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<a href=international journal of chemical and analytical science 4 (2013) 67 e 7 2 Available online at w w w . s c i e n c e d i r e c t . c o m journal homepage: w w w . e l s e v i e r . c o m / l o c a t e / i j c a s Original Article Development and validation of RP-HPLC method for estimation of Tapentadol hydrochloride in bulk and tablet dosage forms Y. Indira Muzib * , J. Ravi Kumar Reddy , K.P.R. Chowdary , E. Swathi Department of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, India Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, India Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, India Department of Pharmaceutics, Andhra University, Vishakhapatnam 530003, Andhra Pradesh, India article info abstract Article history: Objective: To develop a simple, novel, sensitive, precise and specific RP-HPLC method for the Received 6 March 2013 Accepted 27 May 2013 Available online 6 August 2013 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 m m particle size) as a stationary phase using Meth- Keywords: 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. Licrosphere column Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 m g/ml. Methanol The correlation coefficient ( r ) value was found to be 0.9994. Precision study showed % CV Isocratic mode value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro- Quantitative analysis chloride are in the range of 99.96 e 100.01%. The limit of detection and limit of quantifica- Tapentadol hydrochloride tion for Tapentadol hydrochloride were found to be 0.25 m g/ml and 0.75 m g/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 peculiar dual mechanism of action that has demonstrated efficacy in clinical application. Tapentadol is a centrally acting synthetic analgesic, received initial U.S. approval in * Corresponding author . Tel.: þ 91 9441593292, þ 91 7702229333. E-mail address: yindira2002@rediffmail.com (Y.I. Muzib). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.07.001 " id="pdf-obj-0-7" src="pdf-obj-0-7.jpg">

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<a href=international journal of chemical and analytical science 4 (2013) 67 e 7 2 Available online at w w w . s c i e n c e d i r e c t . c o m journal homepage: w w w . e l s e v i e r . c o m / l o c a t e / i j c a s Original Article Development and validation of RP-HPLC method for estimation of Tapentadol hydrochloride in bulk and tablet dosage forms Y. Indira Muzib * , J. Ravi Kumar Reddy , K.P.R. Chowdary , E. Swathi Department of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, India Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, India Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, India Department of Pharmaceutics, Andhra University, Vishakhapatnam 530003, Andhra Pradesh, India article info abstract Article history: Objective: To develop a simple, novel, sensitive, precise and specific RP-HPLC method for the Received 6 March 2013 Accepted 27 May 2013 Available online 6 August 2013 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 m m particle size) as a stationary phase using Meth- Keywords: 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. Licrosphere column Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 m g/ml. Methanol The correlation coefficient ( r ) value was found to be 0.9994. Precision study showed % CV Isocratic mode value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro- Quantitative analysis chloride are in the range of 99.96 e 100.01%. The limit of detection and limit of quantifica- Tapentadol hydrochloride tion for Tapentadol hydrochloride were found to be 0.25 m g/ml and 0.75 m g/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 peculiar dual mechanism of action that has demonstrated efficacy in clinical application. Tapentadol is a centrally acting synthetic analgesic, received initial U.S. approval in * Corresponding author . Tel.: þ 91 9441593292, þ 91 7702229333. E-mail address: yindira2002@rediffmail.com (Y.I. Muzib). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.07.001 " id="pdf-obj-0-66" src="pdf-obj-0-66.jpg">
<a href=international journal of chemical and analytical science 4 (2013) 67 e 7 2 Available online at w w w . s c i e n c e d i r e c t . c o m journal homepage: w w w . e l s e v i e r . c o m / l o c a t e / i j c a s Original Article Development and validation of RP-HPLC method for estimation of Tapentadol hydrochloride in bulk and tablet dosage forms Y. Indira Muzib * , J. Ravi Kumar Reddy , K.P.R. Chowdary , E. Swathi Department of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, India Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, India Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, India Department of Pharmaceutics, Andhra University, Vishakhapatnam 530003, Andhra Pradesh, India article info abstract Article history: Objective: To develop a simple, novel, sensitive, precise and specific RP-HPLC method for the Received 6 March 2013 Accepted 27 May 2013 Available online 6 August 2013 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 m m particle size) as a stationary phase using Meth- Keywords: 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. Licrosphere column Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 m g/ml. Methanol The correlation coefficient ( r ) value was found to be 0.9994. Precision study showed % CV Isocratic mode value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro- Quantitative analysis chloride are in the range of 99.96 e 100.01%. The limit of detection and limit of quantifica- Tapentadol hydrochloride tion for Tapentadol hydrochloride were found to be 0.25 m g/ml and 0.75 m g/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 peculiar dual mechanism of action that has demonstrated efficacy in clinical application. Tapentadol is a centrally acting synthetic analgesic, received initial U.S. approval in * Corresponding author . Tel.: þ 91 9441593292, þ 91 7702229333. E-mail address: yindira2002@rediffmail.com (Y.I. Muzib). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.07.001 " id="pdf-obj-0-68" src="pdf-obj-0-68.jpg">

Original Article

Development and validation of RP-HPLC method for estimation of Tapentadol hydrochloride in bulk and tablet dosage forms

<a href=international journal of chemical and analytical science 4 (2013) 67 e 7 2 Available online at w w w . s c i e n c e d i r e c t . c o m journal homepage: w w w . e l s e v i e r . c o m / l o c a t e / i j c a s Original Article Development and validation of RP-HPLC method for estimation of Tapentadol hydrochloride in bulk and tablet dosage forms Y. Indira Muzib * , J. Ravi Kumar Reddy , K.P.R. Chowdary , E. Swathi Department of Pharmaceutics, Sri Padmavati Mahila Visvavidyalayam, Tirupati 517501, Andhra Pradesh, India Jawaharlal Nehru Technological University Kakinada, Kakinada 500 003, Andhra Pradesh, India Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampet 516126, Andhra Pradesh, India Department of Pharmaceutics, Andhra University, Vishakhapatnam 530003, Andhra Pradesh, India article info abstract Article history: Objective: To develop a simple, novel, sensitive, precise and specific RP-HPLC method for the Received 6 March 2013 Accepted 27 May 2013 Available online 6 August 2013 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 m m particle size) as a stationary phase using Meth- Keywords: 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. Licrosphere column Results: The calibration curve for Tapentadol hydrochloride was linear from 75 to 450 m g/ml. Methanol The correlation coefficient ( r ) value was found to be 0.9994. Precision study showed % CV Isocratic mode value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro- Quantitative analysis chloride are in the range of 99.96 e 100.01%. The limit of detection and limit of quantifica- Tapentadol hydrochloride tion for Tapentadol hydrochloride were found to be 0.25 m g/ml and 0.75 m g/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 peculiar dual mechanism of action that has demonstrated efficacy in clinical application. Tapentadol is a centrally acting synthetic analgesic, received initial U.S. approval in * Corresponding author . Tel.: þ 91 9441593292, þ 91 7702229333. E-mail address: yindira2002@rediffmail.com (Y.I. Muzib). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.07.001 " id="pdf-obj-0-75" src="pdf-obj-0-75.jpg">

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

article info

 

abstract

Article history:

Objective: To develop a simple, novel, sensitive, precise and specific RP-HPLC method for the

Received 6 March 2013 Accepted 27 May 2013 Available online 6 August 2013

 

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-

Keywords:

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.

Licrosphere column

 

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

Methanol

The correlation coefficient (r 2 ) value was found to be 0.9994. Precision study showed % CV

Isocratic mode

 

value less than 2% in all selected concentrations. The % recoveries of Tapentadol hydro-

Quantitative analysis

chloride are in the range of 99.96e100.01%. The limit of detection and limit of quantifica-

Tapentadol hydrochloride

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

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

* Corresponding author. Tel.: þ91 9441593292, þ91 7702229333. E-mail address: yindira2002@rediffmail.com (Y.I. Muzib). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

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 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 inhibition 4 (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 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)

68 <a href=international journal of chemic al and analytical science 4 (2013) 67 e 7 2 2008 and was then placed into the schedule II category of the Controlled Substances Act in May, 2009. 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 inhibition ( Fig. 1 ). To date, only two LC e MS methods to detect Tapentadol in biological matrices (urine and urine and oral fluid) 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 (HPLC e DAD). 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 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) 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 m m particle size). The flow rate was 1.0 ml/min and detector wavelength was kept at 280 nm for monitoring the separation. The column back pressure was maintained at 110 e 115 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- m m membrane filter and degassed by sonication before use. In- jection volume was 20 m L 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 m m membrane filter. From this stock solution Tapentadol hydrochloride calibration standards were freshly prepared prior to analysis prepared at concentrations of 75 e 600 m g/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 m m 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, and mixed well. A portion of the solution was filtered through a 0.45 mm membrane filter, discarding the first 2 e 3 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. " id="pdf-obj-1-41" src="pdf-obj-1-41.jpg">

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 wavelength was kept at 280 nm for 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 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, and mixed well. A portion of the solution was 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 estimation of Tapentadol HCl.

 
 

S. no

Parameter

Description/value

 
  • 1 C18 Licrosphere column (150 mm 4.6 mm inner diameter, 5 mm particle size)

Stationary phase

 
  • 2 Methanol: 0.1 mM Dipotassium Phosphate buffer (pH 4, adjusted with ortho phosphoric acid)

Mobile phase

  • 3 1 ml/min

Flow rate

  • 4 Detection wavelength 280 nm

  • 5 UV (diode array detector)

Detector

  • 6 Elution

Isocratic

  • 7 Injection volume

20 ml

  • 8 Column temperature 25 C

  • 9 Run time

6 min

10

Diluent

Mobile phase

  • 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)

The calibration curves were constructed with six concentrations 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. 8e 10 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:

%Accuracy ¼ 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 study were analyzed using JMP statistical software package by one-way analysis of variance (ANOVA).

<a href=international journal of chemical and analytical science 4 (2013) 67 e 7 2 69 Table 1 e Optimized HPLC conditions for the estimation of Tapentadol HCl. S. no Parameter Description/value 1 C18 Licrosphere column (150 mm 4.6 mm inner diameter, 5 m m particle size) Stationary phase 2 Methanol: 0.1 mM Dipotassium Phosphate buffer (pH 4, adjusted with ortho phosphoric acid) Mobile phase 3 1 ml/min Flow rate 4 Detection wavelength 280 nm 5 UV (diode array detector) Detector 6 Elution Isocratic 7 Injection volume 20 m l 8 Column temperature 25 C 9 Run time 6 min 10 Diluent Mobile phase 3. Method validation The developed method was validated for assay of Tapentadol hydrochloride in accordance with ICH guidelines. 3.1. Detection and quantitation limits (sensitivity) Limits of detection (LOD) and limit of quantitation (LOQ) were estimated from the signal-to-noise ratio. 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) The calibration curves were constructed with six concentrations of Tapentadol hydrochloride ranging from 75 to 450 m g/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. 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: %Accuracy ¼ 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 m g/ mL. 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 study were analyzed using JMP statistical software package by one-way analysis of variance (ANOVA). Fig. 2 e Typical chromatogram of Tapentadol HCL. " id="pdf-obj-2-143" src="pdf-obj-2-143.jpg">

Fig. 2 e Typical chromatogram of Tapentadol HCL.

 

Table 2 e Accuracy (recovery) of Tapentadol hydrochloride.

   
 

Sample

Spiked

Sample

Sample

mg/ml added

mg/ml found

% recovery

% mean

% mean

no.

level

weight (mg)

area

recovery

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

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- ysis were 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 mM Dipotassium 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

70 <a href=international journal of chemic al and analytical science 4 (2013) 67 e 7 2 Table 2 e Accuracy (recovery) of Tapentadol hydrochloride. Sample Spiked Sample Sample m g/ml added m g/ml found % recovery % mean % mean no. level weight (mg) area recovery 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 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- ysis were 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 m m 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 mM Dipotassium 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 r ¼ 0.9999 over a concentration range (75 e 450 m g/ ml), the representative linear regression equation for Tapen- tadol hydrochloride Y ¼ 21349x þ 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 . " id="pdf-obj-3-368" src="pdf-obj-3-368.jpg">

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 r 2 ¼ 0.9999 over a concentration range (75e450 mg/ ml), the representative linear regression equation for Tapen- tadol hydrochloride Y ¼ 21349x þ 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

  • 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 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 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.

 

Table 4 e Precision of Tapentadol hydrochloride.

   
 

S. no

Sample

Inj.

Name

RT

Area

 

name

(20 ml)

 

Precision1

  • 1 Tapentadol

1

3.155

2156575

Precision2

  • 2 Tapentadol

1

3.143

2150804

Precision3

  • 3 Tapentadol

1

3.136

2159065

Precision4

  • 4 Tapentadol

1

3.127

2154633

Precision5

  • 5 Tapentadol

1

3.120

2150334

Precision6

  • 6 Tapentadol 3.114

1

2150547

Mean

2153660

Std. dev.

3676

% RSD

0.2

 

Table 5 e Assay of formulation.

   
 

Sample no.

Sample

weight (mg)

Sample

% Assay e 1

 

area e 1

 
  • 1 8735989

271.0

99.10

  • 2 8809425

271.0

99.93

  • 3 8675493

271.0

98.41

  • 4 8898726

271.0

100.94

  • 5 8852017

271.0

100.41

  • 6 8903936

271.0

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

 

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.

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 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.

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