Development and Validation of RP-HPLC Method for Simultaneous Estimation of Tapentadol and Paracetamol in Bulk Drug and its Pharmaceutical Dosage Form

 

B. Mohammed Ishaq1, Dr. K. Vanitha Prakash2*, Dr. G. Krishna Mohan3

1Research Scholar, Jawaharlal Nehru Technological University, Kakinada, A. P. India and Balaji College of Pharmacy, Anantapur, AP India 515001

2Department of Pharmaceutical Analysis, SSJ College of Pharmacy, Gandipet, Hyderabad (A.P.) India

3Centre for Pharmaceutical Sciences, IST, JNTU Hyderabad, Andhra Pradesh, India

 *Corresponding Author E-mail: prakash.karanam@gmail.com

 

ABSTRACT:

A simple, rapid and sensitive isocratic RP-HPLC method was developed for the simultaneous estimation of Tapentadol and Paracetamol in bulk and their Pharmaceutical dosage form using Waters C18 (250 x 4.6 mm i.d, 5 μ particle size) analytical column in an isocratic mode with mobile phase comprising Acetonitrile: Potassium dihydrogen Phosphate pH 6.8 adjusted with orthophosphoric acid in the ratio of 50:50 (% v/v). The flow rate was 1.0 ml/ min and effluent was monitored at 215nm. The retention times were found to be 2.38 min for Paracetamol and 4.59 min for Tapentadol. The assay exhibited a linear dynamic range of 350-1050 μg/ml for Paracetamol and 50-150 μg/ml for Tapentadol. The calibration curve was linear (r = 1 for Paracetamol and r = 0.9998 for Tapentadol) over the entire linear range. Recovery was found to be 99.00 % + 0.41 for Paracetamol and 100 % + 0.22 for Tapentadol. The proposed method was statistically evaluated and validated as per ICH guidelines and can be applied for routine quality control analysis of Tapentadol and Paracetamol in Pharmaceutical dosage form.                      

              

KEYWORDS: Tapentadol, Paracetamol, isocratic, Acetonitrile, Waters C18 analytical column.

 

 


INTRODUCTION:

Tapentadol (Figure 1) is a novel centrally acting analgesic that was approved for use by the Food and Drug Administration in November 2008. It has structural similarities to tramadol. The drug has a unique mode of action in that it functions as an agonist at the μ-opioid receptor, and as a norepinephrine reuptake inhibitor1. This dual mode of action provides analgesia at similar levels of more potent narcotic analgesics such as hydrocodone, oxycodone and morphine, but with a more tolerable side effect profile. The chemical name is 3 - [(1R,2R) -3 - (dimethylamino) –l - ethyl-2 -methylpropyl] phenol monohydrochloride. The structural formula is: The molecular weight of tapentadol HC1 is 257.80, and the molecular formula is C14H23NO·HCl. The n-octanol: water partition coefficient log P value is 2.87.

 

The pKa values are 9.34 and 10.45.2,3 Chemically, paracetamol is 4-hydroxy acetanilide(Figure 2), used as an analgesic and antipyretic drug. Paracetamol is official in Indian Pharmacopoeia4 and British Pharmacopoeia5. These two pharmacopoeias suggest titrimetric and UV spectrophotometric assay method for paracetamol in bulk and tablet formulations.

 

Figure 1: Chemical Structure of Tapentadol

 

Figure 2: Chemical structure of Paracetamol

Survey of literature revealed that numbers of method have been reported in literature for the individual analysis of Tapentadol and Paracetamol by UV spectrophotometric and RP-HPLC method. UV spectrophotometric method available in literature for simultaneous determination of Paracetamol with other drugs like Aceclofenac, Lornoxicam, Etodolac, Nimesulide.6-9

 

However, to our knowledge, there is no reported simple and convenient RP-HPLC method available for simultaneous estimation of Tapentadol and Paracetamol. The aim of the present work was to develop easy, economic, accurate, specific and precise RP-HPLC methods for simultaneous estimation of Paracetamol and Tapentadol in bulk drugs and combined pharmaceutical formulations and validation of newly developed analytical methods.

 

MATERIALS:

Tapentadol and Paracetamol were received gratis from Hetero drugs, Hyderabad and were used as received. HPLC grade acetonitrile and methanol were purchased from SD Fine Chem Pvt. Ltd. (Mumbai, Maharashtra) and Merck (Darmstadt, Germany), respectively. Ultra-pure water was obtained from ELGA (Bucks, UK) water purification unit. Waters total recovery vials (Waters, Milford, MA, USA) were of glass type 1, class A with 950 μL maximal injectable volumes. All other chemicals were of analytical reagent grade.

 

EXPERIMENTAL WORK:

Chromatographic conditions:

The HPLC system (LC Waters, Milford, MA, USA) consisted of quaternary gradient system (600 Controller), in-line degasser (Waters, model AF), photodiode array detector (Water, 2998 model) and auto sampler (Waters, model 717 plus). Data was processed using Empower Pro software (Waters, Milford, MA, USA).

 

Isocratic elution of the mobile phase Acetonitrile: Potassium dihydrogen Phosphate pH 6.8 adjusted with Orthophosphoric acid in the ratio of 50:50 (% v/v) with the flowrate of 1 ml/min. Separation was performed on a Waters C18 (250 x 4.6 mm i.d, 5 μ particle size) analytical column and a pre-column to protect the analytical column from strongly bonded material. Integration of the detector output was performed using the Waters Empower software to determine the peak area. The contents of the mobile phase were filtered through a 0.45 µm membrane filter and degassed by sonication before use. Mobile phase was used as diluents.

 

The flow rate of the mobile phase was optimized to 1 ml/min which yields a column back pressure of 110–112 kg/cm. The run time was set at 6 min and a column temperature was maintained at 30°C. The volume of injection was 20 µl, prior to injection of the analyte, the column was equilibrated for 30–40 min with the mobile phase. The eluent was detected at 215 nm. The developed method was validated in terms of specificiy, linearity, accuracy, limit of detection (LOD), limit of quantification(LOQ), intra-day and inter-day precision and robustness for the assay of Tapentadol and Paracetamol as per ICH guidelines.10

 

Standard preparation:

Stock solution of tapentadol was prepared by dissolving 10 mg of tapentadol in a 10 ml volumetric flask, and the volume is made up with the diluent. Subsequent dilutions of this solution ranging from 50 to 150 µg/ ml were made with the diluent. 10 mg of paracetamol was dissolved in 10 ml volumetric flask and the volume was made up with the diluents. Subsequent dilutions of this solution ranging from 350 to 1000 µg/ ml were made with the diluent.

 

Sample preparation:

Twenty tablets were taken, and their average weight was calculated. The tablets were crushed to a fine powder, dose equivalent to 10 mg was transferred to a 25 ml volumetric flask, dissolved in a diluent, and then the solution was made up to the mark with the same and filtered through 0.45 µm membrane filter. 2.5 ml of this solution was pipette into a 25 ml volumetric flask and diluted with the diluent to get a concentration of 100 µg/ml.

 

Assay:

A mass of not less than 10 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 25 ml volumetric flask. Approximately, 15 ml milli-Q water was added; the solution was sonicated for 10 min, 10 ml diluent was added to it, and mechanically shaken for 10 more minutes. The flask was equilibrated to room temperature. A portion of the solution was filtered through a 0.45 mm membrane filter, discarding the first 2–3 ml of the filtrate. A portion of the filtered sample (2.5 ml) was diluted into a 25 ml volumetric flask with the mobile phase and mixed well. The resulting solution was injected into the HPLC system for the analysis.

 

RESULTS AND DISCUSSION:

Several systematic trials were performed to optimize the chromatographic conditions for developing a sensitive, precise and accurate RP-HPLC method for the simultaneous estimation of tapentadol and paracetamol in pharmaceutical dosage forms. This method contains the mobile phase Acetonitrile: Potassium dihydrogen Phosphate pH 6.8 adjusted with orthophosphoric acid in the ratio of 50:50 (% v/v) which was found to be the most suitable as the chromatographic peaks obtained with this system were better defined and resolved and all almost free from tailing. Under the above conditions, the retention time obtained for paracetamol was 2.38 min and 4.59 min for tapentadol. A model chromatogram was shown in Figure 3.

 

Figure 3: Typical chromatogram for simultaneous estimation of paracetamol and tapentadol

 

System suitability:

As per the USP 27 System, suitability tests were carried out on a freshly prepared standard solution of paracetamol and tapentadol to check the various parameters such as efficiency, retention time, and peak tailing which was found to comply with USP requirements. The instrumental precisions as determined by six successive injections of the standard solution give RSD below 2% of retention time and area.

 

Linearity:

The calibration curve for paracetamol and tapentadol was drawn by plotting the mean peak area versus concentration yielded a coefficient of regression r = 1 for paracetamol and r= 0.999 for tapentadol over a concentration range of 350 – 1050 µg/ml and 50-150 µg/ml for paracetamol and tapentadol respectively, the representative linear regression equation for paracetamol and tapentadol were y = 17135x + 14069 and y = 21612x - 32341 respectively as shown in Figure 4 and 5.

 

Figure 4: Linearity of Paracetamol

 

Figure 5: Linearity of Tapentadol

 

Accuracy:

The accuracy of the proposed analytical method was determined 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 recovery values from the developed method. The % recovery results of the method are given in Table 1.

 

Table 1: Accuracy (Recovery data) of the method

S. No

Recovery level (Added amount)

Mean Percent recovery #

Paracetamol

Tapentadol

1

50 %

100 ±0.045

101 ±0.032

2

100 %

100 ±0.026

100 ±0.075

3

150 %

98 ±0.041

100 ±0.080

# Average of six estimation at each level of recovery.

 

Precision:

The precision of the method for the determination of simultaneous estimation of paracetamol and tapentadol were studied using the parameters such as system precision, method precision, and intermediate precision. System precision was determined by six replicate injections of a standard solution injected into the HPLC system. The relative standard deviation was less than 2%. The method precision was determined by the six individual sample preparations injected to the HPLC system. The relative standard deviation was less than 2%. Ruggedness of the method was determined by different analysts, different columns, and different instruments and on different days. % RSD was found below 2%. The results indicating that the developed HPLC method was found to be precise. The results of precision were shown in table 2.

 

Robustness:

The robustness of the method was studied by small changes in the method such as altering the mobile phase pH, change in column temperature, flow rate, and changes in wavelength. It was observed that there were no changes in the chromatograms. System suitability and chromatographic parameters were validated such as asymmetry factor and tailing factor and number of theoretical plates were calculated. The results were given in Table 3.


Table 2: Precision of the method

Sample No.

Paracetamol

Tapentadol

Sample Area - 1

%  Assay - 1

Sample Area - 2

%  Assay - 2

1

12508045

99.64

2132304

99

2

12521643

99.75

2150452

100

3

12372333

98.56

2114022

99

4

12372949

98.56

2092453

98

5

12381516

98.63

2086948

97

6

12424701

98.98

2134497

100

Average Assay:

99

Average Assay:

99

STD

0.55

STD

1.17

%  RSD

0.55

% RSD

1.19

 

 


Table 3: Robustness data

S. No

Parameter

Results#

Paracetamol

Tapentadol

1

Tailing factor

1.56

1.32

2

Theoretical plates

3953

6401

3

Retention time

2.38

4.60

# Average of six estimation at each level of recovery.

 

Limit of detection and limit of quantification:

The LOD and LOQ of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (a signal-to-noise ratio of 3). The LOD for both paracetamol and tapentadol was found to be 0.0001µg/ml. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (a signal- to-noise ratio of 10). The LOQ for paracetamol and tapentadol was found to be 0.0003 µg/ml and 0.0005 µg/ml respectively. It was concluded that the developed method is highly sensitive.

 

Assay:

Twenty microliters of standard and sample solutions were injected into an injector of RP-HPLC, peak area of standard amount of drug and the sample were computed. The values are given in Table 2.

 

The UV spectrum of simultaneous estimation of paracetamol and tapentadol was recorded, from which 215 nm was selected as wavelength. The flow rate of 1.0 ml/min was selected. Acetonitrile: Potassium dihydrogen phosphate pH 6.8 adjusted with orthophosphoric acid in the ratio of 50:50 (% v/v) was selected as the mobile phase. The retention times were found to be 2.38 min for Paracetamol and 4.59 min for Tapentadol. The linearity of paracetamol was in the range of 350–1050 µg/ml, and the co-efficient was found to be 0.999 and tapentadol was in the range of 50-150 µg/ml and co-efficient was found to be 0.998. Recovery studies were performed for paracetamol and tapentadol at 50%, 100%, and 150% levels. The results show a mean recovery of 100% for both the drugs. The sensitivity of method LOD and LOQ for paracetamol was 0.0001 and 0.0003 µg/ml and for tapentadol was found to be 0.0001 and 0.0005 µg/ml.

 

The stability at room temperature and refrigeration was found to be 5 and 10.5 h, respectively. From the obtained results, it can be concluded that the proposed method is

 

quite precise and accurate. The low standard deviation and good percentage recovery indicates the reproducibility and accuracy of the method. The absence of additional peaks in the chromatogram indicated that there is no interference of the common excipients used in the tablets. The proposed HPLC method is sensitive and reproducible for the analysis of simultaneous estimation of paracetamol and tapentadol in tablet dosage forms. The method was duly validated by using required statistical parameters. The optimized chromatographic conditions were summarized in Table 4.

 

Table 4: Optimized HPLC conditions for simultaneous estimation of paracetamol and tapentadol

S. No

Parameter

Description/Value

1.

Stationary Phase

Waters C18

(250 x 4.6 mm i.d, 5 μ particle size)

2

Mobile Phase

Acetonitrile: Potassium dihydrogen Phosphate pH 6.8 adjusted with Orthophosphoric acid in the ratio of 50:50 (% v/v)

3

Flow rate

1 ml/min

4

Detection Wavelength

215 nm

5

Detector

Photo diode array

6

Injection

autosampler -Waters, model 717 plus

7

Rt’s

paracetamol: 2.38 min; Vildagliptin: 4.59 min

8

Injection volume

10 μl

9

Column Temperature

30 °C

10

Run time

6 mins

11

Diluent

Mobile Phase

 

CONCLUSION:

A convenient and rapid RP-HPLC method has been developed for simultaneous estimation of paracetamol and tapentadol in the tablet dosage form. The assay provides a linear response across a wide range of concentrations. Low intra-day and inter-day % RSD coupled with excellent recoveries. The proposed method is simple, fast, accurate and precise for the simultaneous quantification of paracetamol and tapentadol in dosage form, bulk drugs as well as for routine analysis in quality control.

 

ACKNOWLEDGMENTS:

The authors are grateful to Hetero drugs, Hyderabad, India, for providing a gift sample of paracetamol and tapentadol.

 

REFERENCES:

1.       Determination of Tapentadol and its Metabolite NDesmethyltapentadol in Urine and Oral Fluid using Liquid Chromatography with Tandem Mass Spectral Detection, Journal of Analytical Toxicology, Volume 34, Issue 8, Pp. 458-463, Cynthia Coulter, Margaux Taruc, James Tuyay and Christine Moore.

2.       Rohtesh, MS, Arnold, RM, Tapentadol. Fast Facts and Concepts, March 2010, http://www.eperc.mcw.edu/fastfact/ff_228.htm

3.       Tzschentke TM, et. al., Tapentadol: with two mechanisms of action in one molecule effective against nociceptive and neuropathic pain, Preclinical overview, Schmerz. 2011, 25(1), 19-25.

4.       Indian Pharmacopoeia. Govt. of India, Ministry of Health and Family Welfare, Delhi. Publication by Controller of Publication, Vol-2, 1996.

5.       British Pharmacopoeia. Her Majesty Stationery Office, London, Vol-I, 2002.

6.       Ashraful Islam SM, Abuzar S and Paul PK: Validation of UV-Spectrophotometric and RP-HPLC methods for the simultaneous analysis of Paracetamol and Aceclofenac in marketed tablets. Int. J. of Pharm. & Life Sci. 2011; 2 (12): 1267-1275.

7.       Sivasubramanian L, Lakshmi K.S and Tintu. T: Simultaneous Spectrophotometric estimation of Paracetamol and Lornoxicam in tablet dosage form. Int J Pharm Pharm Sci. 2010; 2 (4): 166-168.

8.       Balan P, Carolin Nimila I, Lakshmi Prasanna M, Vanaja Rani M and Rajasekar S: Simultaneous estimation of Etodolac and Paracetamol by UV Spectrophotometric method in tablet formulation. Journal of Pharmacy Research 2011; 4(6): 1663-1665.

9.       Kirtawade R, salve P, Seervi C, Kulkarni A and Dhabale P: Simultaneous UV Spectrophotometric Method for Estimation of Paracetamol and Nimesulide in Tablet Dosage Form. Int.J. ChemTech Res. 2010; 2(2): 818-821.

10.    International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use, validation of analytical procedures: definitions and terminology, Geneva 1996.

 

 

 

 

 

Received on 09.12.2013       Modified on 08.01.2014

Accepted on 14.01.2014      © RJPT All right reserved

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page   208-212