Aceclofenac (ACO) is 2-[2-[2-[(2,6diclorophenyl) amino] phenyl] acetyl] oxyacetic acid and non-steroidal anti inflammatory drug [1]. Aceclofenac is used for the relief of pain and inflammation in rheumatoid arthritis, osteoarthritis. Paracetamol (PCM) is N-(4 hydroxyphenyl) acetamide [2] is a potent analgesic and anti-pyretic drug,Tizanidine (TZN) is 5-choloro N(4,5dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4-amine,used as a muscle relaxant [3]. It used to treat the spasms, cramping and tightness of muscles etc. (Figure 1).
A tablet dosage form containing all the three (ACO 100 mg, PCM 500 mg and TZN 2 mg) is commercially available and used as non-steroidal anti-inflammatory, analgesic, muscle relaxant. ACO, PCM and TZN are official in Indian pharmacopoeia. ACO has been analysed separately and in combination with other drugs by UV [4-7] and HPLC [8-12]. PCM has been analysed separately and combination with other drug by UV [13-15], HPLC [16], RP-HPLC [17]. TZN has been analysed separately and combination with other drug by UV [18-20], RP-HPLC [21-22], HPTLC [23-24] and spectrophotometric method has been reported for ACO and TZN [25]. Spectrophotometric method has been reported for estimation of PCM, NIMS (Nimesulide) and TZN [26]. However no spectrophotometric method for simultaneous analysis of ACO, PCM and TZN in combination dosage form has been reported yet, hence the present study is essential to develop simultaneous estimation method for all three drugs in tablet formulation.
Experimental
Reagents and chemicals
The ACO, PCM and TZN reference standards were obtained from Micro labs Pvt. Ltd. Bangalore, Karnataka and Excel Parma Pvt. Ltd, Mehsana, Gujarath. Pharmaceutical dosage forms (Zerodol MRR) containing 500 mg PCM, 100 mg ACO and 2 mg TZN were obtained commercially (Manufactured by IPCA Laboratories Ltd. Mumbai). Acetonitrile and Methanol for chromatography were purchased from Merck and S.D. fine chemicals. HPLC Grade water from Milli-Q Gradient A10 (Milli-pore), were used to prepare the solutions for the UV spectrophotometric experiments.
Apparatus / Instrumentation and analytical conditions
Chromatographic analysis was performed using Shi-madzu LC-20AT HPLC system equipped with LC-20AT pump and SPD-M20A detector, Chromatography was performed on Phenomenex Luna C18 column (250 × 4.6 mm i.d, 5µ) maintained at 40ºC. The mobile phase composition was Methanol: Water 90:10 v/v (Binary flow) which was pumped at flow rate of 1 mL/min without splitter. The mobile phase was prepared freshly before use and filtered through 0.45 µm membrane filter (Milli-pore) and degassed by ultra sonication bath before use .The auto sampler temperature was set at 10ºC. The wavelength of the
*Corresponding author: Chandrashekhar Narajji, Dept of Quality Assurance, Acharya & B.M Reddy College of Pharmacy, Soldevanahali, Bengaluru -560 090, India, E-mail: [email protected]
Received July 13, 2011; Accepted October 03, 2011; Published October 07, 2011
Citation: Chandrashekhar N, Patel HR, Karvekar MD, Suresh BAR (2011) Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC Method. J Anal Bioanal Techniques 2:123. doi:10.4172/2155-9872.1000123
AbstractA rapid and sensitive RP-HPLC method with UV detection and UV spectrophotometric method for routine
Pharmaceutical quality control of aceclofenac, paracetamol and tizanidine in tablets formulation was developed. Chromatography was performed by using Phenomenex-Luna C18 (250 x 4.6 mm, 5µ) with a mobile phase containing Methanol: Water (90:10v/v), flow rate 1mL/min, wavelength at 256 nm. Linearity observed over the concentration range between 5-30µg/mL, 10-60µg/mL and 2-12µg/mL for aceclofenac, paracetamol and tizanidine respectively. The UV spectrophotometric method was performed at 277, 248 and 323 nm by derivative spectrophotometry with simultaneous equation method. The entire three drugs obey Beer’s law in the concentration range between 5-30 µg/mL, 2-16 µg/mL and 2-30 µg/mL respectively. The proposed methods were simple, rapid, precise, accurate and sensitive and can be used for routine quality control in pharmaceuticals.
Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC MethodChandrashekhar Narajji1*, Hardik R Patel1, Manohar D Karvekar2 and Suresh Babu AR3
1Dept of Quality Assurance, Acharya & B.M Reddy College of Pharmacy, Soldevanahali, Bengaluru -560 090, India2Dept of Pharmaceutical Chemistry, Krupanidhi College of Pharmacy, Bengaluru- 560 035, India3G7 Synergon Private Limited, Bengaluru– 560054, India
HN
Cl O
O
COOH
Cl
N
S
N
NH
Cl
N
NH
. HCl HN
OHO
Figure 1: Chemical structure of (a) ACO (b) TZN and (c) PCM.
Journal ofAnalytical & Bioanalytical TechniquesJo
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ical & Bioanalytical Techniques
ISSN: 2155-9872
Volume 2 • Issue 4 • 1000123J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal
Citation: Chandrashekhar N, Patel HR, Karvekar MD, Suresh BAR (2011) Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC Method. J Anal Bioanal Techniques 2:123. doi:10.4172/2155-9872.1000123
Page 2 of 5
UV detector was set to 256 nm, The UV spectrophotometric method was performed on a UV/Visible double beam spectrophotometer (Shimadzu model-1700) at 277 nm, 248 nm and 323 nm using 1cm Quartz cells.
Preparation of the standard solutionFor the HPLC method, ACO, PCM and TZN (10 mg each) were
weighed accurately and transferred to separate 100 mL volumetric flasks. All drugs were dissolved in 100 mL methanol to prepare standard stock solution of 100μg/ mL. For the UV spectrophotometric method, ACO, PCM and TZN (10 mg each ) were weighed and transferred to 100 mL volumetric flask, dissolved and made up to the volume-using methanol. Accurately pipette out 10ml of above solution separately into 100 ml standard flask and the volume was made up to 100 ml using methanol to get a concentration of 100µg/ml of ACO, PCM and TZN respectively.
Preparation of the sample solutionsFor both UV and HPLC analysis of the tablet dosage form, twenty
tablets of Zerodol MRR were weighed individually and their average weight was determined. The tablets were then crushed to a fine powder and powder equivalent to the weight of one tablet was transferred to a 100 mL volumetric flask and dissolved in 50 mL methanol. The solution was shaken vigorously for 15 min and filtered through Whatman #41 filter paper.
Construction of calibration plotsSolutions of ACO, PCM and TZN drugs having different
concentrations were prepared by dilution of the standard solutions. These solutions (20μL) were chromatographed and the peak areas were measured. Peak areas were then plotted against the respective concentrations for ACO, PCM and TZN. From the plots it was found that the linear range for ACO, PCM and TZN was between 5-30, 10-60 and 2-12μg/mL for HPLC method whereas for UV method was between 5-30, 2-16 and 2-30µg/mL.
Method ValidationThe methods were validated according to International Conference
on Harmonization (ICH) guidelines for validation of analytical procedures [27]. The System suitability test was carried through for both the methods evaluating theoretical plates and asymmetry.
LinearityThe calibration curve was obtained with five concentrations of the
standard solution in the range 5 to 30, 10 to 60 and 2 to 12μg/mL for HPLC method and for UV spectrophotometric method the linearity range was optimized with 5 to 30, 2 to 16 and 2 to 30µg/mL for ACO, PCM and TZN respectively (Figure 2-4).
Figure 2: Standard calibration curve for Aceclofenac by RP-HPLC.
Figure 3: Standard calibration curve for Paracetamol by RP- HPLC.
Figure 4: Standard calibration curve for Tizanidine by RP-HPLC.
Figure 5: Typical chromatograms at 256 nm of sample (A) and standard (B) solution by RP- HPLC.
*Average of five readingsTable 1: Result of assay by HPLC and UV method.
Volume 2 • Issue 4 • 1000123J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal
Citation: Chandrashekhar N, Patel HR, Karvekar MD, Suresh BAR (2011) Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC Method. J Anal Bioanal Techniques 2:123. doi:10.4172/2155-9872.1000123
Page 3 of 5
PrecisionThe assay precision was carried out by repeatability (intra-day)
and intermediate precision (inter-day). Repeatability was evaluated by assaying sample solutions of three different concentrations of ACO, PCM and TZN (5, 10 and 15µg/mL, 10, 20 and 30µg/mL, 6, 8 and 10µg/mL for HPLC method and 5, 10 and 15µg/mL, 6, 8 and 10µg/mL, 5, 10 and 15 µg/mL for UV spectrophotometric method) were prepared and assayed at same concentration and during the same day. The Percentage relative standard deviation (% RSD) values of peak area for repeated injections of ACO, PCM and TZN were found to be 0.08 %, 0.17%, 0.023 % and 0.023%, 0.014 %, 0.027% respectively. Intermediate precision of the method was checked by repeating studies on three different days. Percentage relative standard deviation (%RSD) was found to be less than acceptable limit of 2% for within a day and day to day variations, which proves that method is precise.
Accuracy
The accuracy of an analytical method is the closeness of the test results
obtained by the method to the true value. For the spectrophotometric method, the accuracy was determined by recovery of known amounts of ACO, PCM and TZN reference standard added to the samples at the beginning of the process. The accuracy was assessed from five replicate determinations of three concentration levels and the absolute means obtained were shown in Table 3, and it is evident that the method is accurate within the desired range. For the HPLC method, the accuracy was determined by the assay of three concentrations of the sample solution in triplicate and the absolute means obtained were shown in Table 3.
Specificity
The specificity of the HPLC and UV spectrophotometric methods was determined. This parameter was performed to assess and ensure that the impurities, degraded products and diluents do not affect the samples analyzed. ACO, PCM and TZN were injected into the system and chromatograms are recorded. For the UV spectrophotometric method, the specificity was obtained by the absorption spectra of the
*Average of three readingsTable 2: Validation parameter of HPLC and UV Method.
Drug
HPLC METHOD UV METHOD
Amount of sample (µg)
Amount of standard
(µg)
Total conc.found(µg)
% Recovery±%RSD*
Amount of sample (µg)
Amount of standard
(µg)
Total conc.found(µg)
% Recovery±%RSD*
ACO
8 6 14.02 100.04±0.34 10 10 19.8 99.92±0.87
8 8 16.06 100.08±0.74 10 12 21.05 101.15±0.15
8 10 18.15 101.53±0.69 10 14 24.3 101.38±0.84
PCM
40 5 45.03 101.79±0.79 50 10 59.9 99.13±0.07
40 10 50.17 100.30±0.47 50 12 62.6 99.09±0.97
40 15 55.05 100.04±0.34 50 14 63.4 98.94±0.62
TZN
0.16 1 1.15 99.75±0.32 0.2 10 9.8 100.73±1.00
0.16 2 2.14 99.12±0.89 0.2 12 12.09 99.77±0.50
0.16 3 3.13 99.06±0.12 0.2 14 13.8 99.90±0.95
*Average of three readingsTable 3: Result of recovery study.
Volume 2 • Issue 4 • 1000123J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal
Citation: Chandrashekhar N, Patel HR, Karvekar MD, Suresh BAR (2011) Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC Method. J Anal Bioanal Techniques 2:123. doi:10.4172/2155-9872.1000123
Page 4 of 5
ACO, PCM and TZN reference standard 10µg/mL and by the use of the simulated sample of excipients.
Robustness
The effect of small, deliberate variation of the analytical conditions on the peak areas and retention time of the ACO, PCM and TZN were examined, such as small changes in the percentage (±5%) in the mobile phase and flow rate (±0.1mL/min) [28]. The robustness of the UV spectrophotometric method was determined by small changes in the solvent lot.
Limit of detection (LOD) and quantification (LOQ)
The LOD and LOQ of ACO, PCM and TZN by proposed methods were determined using calibration standards. LOD and LOQ of the drugs were calculated as 3.3σ/S and 10σ/S, respectively, where σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot and showed in Table 2.
Results and DiscussionHPLC method
The mobile phase selection was based on peak parameters (symmetry, tailing), run time, ease of preparation and cost. Figure 5 shows a typical chromatogram which was obtained from the analysis of a standard and sample of ACO, PCM and TZN using the proposed method (Table 1) and were eluted forming a symmetrical peak. The retention time observed (1.87, 2.85 and 3.38 min) allowed a rapid determination of the drug, which is important for routine analysis. The calibration curves for ACO, PCM and TZN were constructed by plotting peak area verses concentration and showed good linearity within 5-30, 10-60 and 2-12µg/ mL range. The representative linear equation was y = 18802 x + 8890, y = 99382x + 48412 and y = 65853x + 3314 with a correlation coefficient (r2 = 0.9999) highly significant for the method (Table 2). LOD and LOQ were found to be 0.0194, 0.2246, 0.111µg/mL and 5, 2, 10µg/mL respectively, indicating high method sensitivity. The method precision was determined by repeatability (intra-day) and intermediate precision (inter-day) and was expressed as RSD (%) of a series of measurements. The result obtained showed RSD of 0.08, 0.17 and 0.023% indicating good precision (intra-day). Inter-day variability was calculated in three different days and it showed a mean RSD of 0.35, 0.54 and 0.047% respectively. The accuracy of the method was determined and the mean recovery was found to be 101.68, 100.55 and 100.32%, indicating an agreement between the true value and the found value (Table 3). The HPLC method is specific to the evaluation of ACO, PCM and TZN in tablets. No interfering peaks were observed when simulated samples of excipients were added in the sample solutions.
The method was found to be robust when the mobile phase and flow rate were varied.
UV Spectrophotometric method
The proposed first derivative spectrophotometric method allowed a rapid and accessible quantitation of ACO, PCM and TZN in tablets without any time consuming sample preparation. Moreover, the spectrophotometric method involved simple instrumentation compared with other instrumental techniques. The absorption spectra of ACO, PCM and TZN showed λmax was 277 nm, 248 nm and 323 nm, which was the wavelength used (Figure 3). The calibration curves were constructed in the range of expected concentrations (5-30, 2-16 and 2-30µg/mL). The representative equation analysis was y = 0.033x + 0.006, y = 0.092x - 0.005 and y = 0.0189x + 0.0296 with a correlation coefficient of 0.9999 (Table 2). LOD and LOQ were found to be 0.81, 0.21, 0.29µg/mL and 1.4, 0.71, 0.58µg/mL respectively, showing that the experimental values obtained for the determination of ACO, PCM and TZN in the samples indicated a satisfactory intra-day variability (RSD of 0.023, 0.014 and 0.027%) and inter-day variability (R.S.D. of 0.047, 0.023 and 0.029%). A good accuracy of the method was verified with a mean recovery of 99.78, 99.84 and 99.70% (Table 3). Finally, the method showed to be specific for the determination of ACO, PCM and TZN in tablets.
Comparison between HPLC, UV Spectrophotometric and the official method
The HPLC method and the UV method developed and validated for the analysis of ACO, PCM and TZN in tablets were found to be reliable, simple, fast, precise, accurate and sensitive. Results of UV spectrophotometric method showed no significant difference from those obtained with the method of HPLC and the official method (P > 0.01) but when LOD, LOQ and assay parameter of both the methods were compared and HPLC method was found to be more sensitive than the UV method. Hence these HPLC methods can be used for simultaneous estimation of ACO, PCM and TZN routinely in tablet. In summary, the proposed method can be used for the routine of quality control in pharmaceuticals.
Acknowledgements
The author expresses gratitude to Rajiv Gandhi University of Health science, Bangalore, for providing research grant and also thanks to Micro Lab Pvt. Ltd. and Excel Pharma Pvt. Ltd for providing ACO, PCM and TZN. The authors also thankful to chairman, principal and staff member of Acharya and B.M. Reddy College of Pharmacy, Bangalore, for providing required facilities to carry out this research work.
(a) (b) (c) Figure 6: First derivative spectra of (a) ACO (b) PCM (c) TZN by UV method.
Volume 2 • Issue 4 • 1000123J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal
Citation: Chandrashekhar N, Patel HR, Karvekar MD, Suresh BAR (2011) Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in Their Combined Dosage Forms by Spectrophotometric and RP- HPLC Method. J Anal Bioanal Techniques 2:123. doi:10.4172/2155-9872.1000123
Page 5 of 5
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