Development and Validation of visible Spectrophotometric method of Tapentadol Hydrochloride in bulk and Pharmaceutical dosage form

 

Madhu Babu K., Kathirvel S.*

Department of Pharmaceutical Analysis, Hindu College of Pharmacy,   Amaravathi Road, Guntur, Andhra Pradesh – 522002

 

 

ABSTRACT:

A simple, sensitive, accurate spectrophotometric method has been developed for the estimation of Tapentadol hydrochloride in bulk and pharmaceutical formulation. The method is based on the formation of blue colored chromogen with Folin-Ciocalteu reagent in the presence of NaOH and obeys beer’s law in the range of 5 - 35µg/mL exhibiting the maximum absorbance at 763nm. The limit of detection and quantification were found to be 0.76µg/mL and 2.33µg/mL respectively. The calibration curve demonstrated a linear relationship between the absorbance and concentration, with the correlation coefficient higher than 0.9999. The Regression Equation of the curve was Y=0.0248x-0.0436. The method was fully validated according to ICH guidelines.

 

KEYWORDS: Tapentadol hydrochloride, Folin-Ciocalteu (FC) reagent, NaOH, Validation, ICH guidelines.

 

INTRODUCTION:

Chemically Tapentadol hydrochloride1 is 3-[(1R, 2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl] phenol hydrochloride. It is a centrally acting analgesic with dual mode of action as an agonist at the µ-opioid receptor and as a norepinephrine reuptake inhibitor2. It is used in the treatment of moderate to severe pain3. Analysis plays an important role in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drugs in bulk, in drug delivery systems, in dissolution studies (in vitro), and in biological samples (in vivo). If such a suitable method for a specific need is not available, then it becomes essential to develop a simple, sensitive, accurate, precise and reproducible method for the estimation of drug samples. The literature survey reveals that Tapentadol was analyzed in urine sample by the Ultra Performance Liquid Chromatography method4. More over Visible Spectrophotometric method has not been reported in bulk and pharmaceutical formulation. Thus the present study was undertaken to develop a colorimetric method of Tapentadol by using FC reagent5, and to validate the method as per ICH guidelines6-8.

 

MATERIALS AND METHODS:

An UV-Visible double beam spectrophotometer of Elico, Model: SL-244 with Spectral band width of 2 nm and a pair of 1 cm matched quartz cell was used. A pure drug sample of Tapentadol was supplied by Lupin Pharmaceuticals as gift samples. Tablets of Tapentadol i.e. Tydol (Ranbaxy Laboratories Ltd) was procured from the local market.

Chemicals and Reagents:

0.1N Hydrochloric acid, Folin–Ciocalteu Reagent (FCR) 2N (1:2 Dilution with water), 5% NaOH (w/v) solution (5% solution of sodium hydroxide was prepared by dissolving 5g of NaOH in sufficient quantity of water and finally diluting to 100 ml with water).

 

Figure.1. Structure of Tapentadol Hydrochloride

 

Optimization of parameters for colorimetric method:

All the optimization parameters are estimated at room temperature. Tapentadol  was  found  to yield  a  blue-colored product  with  FCR  and  NaOH  and  has  absorbance maxima (λmax)  at 763 nm. Therefore, investigations were carried out to establish the most favorable conditions for the formation of this colored product. The influence of the concentration as well as volume of reagent on the reaction has been studied.  Different concentrations  and  different  volumes  were  tried  for  all the  reagents,  by  varying  one  parameter  at  a  time.  The optimum concentration of FCR was 2N and of NaOH was 5 %, similarly optimum volume of FCR and NaOH was found to be 1 ml and 1 ml respectively. The optimum concentration and volume were selected on the basis of their ability to give maximum absorbance. Also  the  color  stability  of  newly  formed  complex  was measured  and  the  color  was  found  to  be  stable  for  a period  of  one  hour. 

 

Procedure:

Preparation of the standard stock solution:

A stock solution (1000 µg/mL) of Tapentadol hydrochloride was prepared by dissolving 100 mg of drug in 100 mL of 0.1N HCl (stock solution-I). From the above solution 10 mL was again diluted to 100 mL with 0.1N HCl to get 100 µg/mL solution of tapentadol HCl (stock solution-II). Suitable aliquots of the drug solution (0.5 to 3.5 ml) were taken in a series of 10 ml volumetric flasks. To each flask was added 1ml of 5% NaOH solution. All the flasks were shaken well for at least 3 to 5 min. followed by the addition of 1 ml of FCR solution. Finally volume was made up to the mark with 0.1N HCl to prepare a series of standard solutions containing 5-35 µg/mL tapentadol HCl. The absorbance of blue colored chromogen was measured at 763 nm against reagent blank within one hour and the absorbance were plotted against the respective concentrations to obtain the calibration curve.

 

 

Preparation of sample solution:

The proposed method was applied to analyze the commercially available Tapentadol hydrochloride tablets (Tydol, 100mg Ranbaxy Laboratories Ltd). Ten tablets were weighed and powdered. The amount of tablet powder equivalent to 100 mg of tapentadol was weighed accurately and transferred to a 100 mL volumetric flask containing 10 ml of 0.1N HCl, and sonicated for 20 min for complete dissolution of tapentadol HCl and then the sample solution was filtered and diluted to 100 ml with 0.1N HCl to get concentration of 1000 µg/mL. Appropriate aliquots were then taken in such a way that to produce a final concentration of 10 µg/mL.

 

Method Validation:

Validation is a process of establishing documented evidence, which provides a high degree assurance that a specific activity will consistently produce a desired result, or a product meeting its predetermined specifications and quality characteristics. The method was validated for different parameters like Linearity, Accuracy, Precision, Specificity, Ruggedness, Limit of detection (LOD) and Limit of quantification (LOQ).

 

Linearity:

Various aliquots were prepared from the stock solution-II (100µg/mL) ranging from 5-35 µg/mL. The samples were analyzed with the help of a UV-VIS Spectrophotometer using 0.1N HCl as used as blank. The correlation coefficient of the linearity was found to be 0.999 respectively.

 

Accuracy:

To check the accuracy of the developed methods and to study the interference of formulation excipients, analytical recovery studies were carried out by preparing solutions of different concentrations, i.e., 80, 100, 120% in which the amount of marketed formulation was kept constant and the amount of pure drug was varied respectively. The solutions were prepared in triplicate and the accuracy was indicated by %   Recovery. 

 

Precision:

The precision of the method was demonstrated by intra-day and inter-day variation studies. In the inter-day variation study, the solutions of same concentration were prepared and analyzed thrice, for three consecutive days, and the absorbances were recorded. In the intra-day variation study, the solutions of the same concentration were prepared and analyzed thrice a day (morning, afternoon, and evening). The result was indicated by % RSD.

 

 

LOD and LOQ:                                                                                            

The LOD is the detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected, but not necessarily quantitated as an exact value. The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOD & LOQ were calculated using the formula involving the standard deviation of response and the slope of the calibration curve.

LOD= 3.3 x σ/s

LOQ= 10 x σ/s

Where

σ - Standard deviation of the response,

s – Slope ratio curve

 

RESULTS AND DISCUSSION:

 

Figure: 2. Colorimetry absorption spectra

 

Table: 1. Linearity data

S.No.

Concentration (µg/mL)

Absorbance

1

5

0.1064

2

10

0.2087

3

15

0.3435

4

20

0.4609

5

25

0.5787

6

30

0.6945

7

35

0.8212

 

 

Figure: 3. Calibration graph

 

 

Figure: 4. Stability indicating graph

 

Table: 2. Optical characteristics

Parameter

Result

Absorption maxima λmax (nm)

763nm

Beer’s law limits (µg/mL)

5-35µg/mL

Standard regression equation Y=mx+c

Y=0.0236x-0.0113

Correlation coefficient

0.999

Sandell’s sensitivity (mg/cm2/0.001 absorbance unit)

0.04733

Molar absorptivity (lit.mol-1cm-1)

0.4771×105

LOD

0.769 µg/mL

LOQ

2.33µg/mL

Assay (n=6)

99.23

 

 

 

 

Table: 3. Recovery studies of Tapentadol hydrochloride

Name

Declared value (µg/mL)

Spiked value (µg/mL) (n=3)

Amount found (µg/mL) (n=3)

% Recovery ± S.D

Tapentadol HCl

10

10

10

8

10

12

17.89

19.79

22.26

99.90±0.37

99.58±0.46

100.51±0.55

 

 

 

Table: 4. Precision results showing repeatability of Tapentadol Hydrochloride

Amount of drug taken (µg/mL)

Average absorbance** ± S.D

%R.S.D

10

0.2081±0.00186

0.89

**Average of six readings

 

 

Table: 5. Results for Intermediate Precision of tapentadol HCl for inter and intraday study

Amount of drug (µg/mL)

Average absorbance in

intraday studies** (µg/mL)

Average absorbance in

inter day studies** (µg/mL)

Precision

(intraday)

Precision

(inter day)

10

Sess-I

Sess-II

Sess-III

1st day

2nd day

3rdday

S.D

% R.S.D

S. D

%R.S.D

0.2094

0.211

0.2098

0.212

0.210

0.214

0.00068

  0.32

0.0016

0.77

**Average of six readings

 

The proposed method was simple, rapid and precise. It does not suffer from any interference due to common excipients of tablet. In the presence of alkaline medium, tapentadol hydrochloride reacts instantaneously with the FCR resulting in blue colored product. Formation of blue color product is due to a common reaction mechanism i.e., the oxidation of the drug and the reduction of FCR.  Folin ciocalteu reagent is a mixture of acids and involves the following chemical species:

 

3H2O.P2O5 13WO3.5MoO3.10H2O and 3H2O.P2O5.14WO3 .4MoO3 .10 H2O

 

The  blue  color  formation  by  FCR  with  tapentadol HCl seems analogous  to  Folin  phenol  protein  reaction.  Tapentadol HCl probably  reduce  tungstate  and/or  molybdate  in  FCR producing  reduced  species  which  have  a  characteristic intense blue color with λ max at 763 nm. The  stability  of  colored  complex  was  checked  with respect to time and that color  was  stable  for  1.0  hour (Figure: 4),  so  it is  recommended  that the  readings  should  be  taken  within  the  specified  time range.  The proposed method obeyed beer’s law in the concentration range of 5-35µg/mL with FC reagent respectively. Linearity data for the proposed method is incorporated in Table: 1. the optical characteristics and the data concerning the proposed method were represented in Table: 2. The sensitivity was estimated in terms of limit of quantification (LOQ), the smallest amounts detected were estimated in terms of limit of detection (LOD) The results also shown in Table:2. The recovery studies were carried out for the developed method by the addition of standard drug solution of Tapentadol to pre-analyzed solution. The recovery studies were satisfactory and the percentage of drug recovered (Table: 3) was in the range of 99.90-101.51%, which showed that there was no interference from excipients. The precision of the method expressed as % RSD of intraday and interday validation is given in Table: 4 and 5. The method was validated and found to be simple, sensitive, accurate, and precise. The study has acceptable correlation coefficient and % RSD.  It was successfully applied for determination of drugs in their newly formulated pharmaceutical formulations. 

 

CONCLUSION:

This is for the first time that spectrophotometric methods are being reported for the assay of Tapentadol Hydrochloride in pure form and also in its formulations. Unlike GC and HPLC Techniques, Spectrophotometry is simple and inexpensive. The Spectrophotometric methods require simple reagents, which an ordinary analytical laboratory can afford and the Procedures do not involve any critical reactions. Moreover the proposed method is found to be simple, sensitive, accurate, and with good precision. The amounts obtained by the proposed methods are between 89% and 111% and the acceptance criteria is between 85% and 115%. The recovered percentage values ranges between 99.90 and 101.51. Thus, this approach could be considered for the analysis of this drug in the quality control laboratories.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Lupin Pharmaceuticals for providing the gift sample of tapentadol. The authors are also thankful to the management of Hindu college of Pharmacy for providing necessary facilities to carry out this project.

 

REFERENCES:

1.        Neil M.J.O. (Ed. By)  The merck Index – An Encyclopedia of chemicals, Drugs an Biological.Merck and Co. Inc., 13th Edn. 3626; P, 643.

2.        Tzschentke TM et al. (1R,2R)-3-(3-Dimethylamino-ethyl- 2- methyl-propyl)-phenol Hydrochloride (Tapentadol HCl) ,  a Novel μ-Opioid Receptor Agonist/Norepinephrine Reuptake Inhibitor with Broad-Spectrum Analgesic Properties. Journal of Pharmacology and Experimental Therapeutics. 323(1); 2007: 265-76.

3.        Daniels, S et al. A Randomized, Double-Blind, Phase III Study Comparing Multiple Doses of Tapentadol and Placebo for Postoperative (bunionectomy) Pain.  Current Medical Research and Opinion. 25 (3);   2009: 765-776.

4.        James A Bourland, Ayodele A Collins and Scot A Chester. Determination of Tapentadol (NUCYNTA) and N- desmethyl Tapentadol in authentic urine by UPLC and Tandem Mass Spectroscopy. Journal of analytical toxicology. 34; U.S.A, 2010.

5.        Snell F.D, Snell F.T, and Van Nostrand.D.  Colorimetric Methods of Analysis, Toronto, N. Y., London. III (III); 1953: 104.

6.        ICH. Note for guidance on validation of analytical methods: Definitions and Terminology Q2A; 1994.

7.        ICH. Note for guideline Validation on Analytical Procedures: Methodology Q2B (ICH, Geneva, Switzerland, 1996).

8.       ICH, Q2 (R1) Validation of Analytical Procedures: text and methodology; 2005.

 

 

Received on 10.10.2012

Modified on 17.10.2012

Accepted on 25.10.2012        

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(6): November–December, 2012, 328-331