Analytical Method Development and Validation of Thiocolchicoside and Ibuprofen in Tablet dosage form by UV-Spectrophotometry Method
Jadhav Ankush P.*, Datar P.A., Kedar T.R., Kardile D.P., Shete R.V.
Department of Pharmaceutical Quality Assurance, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor,
Tal. Bhor, Dist. Pune - 412 206 Maharashtra.
*Corresponding Author E-mail: jadhavbrand@gmail.com
ABSTRACT:
A new, simple, accurate, precise and reproducible UV-Spectrophotometric method is being developed for the simultaneous estimation of ibuprofen and thiocolchicoside in tablet dosage form for first time. The stock solutions were prepared in methanol. The λmax for Thiocolchicoside and Ibuprofen were 256nm and 228nm respectively. The Thiocolchicoside, Ibuprofen obeyed Beer’s law in concentration range of 0.4-1.2µg/ml and 0.5-2.5µg/ml respectively. Results of analysis of absorbance ratio method were analysed and validated for various parameters according to ICH guidelines for accuracy, precision, linearity, robustness, LOD and LOQ. The proposed method is highly sensitive, precise and accurate, therefore can be used for intended purpose.
KEYWORDS: Thiocolchicoside, Ibuprofen, Absorbance Ratio Method, Validation, ICH.
INTRODUCTION:
Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) derived from propionic acid. Ibuprofen is a non-selective COX inhibitor and hence, it inhibits the activity of both COX-1 and COX-2. The inhibition of COX-2 activity decreases the synthesis of prostaglandins involved in mediating inflammation, pain, fever, and swelling. The chemical formula for ibuprofen is C13H18O2 and molecular weight is 206.28g/mol. Figure no. 2 shows structure of Ibuprofen6,10.
Figure No. 1: Thiocolchicoside
Figure No. 2: Ibuprofen
MATERIALS AND METHODS:
Chemicals pharmaceutically pure sample of thiocolchicoside was obtained from India Glycols Ltd. (Dehradun) and ibuprofen was obtained from Strides Shasun Ltd. (Puducherry) as gift samples. The commercial tablets Thiocolfen (Thiocolchicoside 400 mg and Ibuprofen 4mg) were procured from the local drug market. All the chemicals and reagents were of analytical grade.
INSTRUMENT:
A double beam UV-Visible spectrophotometer model Jasco V 530PC was used. The spectra were recorded over range 200-400nm against solvent in 1cm quarts cells.
Selection of Solvents:
On the basis of solubility study methanol was selected as the solvent for dissolving Thiocolchicoside and Ibuprofen.
STANDARD SOLUTION PREPARATIONS:
Accurately weighed 10mg of thiocolchicoside and ibuprofen were transferred into volumetric flasks separately and then volume was made up to 10ml with methanol to get a concentration of 1000µg/ml for all two drugs. Standard stock solution (1000µg/ml) was further diluted with methanol to obtain 0.4-1.2µg/ml for thiocolchicoside and 0.5-2.5µg/ml for ibuprofen.
STUDY OF SPECTRA AND SELECTION OF WAVELENGTH:
From the overlain spectrum of Thiocolchicoside and Ibuprofen, two wavelengths were selected one at 256nm (λmax of Thiocolchicoside) and other at 248.5nm (Isoabsorptive point). The method employed Q - values, and the concentrations of drugs in sample solutions were determined using the following equations3.
Qm – Qᵧ A1
C1 = ________ × ___ ……………………….equation 1.
Qₓ – Qᵧ ax1
Qm – Qₓ A2
C2 = ________ × ___ ………………………equation 2.
Qᵧ – Qₓ ay1
Where, A1 and A2 are the absorbances of mixture at 248.5 and 256 nm, Qm = A2 / A1,
Qᵧ = ay2/ay1 and Qₓ = ax2/ax1, ax1 (1.685), ax2 (1.4350), ay1 (2.3012) and ay2 (1.4749) are absorptivities (1%, 1cm) of ibuprofen and thiocolchicoside at 248.5 and 256 nm (Figure No. 3).
Figure No. 3: Overlain spectra of thiocolchicoside and ibuprofen
Preparation for analysis of tablet formulation:
Twenty tablets were taken and their average weight was determined. They are crushed to fine powder; amount equivalent to 100mg of powder was taken in 100ml volumetric flask. The ratio of drugs was 1:100. This was then dissolving in 50ml of methanol by sonication for about 10 minutes. The volume is made up to the mark by methanol and filtered by Whatmann filter paper (no.41) and the filtrate was used to prepare samples of different concentration. Now all the tablet samples was scanned in multi photometric mode and the concentration of all two drugs were obtained. Results of tablet analysis are reported in Table No. 1.
VALIDATION PARAMETERS FOR METHOD:
As per ICH guideline the method is validated and following parameters were evaluated7,13,26.
Linearity:
Its ability (with in a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The calibration curve was constructed between concentration verses absorbance.
Precision:
Precision was determined by repeatability, Interday precision of all two drugs. Repeatability indicates the precision under the same operating condition over short interval time. The interday precision study is expressed within laboratory variation on different days and analyst to analyst variation by different analyst.
Limit of Detection and Limit of Quantification (LOD and LOQ):
Sensitivity of the method was determined with respect to limit of detection (LOD) and limit of quantitation. According to ICH guidelines, the limit of detection is the lowest amount of analyte in a sample that can be detected and the limit of quantitation is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.
Accuracy:
To a preanalysed tablet solution a definite concentration of pure drug was added (80%, 100% and 120% level) and then recovery was studied. A preanalysed tablet solution containing 400µg/ml of ibuprofen and 4µg/ml of thiocolchicoside were taken in 10ml volumetric flasks and known concentrations of pure drug solution was added to them, which were prepared from standard stock solution of ibuprofen and thiocolchicoside.
Robustness:
As per ICH norms, small, but deliberate variations by altering the absorption maxima, pH and ratio of mobile phase solvents were made to check the methods capacity to remain unchanged.
RESULTS AND DISCUSSION:
The absorbance ratio method for estimation of Thiocolchicoside and Ibuprofen in tablet dosage form was found to be simple, precise, accurate and reproducible. The solvent used was 100% methanol and do not shows any significant interference in the spectrophotometric assay of all two drugs.
Linearity:
The proposed method was found to be linear in the range of 0.4-1.2 and 0.5-2.5μg/ml with correlation coefficient 0.998 and 0.944 for Thiocolchicoside and Ibuprofen respectively. The calibration curve of Thiocolchicoside, Ibuprofen is shown in figure 4 and 5 resp. and result of linearity study shown in Table No. 2.
Figure No. 4: Calibration Curve of Thiocolchicoside
Figure No. 5: Calibration Curve of Ibuprofen
Limit of Detection and Limit of Quantification (LOD and LOQ):
The standard deviation of y-intercept of regression line were determined and substituted in the following equation for the determination of detection of limit and quantification limits.
Detection limit= 3.3 σ/s
Quantification limit= 10 σ/s
Where, σ is the standard deviation of y-intercept of regression line and s is the slope of the calibration curve.
The limit of detection (LOD) and limit of quantification (LOQ) data are given in Table no. 2.
Accuracy:
The validity and reliability of proposed methods were assessed by recovery studies. The recovery of added standards (80%, 100% and 120%) was found at three concentrations and 3 replicate levels. The value of mean of recoveries was found to be in ranging from 92.60 to 101.51% for Thiocolchicoside and 99.21 to 102.54% for Ibuprofen. The value of SD and % RSD are less than 2 indicate the accuracy of method. It was repeated at 3 concentrations and 3 replicate levels. Result of recovery study shown in Table no. 3.
Precision:
Precision was determined by repeatability and Interday precision of all two drugs.
I) Repeatability:
The repeatability was performed for six concentrations in linearity range 0.4, 0.6, 0.8, 1, 1.2 and 1.4µg/ml for Thiocolchicoside and 0.5, 1, 1.5, 2, 2.5, and 3µg/ml for Ibuprofen indicates the precision under the same operating condition over short interval time.
II) Interday precision:
Interday precision was also performed within laboratory variation on different days for all two drugs simultaneously in three replicate at three concentrations.
Result of precision shown in Table no. 4.
Robustness:
Standard stock solution of 1000µg/ml of Thiocolchicoside and Ibuprofen were prepared using methanol as a solvent. From standard stock solution, sub stock solution of Thiocolchicoside and Ibuprofen were prepared separately. In this present work the change was made in the ratio of solvent and absorption maxima. Instead of 100%, 95% methanol was used as solvent. And the absorption maxima was decreased and increased 2nm and carried out the process. The RSD% was calculated.
Results of robustness shown in Table no. 5.
Table No. 1: Result of Tablet formulation.
Sr. No. |
Drug Name |
Labelled Amount (mg) |
S.D. |
%COV |
1 |
Thiocolchicoside |
4 |
0.018 |
93.88% |
2 |
Ibuprofen |
400 |
0.077 |
93.50% |
(Where, S.D. = Standard Deviation, %COV= % Recovery)
Table No. 2: Linear regression parameters for Thiocolchicoside and Ibuprofen by both proposed methods.
Sr. No. |
Parameter |
Thiocolchicoside |
Ibuprofen |
1 |
Wavelength (nm) |
256 |
228 |
2 |
Calibration range (µg/ml) |
0.4-1.2 |
0.5-2.5 |
3 |
Correlation coefficient (r²) |
0.9988 |
0.944 |
4 |
Slope (m) |
0.4584 |
0.0207 |
5 |
Intercept (c) |
0.0705 |
0.0234 |
6 |
Limit of detection (µg/ml) |
22.74 |
20.13 |
7 |
Limit of Quantitation (µg/ml) |
68.93 |
61.00 |
Table No. 3: Recovery study at 3 concentration and 3 replicates for Thiocolchicoside and Ibuprofen by both proposed methods.
Sr. No. |
Drug |
Concentration of standard added |
S.D. |
%RSD |
%COV |
1 |
Thiocolchicoside |
80% |
0.0037 |
0.33 |
92.60 |
100% |
0.0008 |
0.13 |
102.69 |
||
120% |
0.0122 |
1.42 |
101.51 |
||
2 |
Ibuprofen |
80% |
0.0025 |
0.85 |
99.12 |
100% |
0.0078 |
0.69 |
101.75 |
||
120% |
0.0020 |
0.51 |
102.54 |
Table No. 4: Precision study for Thiocolchicoside and Ibuprofenby both proposed methods.
Sr. No. |
Parameter |
S.D. |
% RSD |
||
1 |
Drug |
THIO |
IBU |
THIO |
IBU |
2 |
Repeatability |
0.0096 |
0.0035 |
0.52 |
0.20 |
3 |
Interday Precision |
0.0044 |
0.0001 |
0.34 |
0.64 |
(Where, THIO= Thiocolchicoside, IBU = Ibuprofen)
Table No. 5: Robustness study at 3 replicates for Thiocolchicoside and Ibuprofen by both proposed methods.
Sr. No. |
Drug |
Wavelength (nm) |
S.D. |
% RSD |
1 |
Thiocolchicoside |
254 |
0.0153 |
1.20 |
258 |
0.0124 |
0.95 |
||
2 |
Ibuprofen |
226 |
0.0123 |
1.23 |
230 |
0.0077 |
0.73 |
CONCLUSION:
In this work, a new UV-Spectrophotometric method has developed and validated for first time. The results of present study indicate that the proposed UV-Spectrophotometric method is simple, rapid, precise and accurate. The developed UV-Spectrophotometric absorbance ratio method was found suitable for determination of Thiocolchicoside and Ibuprofen in tablet dosage form. They can be easily applied in quality control laboratory tests in the dosage form.
ACKNOWLEDGEMENTS:
The authors would like to acknowledge Principal Dr. R.V. Shete, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor for providing laboratory facilities and encouragement.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 26.02.2020 Modified on 21.04.2020
Accepted on 09.06.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(2):981-985.
DOI: 10.5958/0974-360X.2021.00175.X