Int J Pharm Pharm Sci, Vol 6, Issue 7, 632-637Original Article
RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF DICLOFENAC SODIUM, TRAMADOL HYDROCHLORIDE AND CHLORZOXAZONE FROM THEIR COMBINED TABLET DOSAGE FORM
JIGAR PATEL1*, PINAK PATEL1
1Department of Quality Assurance, Indubhai Patel College of Pharmacy and Research Centre, Amrapali Township, Petlad - Khambhat Road, Dharmaj, Gujarat-388430, India.
Email: jigarpatel2311@gmail.com
Received: 08 May, 2014 Revised and Accepted: 11 June, 2014
ABSTRACT
Objective: The objective of the current study was to develop and validate the RP-HPLC method for the simultaneous estimation of Tramadol Hydrochloride, Chlorzoxazone and Diclofenac sodium from their combined tablet dosage form
Methods: The current method describes RP-HPLC method for the estimation of Tramadol Hydrochloride, Chlorzoxazone and Diclofenac sodium from their combined tablet dosage form. The mobile phase used was Acetonitrile: 0.05M Disodium Hydrogen Phosphate buffer pH 3.5 adjusted with 10% v/v Ortho Phosphoric acid(50:50 v/v) and Hypersil ODS C18 ( x , 5.0 μ particle sizes) was used as a stationary phase with detection wavelength of 220 nm.
Result: Linearity was obtained in the concentration range of 15-75 μg/ml, 100-500 μg/ml and 20-100 μg/ml for TRM, and DIC respectively. The % recovery was found to be 99.41 -99.84%, 99.30 - 99.74 % and 99.6 - 99.97 % for DIC, TRM and respectively. The LOQ was found to be 3.33, 3.95 and 36.71 μg/ml for DIC, TRM and respectively. The proposed method has been validated as per Q2R1 guidelines and successfully applied to the estimation of TRM, DIC and from their combined Tablet dosage form.
Conclusion: The method was found to be simple, accurate, precise, and suitable for the estimation of Tramadol Hydrochloride, Chlorzoxazone and Dicofenac sodium from their combined tablet dosage form.
Keywords: Diclofenac sodium, Tramadol Hydrochloride, Chlorzoxazone, RP-HPLC method, Analytical method validation.
INTRODUCTION
Tramadol Hydrochloride (TRM) is chemically (lRS,2RS)-2-[(dimethyl amino) methyl]-I-(3-methoxyphenyl) cyclohexanol hydrochloride [1] (Figure 1). TRM belongs to analgesic and narcotic category. TRA and its O-desmethyl metabolite (M1) are selective, weak OP3-receptor agonists. It is indicated in the treatment of moderate to severe pain. TRM is used to treat postoperative, dental, cancer, and acute musculosketetal pain and as an adjuvant to NSAID therapy in patients with osteoarthritis. Tramadol Hydrochloride is official in Indian Pharmacopoeia [1], British Pharmacopoeia [2] and United States Pharmacopoeia[3].
Diclofenac sodium (DIC) is chemically Sodium 2-[(2,6-dichlorophenyl)amino] phenyl acetate[4] (Figure 2). DIC is used in acute and chronic treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis. Diclofenac sodium is official in Indian Pharmacopoeia[4], British Pharmacopoeia[5] and United States Pharmacopoeia[6]. Chlorzoxazone (CHL) is chemically 2(3H)-Benzoxazolone, 5-Chloro-2-benzoxazolinone[6] (Figure 3). It is indicated for the relief of discomfort associated with acute painful musculoskeletal conditions. Chlorzoxazone is official in United States Pharmacopoeia [6]. The review of literature revealed that many analytical methods involving UV Spectrophotometric [7-8], RP-HPLC[9-11], HPTLC[12] and UPLC[13] have been reported for TRM individually and in combination with other drugs. Several methods have been reported for the estimation of DIC individually and in combination with other drugs such as UV Spectrophotometric[14], RP-HPLC[15-17], HPTLC[18] and LC-MS[19]. Several methods have been reported for Chlorzoxazone individually and in combination with other drugs namely UV Spectrophotometric[20], RP-HPLC[21] and HPTLC[22]. But no method has been reported for the estimation of TRM, DIC and CHL from their combined dosage form.
So, the present article describes RP-HPLC method for the estimation of TRM, DIC and CHL in their tablet dosage form. The developed method was validated applying ICH Q2R1 guidelines[23] and was used for the assay of three drugs in their tablet dosage form.
Fig. 1: It shows the chemical structure of Tramadol Hydrochloride
Fig. 2: It shows the chemical structure of Diclofenac Sodium
Fig. 3: It shows the chemical structure of Chlorzoxazone
MATERIALS AND METHODS
Materials
Analytically pure samples of TRM, DIC and CHL were procured from Medico Lab, Vatva, Ahmedabad; Medico Lab, Vatva, Ahmedabad and Baroque Pharmaceuticals ltd., Khambhat respectively.
Orthophosphoric acid (A.R. Grade) was purchased fromSD Fines chemicals, Bombay; Water (HPLC Grade) from RFCL limited, New Delhi; Acetonitrile (HPLC Grade) from RFCL limited, New Delhi and Disodium Hydrogen Phosphate (A. R. Grade) from SD Fines chemicals, Bombay. Tablet of TRM, DIC and CHL in combined dosage form (TRICARE-MR) was purchased from the local market.
Instrument and experimental condition
RP-HPLC analysis was carried out on the instrument of Analytical Technologies limited with the injector loop of 20 µl on the isocratic mode. The pump used was P2230 plus HPLC pump and the detector UV 2230 plus detector.
The data was analyzed using Analchrom 2006 software. The column used was Hypersil ODS C18 (250 × 4.6 m, 5 µm particle size). The pH meter used was Chemiline Digital pH meter CL-110.
Preparation of Mobile Phase
Accurately measured 7.089 gm Disodium Hydrogen Phosphate was dissolved in 1000 ml of HPLC grade water. A mixture of 50 ml Acetonitrile and 50 ml of 0.05M Disodium Hydrogen phosphate was prepared and the pH of the mixture was adjusted to 3.5 pH with 10 % orthophosphoric Acid and sonicated for 10 minutes, filtered through 0.45 μm filter paper and sonicated for 10 minutes to degas the mixture and used as mobile phase.
Preparation of standard stock solution
A 100 mg of standard TRM, DIC and CHL were accurately weighed and transferred to each of 100 ml volumetric flask and dissolved in 50 ml methanol. The flask was sonicated for 10 min. The flask was shaken and volume was made up to the mark with methanol to give solutions containing 1000 μg/ml TRM, DIC and CHL.
Analytical Method validation
(1) Linearity and Range
The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.
The range were found to be 20-100 μg/ml for DIC, 15-75 μg/ml for TRM and 100-500 μg/ml for CHL. All the chromatogram was repeated for 6 times. Calibration curves were constructed by plotting average Area versus concentrations for all the three drugs. Straight line equations were obtained from these calibration curves.
(2) Repeatability
Standard solution mixture containing TRM (15-75 μg/ml), DIC (20-100 μg/ml) and CHL (100-500 μg/ml) were prepared and chromatograms were recorded and area were measured and C.V. were calculated. Sample solution containing 30 μg/ml TRM, 40 μg/ml DIC and 200 μg/ml CHL was prepared and chromatogram was recorded. Area was measured of the same concentration solution six times and CV was calculated.
(3) Precision (Inter-day and Intra-day)
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as Coefficient of Variance (CV).
Variations of results within the same day (intra-day), variation of results between days (inter-day) were analyzed. Intraday precision was determined by analyzing TRM, DIC and CHL for three times in the same day. Inter day precision was determined by analyzing the three drugs daily for three days. For this study standard solution mixture of 15, 45 and 75 μg/ml for TRM; 20, 60 and 100 μg/ml for DIC and 100, 300, 500 μg/ml for CHL were prepared and chromatograms were recorded.
(4) Accuracy
Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy sample solution of formulation was prepared and analysis of the same was carried out. Recovery studies were carried out by addition of standard drug to the sample solution at 3 different concentration levels 50, 100 and 150 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed three times and average recoveries were measured.
(5) Reproducibility
The areas were measured using same instrument by twoanalysts for sample solution and the values obtained were evaluated using t-test to verify their reproducibility.
(6) Specificity and Selectivity
Specificity is a procedure to detect quantitatively the analyte inthe presence of component that may be expected to be present in the sample matrix, while selectivity is the procedure to detect qualitatively the analyte in presence of components that may be expected to be present in the sample matrix. Specificity of an analytical method is ability to measure specifically the analyte of interest without interferences from blank and placebo. It was checked for interference from blank.
(7) Limit of detection & Limit of quantification
The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by using the following equations as per International Conference on Harmonization (ICH) guidelines which is based on the calibration curve.
LOD = 3.3 × σ /S
LOQ = 10 × σ /S
Where σ = the standard deviation of y-intercepts of regression lines
S = Slope of calibration curve.
(8) Assay of marketed formulation
Twenty tablets were weighed individually and the average weight of the single tablet was found to be 752 mg. Powder equivalents to 250 mg CHL, 50 mg DIC and 37.5 mg TRM was accurately weighed and transferred to 25 ml volumetric flask. 15 ml of methanol was added to same volumetric flask and sonicated for 20 min. The flask was shaken and volume was made up to the mark with methanol. The above solution was filtered through whatmann filter paper (0.45μm). 5 ml of aliquot was taken and transferred to volumetric flask of 50 ml capacity and volume was made up to the mark with the methanol. Further 2 ml of this solution was transferred to volumetric flask of 10 ml capacity. Volume was made up to the mark with the mobile phase to give a solution containing 30 μg/ml TRM, 40 μg/ml DIC and 200 μg/ml CHL. This solution was sonicated for 5 min. This solution was used for the estimation of TRM, DIC and CHL.
RESULTS AND DISCUSSION
Method optimization
For the selection of mobile phase, various mobile phase systems were tried for thechromatographic separation.Finally, the system containing Acetonitrile and 0.05M Disodium Hydrogen phosphate with a pH 3.5 adjusted with 10 % Ortho Phosphoric Acid (50:50 %v/v) gave well resolved peaks of all the three drugs. The average retention time of TRM, CHL and DIC were found to be 2.11 min., 3.82 min. and 12.39 min. respectively. For the selection of analytical wavelength, the overlain spectra of 50 μg/ml TRM, 50 μg/ml DIC and 50 μg/ml CHL revealed that at 220 nm all the three drugs possess absorbance (Fig. 4).
Analytical method validation
1) Linearity and Range
The method was found to be linear at the concentration range of 15-75 µg/ml for TRM, 100-500 µg/ml for CHL and 20-100 µg/ml for DIC.
Calibration data for TRM, CHL and DIC are shown in Table 2-4 respectively. The calibration curves for TRM, DIC and CHL were prepared by plotting area and concentration(Fig. 6-8).
Fig. 4: It shows the overlain spectra of 50 μg/ml TRM, 50 μg/ml DIC and 50 μg/ml CHL in Methanol
Fig. 5: It shows the chromatogram of mixed standard solution containing 60 μg/ml TRM, 400 μg/ml CHL and 80 μg/ml DIC, using mobile phase Acetonitrile: 0.05M Disodium Hydrogen Phosphate buffer pH 3.5 (50:50 v/v) (Proposed Method).
Table 1: It shows the optimized Chromatographic conditions for TRM, CHL and DIC
| Parameter | Conditions |
| Mobile phase | Acetonitrile: 0.05M Disodium Hydrogen Phosphate buffer pH 3.5 adjusted with 10 % Ortho Phosphoric acid |
| Pump Mode | Isocratic |
| Column | Hypersil ODS C18, 250 x 4.6 mm (5 μm) |
| Detection wavelength (nm) | 220 |
| Flow rate | 1.5 ml/min |
| Run Time ( min) | 20.0 |
| Diluent | Mobile phase |
| Volume of Injection (μl) | 20.0 |
| Retention time | TRM: 2.11 minCHL: 3.82 minDIC: 12.39 min |
Table 2: It shows the result of calibration readings for TRM by HPLC method
| Concentration (µg/ml) | Area Mean ± S.D. | C.V |
| 15 | 109.16 ± 1.4454 | 1.32 |
| 30 | 231.05 ± 2.1030 | 0.91 |
| 45 | 353.11 ± 3.9710 | 1.12 |
| 60 | 476.16 ± 4.3070 | 0.90 |
| 75 | 568.97 ± 4.4632 | 0.78 |
Fig. 6: It shows the calibration curve of TRM by RP-HPLC method.
Table 3: It shows the result of calibration readings for CHL by HPLC method
| Concentration (µg/ml) | Area Mean ± S.D. | C.V |
| 100 | 1340.11 ± 14.8804 | 1.11 |
| 200 | 2827.63 ± 21.1644 | 0.74 |
| 300 | 4278.32 ± 29.2475 | 0.68 |
| 400 | 6243.63 ± 60.5147 | 0.97 |
| 500 | 7968.69 ± 87.2778 | 1.09 |
Fig. 7: It shows calibration curve of CHL by RP-HPLC method
Table 4: It shows the result of calibration readings for DIC by HPLC method
| Concentration (µg/ml) | Area Mean ± S.D. | C.V |
| 20 | 174.065 ± 2.0449 | 1.17 |
| 40 | 601.018 ± 5.9305 | 0.98 |
| 60 | 926.337 ± 8.8130 | 0.95 |
| 80 | 1242.75 ± 9.9257 | 0.79 |
| 100 | 1547.98 ± 13.402 | 0.86 |
Fig. 8: It shows calibration curve of DIC by RP-HPLC method
2) Repeatability:
The developed method was found to be repeatable since C.V. was found to be less than 2 for all the drugs.
3)Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The LOD for TRM, DIC and CHL was found to be 1.30 μg/ml, 1.10 μg/ml and 12.11 μg/ml respectively. The LOQ for TRM, DIC and CHL was found to be 3.95 μg/ml, 3.33 μg/ml and 36.71 μg/ml respectively
4) Accuracy
Accuracy was determined by calculating the % recovery. The method was found to be accurate with % recovery 99.30 - 99.74 % for TRM, 99.41 – 99.84% for DIC and 99.6 - 99.97 % for CHL (Table 5).
5) Precision
Precision was calculated as repeatability and intra- and inter-day variation for all the threedrugs. The method was found to be precise with C.V. 0.86 – 0.99 for intra-day (n=3) and C.V. 0.95 – 1.15 for inter-day (n=3) for TRM; C.V. 0.77 – 0.97 for intra-day (n=3) and C.V. 0.98 – 1.17 for inter-day (n=3) for DIC and C.V. 0.69 – 0.97 for intra-day (n=3) and C.V. 0.97 – 1.31 for inter-day (n=3) for CHL (Table 6-8).
Table 5: It shows the accuracy studies for TRM, CHL and DIC by HPLC method (n=3)
| % Level of Recovery | Amount of drug in sample (µg/ml) | Amount of standard added (µg/ml) | Total amount of Drug (µg/ml) | Amount of drug recovered (µg/ml) ± SD | % Recovery ± SD |
| TRM (µg/ml) | TRM (µg/ml) | TRM (µg/ml) | TRM (µg/ml) | % TRM | |
| Unspiked | 30 | 0 | 30 | 29.55 ± 0.4574 | - |
| 50 % | 30 | 15 | 45 | 14.89 ± 0.1327 | 99.30 ± 0.8722 |
| 100 % | 30 | 30 | 60 | 29.82 ± 0.1681 | 99.39 ± 0.5572 |
| 150 % | 30 | 45 | 75 | 44.88 ± 0.1543 | 99.74 ± 0.3349 |
| DIC (µg/ml) | DIC(µg/ml) | DIC (µg/ml) | DIC (µg/ml) | % DIC | |
| Unspiked | 40 | 0 | 40 | 39.82 ± 0.1541 | - |
| 50 % | 40 | 20 | 60 | 19.95 ± 0.0989 | 99.75 ± 0.4949 |
| 100 % | 40 | 40 | 80 | 39.76 ± 0.1840 | 99.41 ± 0.4581 |
| 150 % | 40 | 60 | 100 | 59.91 ± 0.0917 | 99.84 ± 0.1541 |
| CHL (µg/ml) | CHL (µg/ml) | CHL (µg/ml) | CHL (µg/ml) | % CHL | |
| Unspiked | 200 | 0 | 200 | 199.31 ± 0.7764 | - |
| 50 % | 200 | 100 | 300 | 99.67 ± 0.5800 | 99.67 ± 0.5800 |
| 100 % | 200 | 200 | 400 | 199.93 ± 0.6759 | 99.97 ± 0.3362 |
| 150 % | 200 | 300 | 500 | 298.79 ± 0.6308 | 99.6 ± 0.2118 |
Table 6: It shows the precision study for TRM by RP-HPLC method (n=3)
| Concentration(μg/ml) | Intra-day(Area ± S.D) | C.V. | Inter-day(Area ± S.D) | C.V. |
| 15 | 110.1 ± 0.9543 | 0.86 | 109.95 ± 1.2744 | 1.15 |
| 45 | 352.71 ± 3.3369 | 0.94 | 349.71 ± 3.3459 | 0.95 |
| 75 | 565.17 ± 5.5827 | 0.99 | 567.70 ± 5.6119 | 0.98 |
Table 7: It shows the precision study for CHL by RP-HPLC method (n=3)
| Concentration(μg/ml) | Intra-day(Area ± S.D) | C.V. | Inter-day(Area ± S.D) | C.V. |
| 100 | 1340.13 ± 13.0147 | 0.97 | 1342.22 ± 17.6192 | 1.31 |
| 300 | 4274.11 ± 37.462 | 0.87 | 4258.01 ± 42.0006 | 0.98 |
| 500 | 7928.8 ± 55.2941 | 0.69 | 7927.32 ± 77.3596 | 0.97 |
Table 8: It shows the precision study for DIC by RP-HPLC method (n=3)
| Concentration(μg/ml) | Intra-day(Area ± S.D) | C.V. | Inter-day(Area ± S.D) | C.V. |
| 20 | 173.55 ± 1.6879 | 0.97 | 174.05 ± 2.0439 | 1.17 |
| 60 | 926.78 ± 7.2179 | 0.77 | 925.53 ± 9.0790 | 0.98 |
| 100 | 1538.37 ± 13.8437 | 0.89 | 1537.92 ± 16.0222 | 1.04 |
6) Reproducibility
The method was found to be reproducible (Table 9-11).
Table 9: It shows the reproducibility data for TRM (30 μg/ml)
| Analyst 1Area ± S.D. (n=3) | Analyst 2Area ± S.D. (n=3) | Result of t test* | Inference |
| 232.79 ± 1.9567 | 231.64 ± 2.5402 | 0.341 | No significant difference |
* At 95% confidence interval, (t-Tabulated = 4.30)
Table 10: It shows the reproducibility data for CHL (200 μg/ml)
| Analyst 1Area ± S.D. (n=3) | Analyst 2Area ± S.D. (n=3) | Result of t test* | Inference |
| 2860.9 ± 5.9568 | 2857.97 ± 4.0887 | 0.333 | No significant difference |
* At 95% confidence interval, (t-Tabulated = 4.30)
Table 11: It shows the reproducibility data for DIC (40 μg/ml)
| Analyst 1Area ± S.D. (n=3) | Analyst 2Area ± S.D. (n=3) | Result of t test* | Inference |
| 559.72 ± 3.1038 | 559.21 ± 4.5717 | 0.923 | No significant difference |
* At 95% confidence interval, (t-Tabulated = 4.30)
7) System suitability parameters
Table 12: It shows the system suitability parameters for developed method
| Parameter | Diclofenac sodium | Chlorzoxazone | Tramadol Hydrochloride | Range | Inference |
| Retention time (min) | 12.392 ± 0.3045 | 3.828 ± 0.0348 | 2.116 ± 0.0174 | - | - |
| Resolution (Rs) | 12.142 ± 0.8527 | 6.292 ± 0.0591 | 3.18 ± 0.101 | > 2 | Criteria met |
| Tailing Factor | 1.684 ±0.1335 | 1.3 ± 0.1264 | 1.38 ± 0.1166 | < 2 | Criteria met |
| Theoretical Plates (Plates/Meter) | 8783.42 ± 1409.2 | 7620.15 ± 228.291 | 7678.8 ± 261.282 | > 2000 | Criteria met |
Table 13: It shows the assay result of marketed formulation (n=3)
| Formulation | Drug | Amount Taken (µg/ml) | Amount Found(µg/ml)(n=3) | Labeled claim(mg) | Amount found per Tablet(mg) | %Assay ± SD |
| Tricare-MR | DIC | 40 | 39.95 | 50 | 49.9379 | 99.88 ± 0.2027 |
| TRM | 30 | 29.91 | 37.5 | 37.3833 | 99.69 ± 0.4573 | |
| CHL | 200 | 199.78 | 250 | 249.725 | 99.89 ± 0.5466 |
8) Specificity
The method was found to be specific as no interferencewas observed when the chromatogram was recorded in the presence of excipients.
Fig. 9: It shows the chromatogram of specificity study.
9) Assay
Formulation was analyzed by the proposed method and assay result of formulation is shown in Table 12
CONCLUSION
The RP-HPLC method for the estimation of TRM, CHL and DIC from their combined tablet dosage form was successfully developed. The method was validated as per ICH Q2R1 guidelines for Specificity, Linearity, Accuracy, Precision, Limit of Detection (LOD), Limit of Quantitation (LOQ) and Reproducibility. The method can be successfully applied for the routine analysis of the formulation.
ACKNOWLEDGEMENT
The authors are thankful to Indubhai Patel College of Pharmacy and Research Centre (Dharmaj, India) for providing the necessary facilities for research work.
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