INTRODUCTION:

Tapentadol(3-[(1R,2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl]phenol hydrochloride, C₁₄H₂₃NO, ) is a centrally acting analgesic that exerts its pharmacological effects by two mechanisms of action in a single molecule, that is, mu-opioid receptor agonism and noradrenaline re-uptake inhibition. Its binding affinity to mu-opioid receptors is approximately 18 times less than that of morphine ^1,2 Tapentadol is used to treat moderate to severe chronic pain. It is structurally and pharmacologically related to tramadol. It is a white to off-white powder. It was initially formulated as immediate- release preparation.

Tapentadol was approved as potent Schedule II analgesic by US Food and Drug Administration (FDA). It is also the first analgesic developed in recent 25 years for management of moderate and severe pain ³.

Paracetamol (N- (4-hydroxyphenyl)-acetamide, C8H9NO2) is a widely used over-the-counter analgesic and antipyretic with few, if any, anti-inflammatory properties and no effect on platelet aggregation. It has no irritant effect on the gastric mucosa and can be used safely and effectively in most individuals who are intolerant of aspirin. It is the standard analgesic/antipyretic in pediatrics since, unlike aspirin. The usual adult dose is 0.5–1 g repeated at intervals of four to six hours if needed. . In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post surgical pain and providing palliative care in advanced cancer patients. Paracetamol inhibits prostaglandin biosynthesis under some circumstances (e.g. fever), but not others. The difference from other NSAIDs is still under investigation ^4,5. The chemical structures of the two drugs are shown in Figure 1.

Fig.1. Chemical structures of Paracetamol and Tapentadol

The combination of Tapentadol and Paracetamol is used for moderate to severe acute painful conditions ⁶. Literature survey reveals many analytical methods for determination of Paracetamol such as UV Spectrophotometry ^7-9, HPLC ¹⁰, RP-HPLC ^{11, 12}, and Stability indicating methods ^13-15, from pharmaceutical preparations.

Tapentadol is not official in any Pharmacopoeia .Literature review revealed that limited report on analytical techniques for estimation of Tapentadol from Various biological Fluids^{16, 17}. A few methods have been reported estimation of Tapentadol by RP-HPLC in pharmaceutical dosage forms^18-20. However, to our knowledge there are no reported methods for Stability indicating simultaneous estimation of Tapentadol and Paracetamol in tablet dosage forms.

Therefore, attempts were made in this study to develop and validate a rapid stability indicating reverse-phase high performance liquid chromatographic method for the simultaneous estimation of Tapentadol and Paracetamol in their bulk and tablet dosage form with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time. The method was validated and found to be accurate, precise and reproducible.

EXPERIMENTAL:

Chemicals and Reagents:

Pure samples of Paracetamol and Tapentadol were kindly received as a gift samples from Rainbow Pharma Lab, Kukatpally, Hyderabad, Andhra Pradesh, India. Methanol and Water were of HPLC grade and collected from Merck Specialties private Limited, Mumbai, India. Sodium di hydrogen ortho phosphate is of analytical reagent grade, supplied by Fisher Scientific Chemicals.

Commercial Formulation:

Tapentadol and Paracetamol Tablets available in the market as Vorth®TP Plus in composition of Tapentadol(50mg), Paracetamol(325mg). The samples were properly checked for their manufacturing license numbers, batch numbers, production, expiry dates and stored properly.

Instrumentation:

Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC.

Other Apparatus: Electronic balance, Sonicator, Vacuum Degasser, 0.45µ membrane filter.

Chromatographic Conditions:

The mobile phase is a mixture of buffer (Sodium di hydrogen ortho phosphate, 0.1M, pH adjusted to 7 with ortho phosphoric acid) and methanol (60:40v/v) and was run in Isocratic mode at a flow rate of 0.8 ml/min through the column (Thermo Hypersil BDS C18, 250 mm × 4.6 mm and 5 μm) which is maintained at 40⁰c. The injection volume was 10 µL. The detection wavelength was set at 215 nm. Diluent used was Buffer and Methanol (1:1). The mobile phase was degassed prior to use and filtered through a 0.45μ membrane filter.

Standard Preparation:

Tapentadol (10.30mg) and Paracetamol (65mg) were accurately weighed and was placed in a 100 ml volumetric flask and dissolved using Diluent (Buffer and Methanol, 1:1) to get standard solution containing 100 μg/ml of Tapentadol and 650μg/ml of Paracetamol. This is treated as 100% standard concentration.

Sample Preparation:

A total of 10 tablets were weighed separately. Their average weights were determined. Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric flask. 50 ml of diluent (Buffer and Methanol, 1:1) was added and sonicated for 15 min. Then the volume was made up to mark with diluent and the solution was filtered through 0.45µ membrane filter and marked as sample stock solution. 5 ml was pipette out from the sample stock solution and transferred in to 25 ml volumetric flask and volume was made up to mark with diluent (Buffer and Methanol, 1:1), to give a concentration of 100 μg/ml of Tapentadol and 650μg/ml of Paracetamol. This is treated as 100% sample concentration.

RESULTS AND DISCUSSION:

Optimization of Chromatographic Conditions

The method was optimized to separate the drugs with good resolution, tailing factor and theoretical plates. Initially several attempts were made in achieving these conditions. The samples were run using different stationary phases like C18, C8 and also by changing the column lengths from 250 to 150 mm. Different mobile phase compositions like methanol: water, buffer : acetonitrile, buffer : methanol at various compositions and flow rates were tried. Mobile phases containing phosphate buffers with different pH(3.0–7.0) and using organic modifiers like acetonitrile and methanol in the mobile phase.

Fig. 2. Chromatogram of standard

A mobile phase system comprised of buffer (Sodium di hydrogen ortho phosphate,0.1M) and methanol (60:40 v/v) at flow rate of 0.8 ml/min allowing adequate separation of two drugs with good resolution, tailing factor and theoretical plates, using a C18 Thermo Hypersil BDS C18, 250 mm × 4.6 mm and 5 μm was found to be optimum. The photodiode-array (PDA) spectrum showed good response at 215 nm for two components. Therefore, this wavelength was used for simultaneous determination of drugs. In the optimized conditions the drugs were separated with a resolution of 11.32 and the retention times for Paracetamol and Tapentadol were found to be 2.39min and 4.6min respectively. Figure 2, 3 represents the chromatograms of standard and sample respectively.

System Suitability Tests:

It is the checking of a system, before or during analysis of samples, to ensure system performance. The chromatographic system used for analyses must pass the system suitability limits before sample analysis can commence. A standard solution containing 650μg/ml of Paracetamol and 100 μg/ml of Tapentadol was injected five times. The parameters measured were Peak area, Retention time, tailing factor, theoretical plates and resolution. The results were within the acceptable range indicating that the system was suitable for the analysis. Table 1 represents the system suitability parameters.

Fig.3. Chromatogram of sample

Table.1 System Suitability Parameters

PARAMETERS	PARACETAMOL	TAPENTADOL
Peak area	12553232	2144839
Retention time (min)	2.39	4.6
Tailing factor	1.58	1.35
Theoretical plates	3985	6425
Resolution		11.32

Method Validation:

The proposed method was validated with respect to linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness, as per International Conference on Harmonization (ICH) guidelines ²¹.

Linearity:

Linearity of the method was determined by constructing calibration curves. Standard solutions were prepared at five concentration levels from 50%,-150% of analyte concentrations (325-975 μg/mL for Paracetamol and 50-150 μg/mL for Tapentadol). The peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients. The linearity graph was constructed by plotting the Peak areas of the analytes (in Y-axis) versus analytes concentration (in X-axis). The calibration curves were linear in the range of 325-975 μg/mL for Paracetamol and 50,-150μg/mL for Tapentadol. The linear regression equations were y = 12070x + 52390(R²=0.999) for paracetamol and y = 21427x – 11550 (R² = 0.999) for Tapentadol. Table 2 represents the results of linearity. Figure.4 represents the linearity graphs of both the drugs.

TABLE.2 Linearity Results

Drug	Concentration Level (%)	Concentration (µg/mL)	Equation for regression line	R² value
PARACETAMOL	50-150	325-975	y = 12070x + 52390	0.999
TAPENTADOL	50-150	50-150	y = 21427x – 11550	0.999

For Paracetamol

For Tapentadol

Fig.4. Linearity Graphs

Precision:

Precision of an analytical method expresses the closeness of agreement between a series of measurements obtained from multiple sampling of same homogenous sample. Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was calculated which was found to be less than 2% for within a day (Intra – Day) and day to day (Intra -Day) variations, which proves that method is precise. Table 3 represents the results of precision.

TABLE.3 PRECISION RESULTS:

DRUG

% RSD

(Intra-Day)

%RSD (Inter-Day)

PARACETAMOL

0.58

0.90

TAPENTADOL

0.63

0.72

Accuracy:

To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre‐analyzed sample solution at three different levels 50%, 100% and 150%. The percentages of recoveries were calculated. Mean recoveries for Paracetamol and Tapentadol from the formulation was between 99%-100% and 100%- 102%, indicating that the developed method was accurate for the determination of Paracetamol and Tapentadol in pharmaceutical formulation. Table 4 represents the results of accuracy.

Specificity;

The specificity of an analytical method is the ability of the method to determine the analyte response in the presence of additional components such as impurities, degradation products. A blank was injected to demonstrate the absence of interference of blank with the elution of Paracetamol and Tapentadol. The specificity of the method was also evaluated to ensure there were no interference products resulting from forced degradation.Forced degradation studies were performed under five stress conditions (acid, base, water, oxidation and light).

Acidic degradation studies:

Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric to this 10ml of 0.1 N hydrochloric acid was added and refluxed for 30 min at 60⁰c, cooled and neutralized with 10ml 0.1 N sodium hydroxide then volume made up to mark with diluent (Buffer and Methanol, 1:1) and the solution was filtered through 0.45µ membrane filter. 5 ml was pipette out from the above solution and transferred in to 25 ml volumetric flask and volume made up to mark with diluent (Buffer and Methanol, 1:1). Figure.5 represents the chromatograms of acidic degradation.

Fig.5. Chromatogram of acidic degradation

Alkali degradation studies:

Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric to this 10ml of 0.1 N sodium hydroxide was added and refluxed for 30 min at 60⁰c, cooled and neutralized with 10ml 0.1 N hydrochloric acid then volume made up to mark with diluent (Buffer and Methanol, 1:1) and the solution was filtered through 0.45µ membrane filter. 5 ml was pipette out from the above solution and transferred in to 25 ml volumetric flask and volume made up to mark with diluent (Buffer and Methanol, 1:1). Figure.6 represents the chromatograms of basic degradation

Degradation with water:

Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric to this 10ml of Water was added and refluxed for 30 min at 60⁰c, cooled and then volume made up to mark with diluent (Buffer and Methanol, 1:1) and the solution was filtered through 0.45µ membrane filter. 5 ml was pipette out from the above solution and transferred in to 25 ml volumetric flask and volume made up to mark with diluent (Buffer and Methanol, 1:1). Figure.7 represents the chromatograms of water degradation.

Fig.6. Chromatogram of basic degradation

Oxidative degradation

Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric to this 10ml of 3% hydrogen peroxide was added and refluxed for 30 min at 60⁰c, cooled and then volume made up to mark with diluent (Buffer and Methanol, 1:1) and the solution was filtered through 0.45µ membrane filter. 5 ml was pipette out from the above solution and transferred in to 25 ml volumetric flask and volume made up to mark with diluent (Buffer and Methanol, 1:1). Figure.8 represents the chromatograms of oxidative degradation.

Fig.7. Chromatogram of water degradation

Fig.8. Chromatogram of oxidative degradation

Degradation in presence of light:

Degradation in presence of light was performed by taking powder of tablets equivalent to one tablet weight was weighed and were exposed to sunlight for 48 hrs. Then this exposed powder is transferred in to100 ml volumetric flask and then few ml of diluent was added to the volumetric flask and the sonicated for 15 min. After the sonication, volume made up to mark with diluent (Buffer and Methanol, 1:1) and the solution was filtered through 0.45µ membrane filter. 5 ml was pipette out from the above solution and transferred in to 25 ml volumetric flask and volume made up to mark with diluent (Buffer and Methanol, 1:1). Figure.9 represents the chromatograms of oxidative degradation.

After the stress assays, no changes in retention times of drug and no degradation peaks were observed. Therefore, this confirms the specificity of the method.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOQ refers to the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy. There are different approaches to determine the LOQ and LOD.

Typically, the concentration level that generates a signal-to noise(S/N) of 10 is regarded as the LOQ and the concentration level that generates S/N=3 is regarded as the LOD. The Limit of Detection (LOD) and Limit of Quantification (LOQ) for Paracetamol were found to be 0.9 μg/mL and 0.27μg/mL respectively. The Limit of Detection (LOD) and Limit of Quantification (LOQ) for Tapentadol were found to be 0.38μg/mL and 1.2μg/mL respectively. At the selected LOQ and LOD concentrations, S/N ratio for Paracetamol was found to be 4 and 16 respectively and for Tapentadol was found to be 5 and 12 respectively. This indicates that the method can be used for simultaneous detection and quantification of these drugs over a wide concentration range.

Fig.9. Chromatogram of light degradation

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. To evaluate the robustness of the developed RP-HPLC method, flow (±0.2ml/min) and temperature (±50c) variations were done. The effect of changes in the flow and temperature were studied. The method was found to be unaffected by flow and temperature variations. Table-5 represents the results of robustness.

Table.5 Robustness results

DRUG	PARAMETER	MODIFICATION	RETENTION TIME	TAILING FACTOR	PLATE COUNT
PARACETAMOL	FLOW (0.8ml/min)	0.6ml/min	3.181	1.54	5271
	FLOW (0.8ml/min)	1.2ml/min	1.9	1.50	3811
	TEMPETATURE (40⁰C)	35⁰C	2.39	1.6	3867
	TEMPETATURE (40⁰C)	45⁰C	2.40	1.6	4195
TAPENTADOL	FLOW(0.8ml/min)	0.6ml/min	6.09	1.39	7467
	FLOW(0.8ml/min)	1.2ml/min	3.75	1.31	6250
	TEMPERATURE (40⁰C)	35⁰C	4.66	1.37	6330
	TEMPERATURE (40⁰C)	45⁰C	4.64	1.32	7381

CONCLUSION:

A validated stability-indicating RP-HPLC method has been developed for determination of Tapentadol and Paracetamol in their bulk and combined tablet dosage forms. The results obtained by the stress degradation conditions of the drugs show that the method is specific and stability-indicating. The method was found to be simple, accurate, precise and sensitive. The method can be suitable for quality control of pharmaceutical preparations containing Tapentadol and Paracetamol either alone or in combination.

ACKNOWLEDGMENT:

The authors would like to thank Rinbow Pharma Lab, Kukatpally, Hyderabad, Andhra Pradesh, India for providing the gift samples and for providing the research facilities.

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21. ICH; Validation of analytical procedures; text and methodology Q2(R1)

Received on 23.09.2012 Modified on 01.10.2012

Asian J. Research Chem. 5(10): October, 2012; Page 1255-1261

DRUG	LABLE CLAIM (mg)	CONCENTRATION LEVEL (%)	AMOUNT ADDED (µg/ml)	AMOUNT RECOVERED (µg/ml)	% RECOVERY
PARACETAMOL	325	50	325	326.36	100.42
		100	650	651.56	100.24
		150	975	962.71	98.74
TAPENTADOL	50	50	50	50.69	101.38
		100	100	100.08	100.08
		150	150	153	102