STABILITY INDICATING SPECTROFLUORIMETRIC QUANTIFICATION OF TAPENTADOL HCL AND APPLICATION TO IN-VITRO DISSOLUTION STUDIES
Panikumar.D.Anumolu*, Haripriya A, Sirisha N, ...
Department of Pharmaceutical Analysis, Gokaraju Rangaraju College of Pharmacy, Osmania University, Hyderabad, Andhra Pradesh-500090 India
Keywords: Emission Wavelength – 592 nm, Excitation Wavelength – 272 nm, Fluorimetry, Forced degradation, tapentadol HC
Abstract

Purpose: In this present work a simple, rapid, specific and highly sensitive spectrofluorimetric method was developed and validated for the quantification of tapentadol HCl bulk drug and pharmaceutical dosage forms and proposed method was also applied to study of forced degradation and in-vitro dissolution studies. Materials and Methods: The fluorescence intensity of tapentadol HCl in distilled water was measured at emission wavelength 592 nm after excitation at 272 nm by using a Shimadzu (Japan) RF-5301 PC spectrofluorophotometer. A linear relationship was found between fluorescence intensity and concentration in the range of 1-6 µg/mL with a good correlation coefficient – 0.999. Results: The detection and quantification limits were found to be 23.01 and 76.72 ng/mL, respectively. The proposed method was applied for quantification of tapentadol HCl in tablets, with percentage recovery of 99.95–101.45% and percentage RSD values were found to be less than 2 for accuracy and precision studies. Statistical analysis of the results revealed high accuracy and good precision. Conclusion: The suggested procedures could be used for the determination of tapentadol HCl in drug substance and drug products as well as in presence of its degradation products.

Article Information

Identifiers and Pagination:
Year:2013
Volume:5
First Page:89
Last Page:99
Publisher Id:JAppPharm (2013 ). 5. 89-99
Article History:
Received:May 14, 2013
Accepted:June 21, 2013
Collection year:2013
First Published:July 1, 2013

INTRODUCTION

Tapentadol hydrochloride (Tapentadol HCl) is centrally acting synthetic analgesic [1, 2]. It is chemically 3-[(1R, 2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl] phenol hydrochloride [Figure 1]. After thorough literature survey, we came to know that there is no stability indicating method available for the estimation of tapentadol HCl either in bulk drug or in formulation by spectrofluorimetry.

Spectrofluorimetry has assumed a major role in drug analysis because of its greater sensitivity and selectivity than absorption spectrophotometry [3-9]. Hence, we aimed to develop and validate a simple, precise, economical and accurate spectrofluorimetric method for the estimation of tapentadol HCl and application of the proposed method for forced degradation and in-vitro dissolution studies of tapentadol HCl bulk drug and marketed formulations.

 

Figure 1. Structure of tapentadol HCl

 

MATERIALS AND METHODS

Instruments

The fluorescence spectra and measurements were recorded using a Shimadzu (Japan) RF-5301 PC Spectrofluorophotometer, equipped with 150 watt Xenon arc lamp, 1cm quartz cell was used, connected to RFPC software. The instrument was operated both at low and high sensitivity with excitation and emission slit width set at 5 nm, dissolution apparatus (Electro lab TDT-08L), analytical balance (Shimadzu AUX 220, Japan) and pH meter (Elico) were used for the study.

Materials

Tapentadol HCl active ingredient sample was gifted by MSN Laboratories Private Limited (Hyderabad, India). Tapenta-50 and Tydol-50 both commercial formulations containing
50 mg of the drug were obtained from the local pharmacies. The analytical grade solvents such as methanol, ethanol, acetonitrile, acetone, dimethyl Formamide (DMF), dimethyl sulphoxide (DMSO) were obtained from
Sd Fine-Chem Ltd., Mumbai.

Preparation of standard solutions

 A stock solution of tapentadol hydrochloride was prepared by dissolving 10 mg of the drug in 10 mL of distilled water to get 1mg/mL stock solution [10, 11]. Standard solutions were prepared by appropriate dilution of the stock solution with the same solvent.

Analysis of tapentadol hydrochloride in tablet dosage form

Twenty tablets of each marketed formulation (Tapenta-50 and Tydol -50) were taken and accurately weighed. An accurately weighed powder equivalent to 10 mg tapentadol HCl was transferred to volumetric flask of 10 mL capacity. The flask was shaken and volume was made up to the mark with water. The solution was filtered through whatmann filter paper (No.41). From the filtrate a final concentration of 5 µg/mL solution was prepared for the estimation of tapentadol HCl.

The amount of tapentadol HCl present in the sample solution was determined by substituting fluorescence intensity into the equation of the straight line representing the calibration curves for tapentadol HCl, with correction for dilution.

Method validation

The method was validated linearity, precision, accuracy, selectivity and sensitivity by the following procedures [12, 13].

Linearity

Aliquots of tapentadol HCl stock solutions were transferred into a series of 10 mL volumetric flasks so that the final concentration was in the range of 1.0-6.0 µg/mL. Then, the volumes were completed to the mark with distilled water and mixed well. The fluorescence intensities of the solutions were measured at 572 nm after excitation at 272 nm. The calibration curve was constructed by plotting the analyte intensity against the concentration (µg/mL).

Sensitivity

The sensitivity of the method was determined with respect to LOD and LOQ. The LOD and LOQ were separately determined based on standard calibration curve.

Selectivity

Selectivity is the ability of the method to accurately measure a compound in the presence of other components such as impurities, degradation products and matrix components. The selectivity of the proposed method was evaluated through the analysis of a placebo solution, which it was prepared with the common excipients (lactose, starch, microcrystalline cellulose, magnesium stearate, titanium dioxide and talc) of the pharmaceutical formulation. Thus, the mixture of component inert was prepared in their usual concentration employed in tablets (concentrations were determined based in Handbook of pharmaceutical Excipients and calculated for medium weight of content). The developed method was applied to in order to check if any component of the formulation could generate a response or a read with emission band similar to the drugs.

Precision

Precision of the method was determined by intra-day precision and inter-day precision variations as per ICH guidelines. For both intra-day precision and inter-day precision of the samples containing  tapentadol HCl 2,4, and 6 µg/mL were analyzed six times on the same day (intra-day precision) and for three consecutive days (inter day precision). The % RSD was calculated.

Accuracy

The accuracy of the method was determined by calculating recovery of tapentadol HCl by the method of standard additions. Tablet powder (Tapenta and Tydol) equivalent to 10 mg of tapentadol HCl was transferred into three different 10 mL volumetric flasks and to it 80%, 100% and 120% of pure bulk drug was added respectively and made the volume up to the mark with distilled water. These solutions were further diluted with distilled water and analyzed by using water as blank. The amount of tapentadol HCl was estimated by measuring fluorescence intensity at the appropriate wavelengths. The recovery was verified by estimation of drug in triplicate preparations at each specified concentration level.

Forced degradation studies

Forced degradation studies were carried out by exposing the sample solution to stress conditions such as acidic, alkaline, oxidative, thermal and UV effect.

Stock solution was prepared by dissolving 10 mg of tapentadol HCl in 10 mL of
1N HCl. From the above stock solution 1mL was taken in 10 mL volumetric flask and to this 1mL of 1N NaOH was added to neutralize the solution and the volume was made up to the mark with distilled water. One of the concentrations in the range of linearity was prepared by appropriate dilution and scanned in spectrofluorimeter.

This procedure was repeated for every 1 hr. Similarly alkaline degradation (1N NaOH), oxidative degradation (3% H2O2), thermal degradation (hot air oven at 1050C) was performed. For UV degradation 50 mg of accurately weighed drug was placed in UV cabinet at short wavelength (254 nm) and readings for intensity were taken hourly.  

Dissolution studies

Procedure for dissolution testing of Tapentadol HCl tablet formulation

Dissolution testing [14] of tapentadol HCl tablet formulations (Tapenta-50 and Tydol-50) were carried out using USP apparatus type-1 (basket), 900mL of 0.1 N HCl as dissolution medium with a speed of 75 rpm at 37±0.50C temperature and sampling intervals were 10, 20, 30 , 45, 60 min as recommended by FDA guidelines [14, 15]. Sampling aliquots of 5.0 mL were withdrawn at specified intervals and replaced with an equal volume of fresh medium to maintain constant total volume. The amount of drug dissolved was sampled, pooled (n=3) and analyzed by the developed and validated spectrofluorimetric method. Amount of the dissolved drugs were calculated using their respective calibration curves. The content results were used to calculate the percentage release on each time of dissolution profile. The cumulative percentage of drug dissolved was plotted against time.

 

RESULTS AND DISCUSSION

Selection of optimum ??

Tapentadol HCl solution (10 µg/mL) was prepared and placed in spectrofluorimeter for the determination of excitation and emission wave lengths. Emission wavelength was found to be at 592 nm after excitation at 272nm.The emission spectrum of tapentadol HCl in water is shown in Figure 2.

Effect of pH

The influence of pH on fluorescence intensity of the tapentadol HCl was investigated using different kinds of buffers covering the whole pH range (1.0-9.0). The native fluorescence intensity of tapentadol HCl was higher in water (neutral pH) than other buffers as shown in Figure 3. Hence, for simplicity of the method no buffer was used throughout the study.

Effect of diluting solvent

Dilution with different solvents such as water, methanol, ethanol, acetonitrile, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) was employed. The fluorescence intensity of tapentadol HCl was higher in water and methanol compared with other solvents as shown in Figure 4. Hence, in this method water was the best solvent for dilution as it gave the highest fluorescence intensity with no spectral interference, economical and eco-friendly.


Figure 2: Emission spectrum of tapentadol HCl in distilled water at 592 nm after excitation at 272 nm

 


Figure 3: Effect of pH on fluorescence intensity of tapentadol HCl

 


Figure 4: Effect of solvents on fluorescence of tapentadol HC


Figure 5: Spectrofluorimetric linearity range of tapentadol HCl
(1-6 µg/mL)


Figure 6: Standard plot of tapentadol HCl in distilled water at 592 nm ((?EM)

Effect of stability time

The effect of time on the development and stability of the fluorescence intensity of tapentadol HCl was also studied. It was found that the fluorescence readings remained stable for more than 24 hours.

Method validation

The calibration curve was linear in the range of 1 – 6.0 µg/mL with a correlation coefficient of 0.999 (Figure 5 & 6). Limit of detection (LOD) and Limit of quantification (LOQ) were experimentally verified to be 23.01 and 76.72 ng/mL, respectively, which indicated that the method shows high sensitivity. Optimized conditions for proposed method are given in Table 1.The percentage recoveries were found to be in the range of 99.5-102 %. This indicates that the method is accurate (Table 2). Results from precision expressed in terms of %RSD, found to be less than 2. No significant differences between intraday and inter day precision, which indicated that the method is reproducible (Table 3).

Table 1: System suitability Parameters

Parameter

Value

Excitation Wavelength (nm)

272

Emission Wavelength (nm)

592

Range (µg/mL)

1-6

Limit of Detection (ng/mL)

23.01

Limit of Quantification (ng/mL)

76.72

Correlation Coefficient (r2)

0.999

Slope (m)

1.9555

Intercept ( c )

0.6366

Regression equation

Y=1.955x+0.6366

 

 

 

 

 

 

 

 

Table 2: Accuracy of the method (Recovery studies)

Formulation

Recovery Level (%)

Amount of the drug in tablet powder (mg)

Tapentadol  pure drug added (mg)

Mean (mg/mL) ± S.D. (n=3)

% Amount Recovered

% RSD

TAPENTA-50

80

10

8

17.872 ±0.236

99.95

1.3681

100

10

20.667 ±0.153

101.35

0.738

120

12

22.232 ±0.252

101.41

1.117

TYDOL-50

80

8

18.12±0.125

100.66

0.689

100

10

20.09±0.245

100.45

1.219

120

12

22.32±0.364

101.45

1.63

 

Applications

Assay

The proposed method was applied to the quantification of tapentadol HCl in tablet dosage forms. The assay results show that the proposed method was selective for tapentadol HCl without interference from the excipients used in the tablet dosage form. The values were shown in Table 4.

 

Table 3: Precision of the method

Concentration (mcg/ml)

Intra-day precision

Inter day precision

Mean (µg/mL) ± S.D. (n=3)

% recovery

% RSD

Mean (µg/mL) ± S.D. (n=3)

% recovery

% RSD

2

1.985 ±0.013

99.25

0.654

1.991 ±0.011

99.55

0.578

4

4.056 ±0.0645

101.45

1.55

3.996 ±0.055

99.9

1.397

6

6.047 ±0.063

100.78

1.04

5.886 ±0.009

99.76

0.154

 

Table 4: Analysis of commercial tablets (assay)

Formulation

Tapentadol HCl

Label claim (mg)

Amount found (mg)

 (AM ± SD) (n=3)

% RSD

Tapenta

50

51.46 ±0.971

1.89

Tydol

50

50.09 ± 0.462

0.922

 

Table 5: Cumulative % drug release data in zero order, first order, Higuchi and Hixon-Crowell cube root equations

Fitting of release data  into zero order, first order, Higuchi and Hixon-Crowell cube root equations

Formulation

Equations And r2

Zero

First

Higuchi

Hixon-Crowell Cube root

Tydol -50 mg tablet

y = 0.523x + 19.37

y = 0.007x + 1.292

y = 6.4797x + 0.7711

y = 0.017x + 2.695

r2= 0.723

r2= 0.659

r2= 0.9375

r2= 0.681

Tapenta 50mg tablet

y = 0.247x + 33.54

y=0.002x + 1.520

y = 2.992x + 25.21

y = 0.007x + 3.216

r2= 0.531

r2= 0.511

r2=  0.640

r2= 0.517










In-vitro dissolution studies

Dissolution studies of two tapentadol tablets (Tapenta 50 and Tydol 50) conducted as per FDA guidelines and analysed by proposed spectrofluorimetric method. The cumulative percentage of drug dissolved was plotted against time from the dissolution studies.

 


Figure 7:  In-vitro dissolution studies of tapentadol tablet formulations


Figure 8: Stability profile of tapentadol HCl in acid (1N HCl), basic (1N NaOH) and oxidative (3% H2O2)

The percentage drug release was found to be more than 90% for both formulations, which is correlating with FDA guidelines [Figure 5]. Release of tapentadol formulations in 0.1N HCl during dissolution was processed for regression analysis and is it plotted into graphs to understand the linear relationship or kinetic principles (Table 5). From the data it is clear that the dissolution phenomenon of tapentadol HCl follows zero order kinetics and it is controlled by diffusion process

Forced degradation studies

 The stability of tapentadol HCl was studied in terms of fluorescence intensity in the presence of acid, alkaline, oxidative agent, photo and thermal degradation and construct the plot between amounts of drug degraded in terms of (%) Vs time interval. Considerable degradation of the drug was observed in 1N HCl (99.02% in 4 hrs), 1N NaOH (95.2% in 4 hrs), slow degradation in 3% H202 (80% in 24 hrs) [Figure 8] and no significant difference was observed in thermal and UV cabinet.

 

CONCLUSION

A simple, rapid, sensitive, precise and accurate new spectrofluorimetric method has been developed and validated as per ICH guidelines for the quantification of tapentadol HCl in bulk drug and formulations. The assay values were in good agreement with their respective labeled claim. This spectrofluorimetric method has been found to be better because of its high specific, sensitivity, readily available solvent, economical, eco-friendly and also utilized for forced degradation studies and in-vitro dissolution studies. The percentage drug release was found to be more than 90% for both formulations. Forced degradation revealed that the drug was not susceptible to thermal and UV effect and it was degraded in acidic, alkaline, and oxidative degradation methods. These advantages encourage that; the proposed method can be routinely employed in quality control for analysis of tapentadol HCl in tablet dosage forms, dissolution studies and in the presence of degradation products.

 

ACKNOWLEDGEMENTS

The authors sincerely express gratefulness to the management and Prof.C.V.S.Subrahmanyam, Principal, Gokaraju Rangaraju College of Pharmacy.

 

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Bibliography

Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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