ww.sciencedirect.com

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 2

Available online at w

journal homepage: www.elsevier .com/locate/ i jcas

Original Article

Development and validation of RP-HPLCmethod forestimation of Tapentadol hydrochloride in bulkand tablet dosage forms

Y. Indira Muzib a,*, J. Ravi Kumar Reddy b,c, K.P.R. Chowdary d, E. Swathi c

aDepartment of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, Indiab Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, IndiacDepartment of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, IndiadDepartment 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

* Corresponding author. Tel.: þ91 9441593292E-mail address: yindira2002@rediffmail.c

0976-1209/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijcas.2013.07.001

a b s t r a c t

Objective: To develop a simple, novel, sensitive, precise and specific 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 flow rate of 1 ml/min.

Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 mg/ml.

The correlation coefficient (r2) 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 quantifica-

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 specificity 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 ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction previously characterized centrally acting analgesics in that a

Tapentadol, 3-[(1R,2R)-3-(dimethylamino)-1-ethyl-2-methyl]-

propylphenol hydrochloride (TAP), differs distinctly from

, þ91 7702229333.om (Y.I. Muzib).2013, JPR Solutions; Publi

peculiar dual mechanism of action that has demonstrated

efficacy in clinical application.1 Tapentadol is a centrally

acting synthetic analgesic, received initial U.S. approval in

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 268

20082 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 profile 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 inhibition4 (Fig. 1).

To date, only two LCeMS methods to detect Tapentadol in

biological matrices (urine and urine and oral fluid)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 purification. 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 filtered through 0.45

micron membrane filters purchased from Pall Pharmalab

Filtration Pvt. Ltd. (Mumbai, India). All chemicals were of

analytical grade unless stated otherwise and used as

received. Purified HPLC grade water was obtained by reverse

osmosis and filtration 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 Shimadzumodel 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)

Fig. 1 e Structure of Tapentadol.

with the flow rate of 1 ml/min. Separation was performed on

C18 Licrosphere column (150 mm � 4.6 mm inner diameter,

5 mm particle size). The flow rate was 1.0 ml/min and detector

wavelengthwas kept at 280 nm formonitoring 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 filtered through a 0.45-mm

membrane filter 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 final concentration of 1.0 mg/mL. The prepared

stock solution was stored at specified temperatures in amber

glass scintillation vial. The diluted solutions were filtered

through 0.45 mm membrane filter. 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 fine powder. A

portion of powder equivalent to the weight of one tablet was

accurately weighed into each of six 25 ml volumetric flasks

and 10 ml of mobile phase was added to each flask. The

volumetric flasks 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 filtered through a

0.45 mm nylon filter. One microlitre of the filtered solution

was transferred to a volumetric flask 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 fine, 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 flask.

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 flask was

equilibrated to room temperature, carefully filled to volume

with the diluent, andmixedwell. A portion of the solutionwas

filtered through a 0.45 mm membrane filter, discarding the

first 2e3 ml of the filtrate. A portion of the filtered sample

(5.0 ml) was diluted into a 50 ml volumetric flask with the

mobile phase and mixed well.

Table 1 e Optimized HPLC conditions for the estimationof 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 �C9 Run time 6 min

10 Diluent Mobile phase

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 2 69

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 defined as

the lowest concentration resulting in a peak area of three

times the baseline noise. LOQ is defined 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)

Thecalibrationcurveswere constructedwith six concentrations

of Tapentadol hydrochloride ranging from 75 to 450 mg/mL.

Calibration curves were constructed by plotting the ratio of the

Fig. 2 e Typical chromatogr

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 defined as the nearness of

the true value and found value, was evaluated as % bias for

Tapentadol hydrochloride according to the following equation:

%Accuracy¼ observedconcentration=nominalconcentration

�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 coeffi-

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 studywere analyzed using JMP statistical

software package by one-way analysis of variance (ANOVA).

am of Tapentadol HCL.

Table 2 e Accuracy (recovery) of Tapentadol hydrochloride.

Sampleno.

Spikedlevel

Sampleweight (mg)

Samplearea

mg/ml added mg/ml found % recovery % meanrecovery

% meanrecovery

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

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 270

Univariate linear regression analysis using least square

method was applied to test the fitted model. Correlation co-

efficient was calculated and the results of the statistical anal-

ysiswere considered significant 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 first 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 mMDipotassium Phosphate

buffer (pH 4, adjusted with ortho phosphoric acid) at a flow

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

Fig. 3 e Linearity of Tapentadol.

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 coefficient of

regression r2 ¼ 0.9999 over a concentration range (75e450 mg/

ml), the representative linear regression equation for Tapen-

tadol hydrochloride Y¼ 21349xþ 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, five 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.

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 5 e Assay of formulation.

Sample no. Sampleweight (mg)

Samplearea 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 n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 2 71

4.6. Determination of the main drug in bulk and tabletdosage form (assay)

Six solutions of Tapentadol hydrochloride prepared from the

bulk drug and tablet dosage formwere prepared and analyzed

with the same experimental conditions and found to be drug

content within the specified 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

influential 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, five standard solutions of Tapentadol hydro-

chloride (at 0.05 mg ml�1) were assessed on each of two

occasions and the results showed no significant 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 ¼ 5) respectively, and for

measurements at two months the RSDs were 0.26% and

1.05%, respectively.

4.8. Limits of detection and quantification

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 timewas 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.

Table 4 e Precision of Tapentadol hydrochloride.

S. no Samplename

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

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 LOQmeasurements

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.

Conflict 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 theManagement 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. Quantification of tapentadol incanine plasma by HPLC with spectrofluorimetric detection:

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 6 7e7 272

development and validation of a new methodology. J PharmBiomed Anal. 2012;67e68:148e153. PBA-8601 http://dx.doi.org/10.1016/j.jpba.2012.04.020.

2. Physician’s Desk Reference, LLC. 64th ed. vol. 8. Montvale, NJ:PDR Network; 2009:2643e2648.

3. Schedules of controlled substances: placement of tapentadolinto schedule II. FR Doc E9-11933. Fed Regist.2009;74:23790e23793.

4. TzschentkeMThomas, Christoph Thomas, Kogel Babette, et al.(e)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methyl-propyl)-phenol hydrochloride (tapentadol HCl): a novel m-opioidreceptor agonist/norepinephrine reuptake inhibitor withbroad-spectrum analgesic properties. J Pharmacol Exp Ther.2007;323:265e276. http://dx.doi.org/10.1124/jpet.107.126052.

5. Coulter C, Taruc M, Tuyay J, Moore C. Determination oftapentadol and its metabolite N-desmethyltapentadol inurine and oral fluid using liquid chromatography withtandem mass spectral detection. J Anal Toxicol.2010;34:458e463.

6. “Validation of analytical procedures: text and methodology”;International Conference on Harmonization, draft revisedguidance on Q2(R1). Fed Regist. March 1, 1995;60.

7. Ermer J. Validation in pharmaceutical analysis. Part I: anintegrated approach. J Pharm Biomed Anal. 2001;24:755e767.

8. Shabir GA. Validation of high-performance liquidchromatography methods for pharmaceutical analysis.Understanding the differences and similarities betweenvalidation requirements of the US Food and DrugAdministration, the US Pharmacopeia and the InternationalConference on Harmonization. J Chromatogr A.2003;987:57e66.

9. ICH draft guidelines on validation of analytical procedures:definitions and terminology. Fed Regist. 1995;60. IFPMA,Switzerland.

10. Anonymous Guidelines for Validation of AnalyticalProcedures: Methodology e International Conference ofHarmonization of Technical Requirements for Registration ofPharmaceutical for Human Use, 1996.