VALIDATED STABILITY INDICATING LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF PARACETAMOL, TRAMADOL AND DICYCLOMINE IN TABLETS

VALIDATED STABILITY INDICATING LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF PARACETAMOL, TRAMADOL AND DICYCLOMINE IN TABLETS

Shivakumar Reddy L. ¹*, Prasad Reddy S. L. N ², Srinivas Reddy G. ³ and Surender Reddy L. ⁴

Department of Pharmaceutical Sciences ^{1, 3}, Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India.

Sanskruthi College of Pharmacy, Ghatkesar ², Hyderabad, Andhra Pradesh, India.

Department of Pharmaceutical Sciences, Osmania University ⁴, Hyderabad, Andhra Pradesh, India.

ABSTRACT: A new simple stability indicating reversed-phase liquid chromatographic method has been developed and validated for simultaneous estimation of Paracetamol, Tramadol and Dicyclomine in combined pharmaceutical dosage form. An Agilent Zorbax SB C18 (250mmx4.6mm, 5microns) column with mobile phase containing ammonium acetate: acetonitrile (700:300 v/v) was used. The flow rate was maintained at 1.0 mL/min, column temperature was 30°C and effluents were monitored by using a photodiode array detector at 233 nm. The retention times of Paracetamol, Tramadol and Dicyclomine were found to be 2.331, 3.182 and 7.650 min, respectively. Correlation co-efficient for Paracetamol, Tramadol and Dicyclomine were found to be 0.99, 0.99 and 0.99 respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity, and robustness. Recovery of Paracetamol, Tramadol and Dicyclomine in formulations was found to be in a range of 98-102%, 98-102% and 98-102% respectively. Paracetamol, Tramadol and Dicyclomine dosage form is also subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved and peak purity test results confirmed that Paracetamol, Tramadol and Dicyclomine peaks were homogenous and pure in all stress samples, thus proving stability-indicating power of the method. Due to its simplicity, rapidness and high precision, this method can be applied for regular analysis.

Paracetamol, Tramadol, Dicyclomine, Liquid chromatography,

Method validation

INTRODUCTION: Paracetamol (INN) or acetaminophen (USAN) chemically named N-acetyl-p-aminophenol, is a widely used over-the-counter analgesic (pain reliever) and antipyretic (fever reducer).  Paracetamol is classified as a mild analgesic. It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies.

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. Though Paracetamol is used to treat inflammatory pain, it is not generally classified as an NSAID because it exhibits only weak anti-inflammatory activity.

The onset of analgesia is approximately 11–29.5 minutes after oral administration of Paracetamol, and its half-life is 1–4 hours. Tramadol is a centrally acting opiod analgesic used to treat moderate to moderately severe pain. The chemical name for Tramadol hydrochloride is (±) cis-2-[(dimethylamino) methyl]-1-(3methoxyphenyl) cyclohexanol hydrochloride. Structurally, Tramadol closely resembles a stripped down version of codeine. Both codeine and Tramadol share the 3-methyl ether group, and both compounds are metabolized along the same hepatic pathway and mechanism to the stronger opioid, phenol agonist analogs. For Codeine, this is Morphine, and for Tramadol, it is the O-desmethyltramadol. The drug has a wide range of applications, including treatment of rheumatoid arthritis, restless legs syndrome, motor neuron disease and fibromyalgia. Dicyclomine, also known as Dicycloverine is an anticholinergic that blocks muscarinic receptors.

It is chemically (bicyclohexyl)-1-carboxylic acid, 2-(diethylamino) ethyl ester, hydrochloride. Dicyclomine is used to treat intestinal hypermotility and the symptoms of irritable bowel syndrome (IBS) (also known as spastic colon). It relieves muscle spasms and cramping in the gastrointestinal tract by blocking the activity of acetylcholine on cholinergic (or muscarinic) receptors on the surface of muscle cells. It is a smooth muscle relaxant ^1-2. Structures of Paracetamol, Tramadol and Dicyclomine are depicted in Figure 1.

A literature survey revealed few liquid chromatography (LC) assay methods that have been reported for the determination of Paracetamol and Tramadol in bulk dug and pharmaceutical dosage forms, but there are no reported methods for simultaneous estimation of Paracetamol, Tramadol and Dicyclomine in combined pharmaceutical dosage forms ^3-23.

The present International Conference on Harmonization (ICH) drug stability guidelines  suggest that stress studies should be conducted on the drug product to establish its inherent stability characteristics, and the analytical method should able to separate all degradation impurities formed under stress studies to prove its stability-indicating power. In order to monitor possible changes to a product over time, the applied analytical chromatogra­phic method must be stability-indicating. The best case for testing the suitability of a method is using real-time stability samples containing all relevant degra­dation products that might occur. But due to product development timelines, process characteristics, excipients, and other environmental factors, a forced degradation study (stress test) can serve as an alternative.

In a typical study, relevant stress condi­tions are light, heat, humidity, hydrolysis (acid / base influence) and oxidation or even a combination of described parame­ters. If it is necessary to form degra­dation products, the strength of stress conditions can vary due to the chemical structure of the drug substance, the kind of drug product, and product specific storage requirements. An individual pro­gram has to be set up in order to reach a target degradation of 5 to 20%.

A higher level of degradation will be out of the scope of product stability requirements and therefore unrealistic. The scope of the test is to generate degradation products in order to facilitate a method development for determination of the relevant products.

Therefore, samples will be stressed in a solid form and/or in solution. Typi­cally, stress tests are carried out on one batch of material. For drug products the placebo should be stressed in a similar way in order to exclude those impurities that are not degradation products (e.g. impurities arising from excipients). The stability studies were determined by applying the physical stress (acid, base, peroxide, heat and light) to the product ^24-33.

The aim of the present work is to focus on the development of an efficient stability indicating liquid chromatographic method for simultaneous estimation of Paracetamol, Tramadol and Dicyclomine in combined pharmaceutical dosage form such as Tablets in presence of its excipients and degradation products in a short chromatographic run.

The present work concerns the method development, method validation and forced degradation studies of Paracetamol, Tramadol and Dicyclomine in combined pharmaceutical dosage form. The developed Liquid Chromatographic method was validated with respect to specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy and robustness. Forced degradation studies were performed on the placebo and drug products to show the stability-indicating nature of the method. These studies were performed in accordance with established ICH guidelines.

MATERIALS AND METHODS:

Instrumentation:

Samples were analyzed on Waters alliance 2695 HPLC system (Waters Corporation, Milford, MA) equipped with a with binary HPLC pump, Waters 2998 PDA detector and  Waters Empower2 software. The separation was achieved on Agilent Zorbax SB C18 (250mmx4.6mm, 5microns) column.

Chemicals and Reagents:

Paracetamol, Tramadol and Dicyclomine standards were supplied by Dr. Reddy’s Laboratories Ltd., Hyderabad. Acetonitrile of HPLC grade was purchased from E. Merck (India) Ltd., Mumbai.  Ammonium acetate of AR grade was obtained from S.D. Fine Chemicals Ltd., Mumbai and milli Q water. Paracetamol, Tramadol and Dicyclomine tablets (Ultraspas: Aristo and Topspas: psychotropic’s India) were procured from market.

 HPLC Conditions:

The mobile phase consisting of Ammonium acetate: Acetonitrile (HPLC grade) were filtered through 0.45 µm membrane filter before use, degassed and were pumped from the solvent reservoir in the ratio of 700:300 v/v into the column at a flow rate of 1.0 ml/min. The column temperature was maintained at 30°C. The detection was monitored at 233 nm and the run time was 10 minutes. The volume of injection loop was 20 µl prior to injection of the drug solution.

 Preparation of standard solution:

Take accurately 75 mg of Paracetamol, 650 mg of Tramadol and 40 mg of Dicyclomine HCl working standard and transfer it into 50 ml volumetric flask dissolve and diluted to volume with Mobile Phase. From the above solution take 5 ml into 25 ml volumetric flask make up the volume with Mobile Phase. (Concentration of Paracetamol : 0.3 mg/ml, Concentartion of Tramadol : 2.6 mg/ml, Concentration of Dicyclomine: 0.16 mg/ml).

 Preparation of sample (drugs from marketed formulations) solution:

Take accurately sample powder equivalent to one and transfer it into 100 ml volumetric flask dissolve and diluted to volume with mobile phase. From the above solution take 5 ml into 25 ml volumetric flask make up the volume with Mobile Phase. (Concentration of Paracetamol: 0.3 mg/ml, Concentartion of Tramadol: 2.6 mg/ml, Concentration of Dicyclomine: 0.16 mg/ml).

Forced degradation studies:

Forced degradation studies were performed at a 1617.90 mg/mL concentration of Paracetamol, Tramadol and Dicyclomine in tablets to provide an indication of the stability-indicating property and specificity of the proposed method. A peak purity test was conducted for Paracetamol, Tramadol and Dicyclomine peaks by using a PDA detector on stress samples. All solutions used in forced degradation studies were prepared by dissolving the drug product in a small volume of stressing agents. After degradation, these solutions were diluted with mobile phase to yield a stated concentration approximately. Conditions employed for performing the stress studies are described below.

 Acid degradation:

Tablet powder equivalent to 1617.90 mg was accurately weighed and dissolved in 5 ml of mobile phase, 5 ml of 5 N HCl was added and the mixture was kept at 70⁰C for 5 min. The solution was brought to ambient temperature, neutralized by the addition of 5 ml of 5 N NaOH and diluted to 25 ml with mobile phase.

To prepare the blank, 5 mL of 5 N HCl and 5 mL of 5 N NaOH were diluted to 25 mL with mobile phase.

 Base degradation:

Tablet powder equivalent to 1617.90 was accurately weighed and dissolved in 5 ml of mobile phase, 5 ml of 5N NaOH was added and the mixture was kept at 70⁰C for 5 min. The solution was brought to ambient temperature, neutralized by the addition of 5 ml 5 N HCl and diluted to 25 mL with mobile phase. To prepare the blank, 5 mL of 5 N NaOH and 5 mL of 5 N HCl were diluted to 25 mL with mobile phase.

Oxidation degradation:

Tablet powder equivalent to 1617.90 mg was accurately weighed and dissolved in 5 mL of mobile phase, 5 mL of 3% hydrogen peroxide was added and the mixture was kept at 70⁰C for 10 min. The solution was brought to ambient temperature and diluted to 25 mL with mobile phase.

To prepare the blank, 5 ml of 3% hydrogen peroxide was diluted to 25 mL with mobile phase.

 Thermal degradation:

Tablet powder equivalent to 1617.90 was stored at 105⁰C for 9 h, dissolved and diluted to 25 mL with mobile phase.

 Photolytic degradation:                       

The susceptibility of the drug product to the light was studied; Tablet powder for photo stability testing was placed in a photo stability chamber and exposed to a white florescent lamp with an overall illumination of 1.2 million lux hours and near UV radiation with an overall illumination of 200 watt/m²/h at 25⁰C. Following removal from the photo stability chamber, the sample was prepared for analysis as previously described.

 RESULTS:

Method Development:

The analytical procedure for the estimation of Paracetamol, Tramadol and Dicyclomine in marketed formulation was optimized with a view to develop a simple, precise and accurate assay method. Agilent Eclipse XDB (4.6*150 mm*3.5 mic), Agilent Zorbax SB C18 (4.6*250mm*5 mic) and Inertsil-ODS (4.6*250 mm*5 mic) were used to provide an efficient separation but appropriate chromatographic separation was achieved on Agilent Zorbax SB C18 (4.6*250mm*5 mic).

Various mobile phase systems were prepared and used to provide an appropriate chromatographic separation, but the proposed mobile phase containing Ammonium acetate: acetonitrile in the ratio of 700:300 (v/v) gave a better resolution. Using UV-visible PDA detector at 233 nm carried out the detection. Amongst the several flow rates tested, the flow rate of 1 ml/min was the best suited for both the drugs with respect to location and resolution of peaks. The retention time of Paracetamol, Tramadol and Dicyclomine was found to be 2.331, 3.182 and 7.650 min respectively. The chromatograms of standard and sample solution of Paracetamol, Tramadol and Dicyclomine were shown in Figure 2. The asymmetry factor of Paracetamol, Tramadol and Dicyclomine was 1.391, 1.410 and 1.246 found to be respectively, which indicates symmetrical nature of the peak.

The USP resolution of 5.884 was achieved between Paracetamol, Tramadol and 16.232 was achieved between Paracetamol and Dicyclomine. The USP plate count of Paracetamol, Tramadol and Dicyclomine was 7268, 5094 and 7277 found to be respectively, which indicates column efficiency for separation. System suitability parameters such as Peak asymmetry, Resolution and Number of theoretical plates are meeting ICH requirements.   The percentage label claim of individual drugs found in formulations was calculated. The results of analysis shows that the amounts of drugs estimated were in good agreement with the label claim of the formulations ^22-23.

Method Validation:

System Suitability Studies:

System suitability was determined before sample analysis from duplicate injections of the standard solutions of Paracetamol, Tramadol and Dicyclomine. The column efficiency, resolution and peak asymmetry were calculated for the standard solutions. The USP resolution of 5.884 was achieved between Paracetamol, Tramadol and 16.232 was achieved between Paracetamol and Dicyclomine.  USP tailing (Peak Asymmetry) for Paracetamol, Tramadol and Dicyclomine were found to be 1.391, 1.410 and 1.246 respectively. Number of theoretical plates (USP plate count) for Paracetamol, Tramadol and Dicyclomine were found to be 7268, 5094 and 7277 respectively.

The values obtained demonstrated the suitability of the system for the analysis of this drug combinations, system suitability parameters may fall within ± 3 % standard deviation range during routine performance of the method ^24-25.

 Specificity: Specificity is the ability to assess unequivocally the analyte in presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. Placebo interference was evaluated by analyzing the placebo prepared by the test method. No peak due to placebo was detected at the retention time of Paracetamol, Tramadol and Dicyclomine. The specificity of the developed method was also conducted in presence of its degradation products.

 Precision: The precision of method was verified by repeatability and intermediate precision. Repeatability was checked by injecting six individual sample preparations of Paracetamol, Tramadol and Dicyclomine tablets. Percent relative standard deviation (RSD) of the area for each drug was calculated. The intermediate precision of the method was also evaluated using different analysts and different instruments and performing the analysis on different days. The results of precision study are provided in Table 1.

  TABLE 1: PRECISION STUDIES OF PARACETAMOL, TRAMADOL AND DICYCLOMINE

 Accuracy: The accuracy of the method was determined by recovery experiments. The recovery studies were evaluated in triplicate using three concentration levels 50%, 100% and 150%. The percentage recovery data was obtained, added recoveries of standard drugs were found to be accurate (Table 2, 3 and 4).

 TABLE 2: ACCURACY FOR PARACETAMOL

 TABLE 3: ACCURACY FOR TRAMADOL

 TABLE 4: ACCURACY FOR DICYCLOMINE

 Linearity and Range:

The linearity of the method was determined at five concentration levels (50%, 75%, 100%, 125% and 150%). Linearity test solutions were prepared by diluting the stock solutions to the required concentrations. The calibration curves were plotted between the responses of peak area versus concentration of analyte. The slope and intercept value for calibration curve was y = 43900 x (r2=0.99) for Paracetamol, y = 43363 x (r2=0.99) for Tramadol and y = 12428 x (r2=0.99) for Dicyclomine. The result (Table 5) shows that an excellent correlation exists between areas and concentration of drugs within the concentration range. Calibration curves are presented in Figure 3.

 TABLE 5: LINEARITY OF PARACETAMOL, TRAMADOL AND DICYCLOMINE

FIGURE 3: LINEARITY CURVES (A) PARACETAMOL (B) TRAMADOL (C) DICYCLOMINE

 Limit of detection & Limit of quantification (LOD & LOQ):

Limit of quantification and detection were predicted by plotting linearity curve for different nominal concentrations of Paracetamol, Tramadol and Dicyclomine (Table 5). Relative standard deviation (σ) method was applied, the LOQ and LOD values were predicted using following formulas. Precision was established at these predicted levels.

(a) LOQ = 10 σ / S

(b) LOD = 3.3 σ / S

Where σ = residual standard deviation of response

S = slope of the calibration curve.

LOQ and LOD values for Paracetamol, Tramadol and Dicyclomine were found to be 2.922, 9.740; 2.846, 9.486 and 2.759, 9.195 respectively.

Robustness:

Robustness of the method was determined by making slight changes in the chromatographic conditions and system suitability parameters for Paracetamol, Tramadol and Dicyclomine standard and the resolution, USP Tailing and USP Plate count were recorded. The variables evaluated in the study were column temperature (±5⁰C), flow rate (±0.2 mL/min).  It was observed that there were no marked changes in the chromatograms, which demonstrates that the method developed is rugged, and robust (Table 6, 7 and 8).

TABLE 6: ROBUSTNESS-PARACETAMOL

 TABLE 7:    ROBUSTNESS-TRAMADOL

TABLE 8:  ROBUSTNESS-DICYCLOMINE            

Forced Degradation Studies:

Based on the results of the stress studies, the degradation behavior of Paracetamol, Tramadol and Dicyclomine is as follows.

 Acid degradation:

Paracetamol, Tramadol and Dicyclomine were undergoing degradation in 5 N HCl at 70⁰C for 10 min moderately. The impurities formed during this study are well separated from main drug peaks and mass balance is found to be in acceptable limit. Peak purity of drugs was also matches (Table 9, Figure 4(A).

 Base degradation:

Paracetamol, Tramadol and Dicyclomine were undergoing degradation in 5 N NaOH at 70⁰C for 5 min moderately. The degradation peaks are well separated from main drug peaks and well resolved. Mass balance is found to be in acceptable limit. Peak purity of drugs was also matches (Table 9, Figure 4(B).

 Oxidation degradation:

Paracetamol, Tramadol and Dicyclomine were undergoing degradation in 3% hydrogen peroxide at 70⁰C for 10 min. The degradation peaks were well resolved from main drug peaks. Mass balance is found to be in acceptable limit. Peak purity of drugs was also matches (Table 9, Figure 4 (C).

 Thermal degradation

Paracetamol, Tramadol and Dicyclomine were undergoing degradation upon thermal exposure moderately. Degradation peaks formed were well resolved from main drug peaks. Mass balance is found to be in acceptable limit. Peak purity of drugs was also matches (Table 9, Figure 4 (D).

 Photolytic degradation

Upon subjecting the Paracetamol, Tramadol and Dicyclomine sample to both UV and visible light, only partial degradation of was observed. Testing of a placebo containing preserva­tive leads to formation of number of different ­im­purities with respect to an unstressed placebo. The amount of preservative decreased mainly by influence of oxidation, light and acid. Mass balance of preservative shows almost 100%. The active ingredients remain almost stable within tested period and mass balance matches (Table 9, Figure 4(E).

 TABLE 9:  STRESS STUDIES (A) PARACETAMOL (B) TRAMADOL (C) DICYCLOMINE

(A)

(B)

FIGURE 4: TYPICAL CHROMATOGRAMS (A) ACID DEGRADATION (B) ALKALI DEGRADATION (C) OXIDATIVE DEGRADATION (D) THERMAL DEGRADATION (E) PHOTOLYTIC DEGRADATION

 DISCUSSION: System suitability parameters such as retention time, resolution, tailing and plate count were shown uniformity and %RSD was less than 1 so we can say system is suitable for analysis method specificity was concluded by Figure 2.  Those figures are Paracetamol, Tramadol and Dicyclomine standard chromatogram and other one is formulation they were not observed in placebo and excipients peaks interference with standard and analytic peak so it proves method is selective. The result given in Table 2 says that the method precision is passed for Paracetamol, Tramadol and Dicyclomine studies. The method accuracy was evaluated by recovery studies. Paracetamol, Tramadol and Dicyclomine recovery is meeting the ICH requirements and USP requirements 97%-103% and 98-102% respectively.

Percentage RSD is also very low so the method is accurate as shown in Table 3, 4 and 5. Linearity calibration curve was plotted for Paracetamol, Tramadol and Dicyclomine, the graph of concentration versus peak areas to construct the linear regression equation and to calculate the value of correlation coefficient. The slope and intercept value for calibration curve was y = 43900 x (r²=0.99) for Paracetamol, y = 43363 x (r²=0.99) for Tramadol and y = 12428 x (r²=0.99) for Dicyclomine. Method robustness results were also studied. Forced degradation studies of Paracetamol, Tramadol and Dicyclomine proves the method is also stability indicating and can be used for stability monitoring of the dosage forms to establish the re-test period.

 CONCLUSION: The proposed HPLC method for the simultaneous estimation of Paracetamol, Tramadol and Dicyclomine in pharmaceutical dosage forms was found to be simple, sensitive, precise, accurate, linear, robust and rugged during validation. Further this method is stability indicating and can be used for routine analysis of production samples. Hence this method can easily and conveniently adopt for routine quality control of Paracetamol, Tramadol and Dicyclomine in pure and its pharmaceutical dosage forms.

 ACKNOWLEDGEMENT: Authors are thankful to Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Hyderabad and Rainbow Pharma training lab, Kukatpally, for providing instruments and analytical support. Authors are also thankful to Dr. Reddy’s Laboratories Ltd. for providing Paracetamol, Tramadol and Dicyclomine standards as gift samples.

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    How to cite this article:

    Reddy LS, Prasad Reddy SLN, Srinivas Reddy G and Surender Reddy L: Validated Stability Indicating Liquid Chromatographic Method for Simultaneous Estimation of Paracetamol, Tramadol and Dicyclomine in Tablets. Int J Pharm Sci Res 2015; 6(3): 1230-40.doi: 10.13040/IJPSR.0975-8232.6(3).1230-40.

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