP HPLC Method
SD 6045.85 0.017
% RSD 0.666 0.519 Standard no Area Retention time Theoretical plates Tailing

Linearity 1 902565 3.23 10461 1.26

The linearity of measurement was evaluated by analyzing different concen- 2 903494 3.19 10021 1.25
3 900789 3.22 10513 1.30
trations of standard solutions of Tapentadol. After analysis, the areas of the 4 908775 3.31 10209 1.31
peaks were recorded. Calibration curve was constructed by plotting average 5 903393 3.23 10237 1.28
peak area versus and concentrations. The regression equation is y = 8351.x + Avrg 903803.2 3.23
SD 2983.33 0.023
4826 and regression coefficient r 2 is 0.999.
%RSD 0.33 0.712

Table 7: System Suitability parameters of proposed RP-HPLC method

S. No Parameters Values

1 Retention time (min) 3.31

2 Theoretical plates 10579
3 Tailing factor 1.31
4 Wavelength (?max) 272 nm
5 Regression equation y = 8351.x + 4826
6 Correlation coefficient(r2) 0.999
7 Capacity factor (k) 0.122

CONCLUSION
The results suggest that a simple, easy and accurate RP-HPLC method was
developed for the estimation of Tapentadol. The method was validated suc-
Figure 4: Linearity curve

Journal of Pharmacy Research Vol.5 Issue 12.Decemeber 2012 5489-5492

 Chinnalalaiah Runja et al. / Journal of Pharmacy Research 2012,5(12),5489-5492
cessfully using parameters like accuracy, precision, linearity and robustness. 3. Thomas C and Babette K. Tapentadol HCl: a Novel ì-Opioid
The %RSD for all parameters was found within the limits, which indicates Receptor Agonist/Nor epinephrine Reuptake Inhibitor with
the validity of the method and therefore the proposed method can be used for Broad-Spectrum Analgesic Properties, Journal of Pharmacology
routine analysis for Tapentadol in bulk and pharmaceutical formulation. & Experimental Therapeutics, 323 (1265); 2007,276.
4. Vadivelu N, Timchenko A, Huang Y, Sinatra R. Tapentadol ex-
ACKNOWLEDGEMENT tended-release for treatment of chronic pain: a review, Journal of
The authors are wish to thanks MSN laboratory for providing gift sample. Pain Research, 4; 2011, 211–218.
5. Martindale- The complete drug reference, 37th edition, pharma-
REFERENCES ceutical press, London, 2011, 133.2
1. Wade WE, Spruill WJ. Tapentadol hydrochloride: a centrally act- 6. Bourland, James A. and Collins. Determination of Tapentadol
ing oral analgesic, Clin Ther, 31(12); 2009, 2804-18. and N-Desmethyltapentadol in Authentic Urine Specimens by
2. Ramanath Royal B. and Narayan Pandurang B. Evaluation of Ultra-Performance Liquid Chromatography-Tandem Mass Spec-
antidepressant -like activity of tapentadol in swiss albino mice, trometry, J Analytical Toxicology, 34(8); 2010, 450-457.
International Journal of Pharma and Bio Sciences, 2(4); 2011, 7. Giorgi M, Meizler A, Mills PC. Quantification of tapentadol in
442-447. canine plasma by HPLC with spectrofluorimetric detection: de-
velopment and validation of a new methodology, J Pharm Biomed
Anal, 2012, 148-53.
Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 12.Decemeber 2012 5489-5492