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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2022 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                                       Research Article 

Stability-Indicating Analytical Method Development and Validation of Thiocolchicoside and Ibuprofen in Tablet Dosage Form by RP-HPLC Method

Jadhav Ankush P.¹*, Datar Prasanna A.¹, Kedar Tejashree R.¹, Kardile Deepak P.² 

¹ Department of Pharmaceutical Quality Assurance, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor- Pune, Maharashtra, India. (Pin. 412 206)

² Department of Pharmaceutical Chemistry, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor- Pune, Maharashtra, India. (Pin. 412 206)

1. INTRODUCTION:

1.1 Introduction of Drugs:

THIO is a glycoside of natural anti- inflammatory moiety which is derivative of colchicine from semi- synthetic process. It procured from the Superba Gloriosa flower seeds. It gives anti-inflammatory as well as analgesic with muscle relaxant effects. It works through selective binding to the Gamma (y) Amino Butyric Acid i.e. GABA - A receptor. By activating the GABA inhibitory motor pathway it prevents muscle contractions so mainly it has major role in the cure of orthopedic and disorders of rheumatology ^{2- 4}. The molecular formula of THIO is C₂₇H₃₃NO₁₀S and molecular weight is 563.6 g/mol. Fig.1-A shows structure of THIO.

IBU is derived from propionic acid which has a non-steroidal anti-inflammatory drug (NSAID) activity. IBU is a non-selective cyclooxygenase i.e. COX inhibitor therefore this drug inhibits the activity of both cyclooxygenase i.e. COX-1 and COX-2. The inhibition of cyclooxygenase-2 activity minimizes the overall synthesis of prostaglandins which involved in mediating inflammation, pain, fever, and swelling ^{5, 6}. The chemical formula for IBU is C₁₃H₁₈O₂ and molecular weight‎ is ‎206.28 g/mol. Fig.1-B shows structure of IBU.

Figure 1-A: Thiocolchicoside                                         Figure 1-B: Ibuprofen

1.2 Background of study:

To the best of our knowledge, there is no reported RP-HPLC method for simultaneous estimation of THIO and IBU in pharmaceutical formulations, previous to our work. Thus, efforts were made to develop fast, selective and sensitive stability-indicating analytical method for the estimation of THIO and IBU in their tablet dosage form using Reverse Phase Chromatography i.e. RPC method. In the current work author developed a simple, accurate, reliable and reproducible stability-indicating RP-HPLC method which was duly validated by statistical parameters precision, accuracy and recovery as per ICH Q2 (R1) and Q1A (R2) guideline within all acceptance criteria.

2. MATERIALS AND METHODS:

2.1. Chemicals and reagents:

The commercial tablets Thiocolfen (Thiocolchicoside 400 mg and Ibuprofen 4 mg) were procured from the local drug market. Chemical is used as Acetonitrile (HPLC Grade), Methanol (HPLC Grade) and Dist. water (HPLC Grade) etc.

2.2 Instrumentation:

Instruments were as follows UV-Spectrophotometer (Jasco V 530 PC), HPLC- Schimadzu Model consisting of Inertsil, 3V ODS (C18 4.6 mm x 250 mm, 5μ columns), pH Meter (Chemiline). 

2.3 Preparation of standard stock solutions:

2.3.1 Ibuprofen standard stock:

100.1 mg IBU standard was weighed and dissolved in 25 ml with diluent (use mobile phase as a diluent).

2.3.2 Final standard solution:

20.1 mg of THIO drug was weighed and add 5.0 ml of IBU standard stock dissolved in 25.0 ml with diluent.

2.4 Preparation of sample solutions:

The mean weight of twenty tablets was taken and after that it crushed to fine powder; amount equal to (powder) 100 mg was kept in flask of volumetric (100 ml). The drugs ratio was 1:100. This was then dissolving in 50 ml of diluent by sonication for about 10 min. The volume is made to the mark by diluent and filtered by Whatmann filter paper (no. 41) to forms 1000 µg/ml of sol^n. and the this sol^n. was utilized to prepare samples of various attentiveness.

2.5 Selection of detection wavelength:

From the standard stock solution further dilutions were done using diluent and scanned over the range of 200-400 nm and the spectra were overlain. It was observed that maximum wavelength of THIO and IBU 256 nm and 228 nm resp. and these drugs showed considerable absorbance at 248 nm and their overlain spectra of is given in fig. 2.

Figure 2: Overlain spectra of Thiocolchicoside & Ibuprofen

2.6 Chromatographic conditions:

HPLC experiment was carried out on a Schimadzu LC-2030 PLUS (IND) System separation module, with photodiode array detector using Auto sampler. The analytical column used for the separation was Inertsil, 3V ODS (C18 4.6 mm x 250 mm, 5μ columns). Optimized chromatographic conditions as shown in the table 1-A, Characteristic chromatogram was shown in fig. 3 and system suitability parameters of THIO and IBU shown in table 1-B.

Table 1-A: Optimized Chromatographic conditions

Figure 3: The chromatogram of optimized standard mixture

Table 1-B: System suitability parameters of optimized standard mixture 

3. METHOD DEVELOPMENT:

The objective of this experiment was to achieve good separation with good resolution peaks between all the components by trying different proportions of solvents like Methanol, Acetonitrile and Dist. water testing. For that we have tried different mobile phases and we obtained optimized chromatogram by using Methanol (MeOH) and Dist. water (H₂O) in 50:50, v/v ratios at 1 ml/min flow rate with 20 μl injection volumes in 5 min. run time and it was detected at 248 nm wavelengths. This trial gives more asymmetry & sharp peaks with good resolutions which is shown in fig. 3 and table 1-B.

4. METHOD VALIDATION:

This method was validated according to ICH validation parameters which gives acceptable results ^{16- 19};

5. FORCED DEGRADATION STUDIES:

Sample is stressed by different conditions to assess stability indicating aspect of the method. The degraded sample is analyzed using a HPLC ^{23, 24};

Table 2: Sample and percentage impurity detection in forced degradation studies

6. RESULTS AND DISCUSSION:

6.1 Accuracy:

Accuracy study was performed; in that drug Assay was conducted in duplicate as per desired test method with equivalent amount of THIO & Ibuprofen into flask of volumetric each for level of spike to become the mass of THIO & IBU equivalent to 25 %, 50 %, 75 %, 100 %, 125 %and 150 %for amount labelled as per desired method of test. The mean % recovery of the THIO & IBU at each apex level is not less than 90.0% and not more than 110.0%. Result of recovery study shown in table 3-A and 3-B.

Table 3-A: Data of Accuracy for THIO

Table 3-B: Data of Accuracy for IBU

6.2 Precision:

The assays of THIO & IBU are not less than 90.0 % and not more than 110.0 %. The test results of combination gives that the method for test is precise. Result of recovery study for method and system precision are shown in table 4-A and 4-B.

Table 4-A: Method precision studies of THIO and IBU

Table 4-B: System precision studies of THIO and IBU

6.3 Linearity:

A series of solutions are prepare using IBU & THIO working standard at concentration levels for IBU from 400 ppm  to 2400 ppm of target concentration & concentration levels for THIO from 100 ppm to 600 ppm of target concentration. The line are fit of the system was illustrated graphically. The calibration curve (Concentration V/s. Response) of THIO and IBU are shown in fig. 4-A and 4-B resp. The result of linearity study shown in table 5-A, and optical characteristics are shown in 5-B.

Table 5-A: Linearity studies of THIO and IBU

Figure 4-A: Linearity Plot of THIO

Figure 4-B: Linearity Plot of IBU 

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

From the linearity data, the limit of detection and quantitation are calculated using the formula is as follow; 

             LOD = 3.3 × SD / Slope = 3.3 × 5843.029/ 35839 = 0.54

             LOQ = 10 × SD / Slope = 10 × 5843.029/ 35839 = 1.63

             LOD = 3.3 × SD / Slope = 3.3 × 3493.175/ 5749.7 = 2.00

             LOQ = 10 × SD / Slope = 10 × 3493.175/ 5749.7 = 6.08

Table 5-B: Optical characteristics of THIO and IBU

6.5 Robustness:

To check robustness, we made alteration in rate of flow and mobile phase composition. 

THIO & IBU was resolved from peaks of all other and there retention times were comparable with peaks obtained for mobile phase having flow rates 1.0 ml/min. The factor of symmetry for THIO & IBU for alteration in rate of flow was within the limits which are reported in table 6-A & 6-B.

THIO & IBU was resolved from all other peaks and there retention times were comparable with obtained for mobile phase having composition MeOH: H₂O (50:50 v/v). The factor of symmetry for THIO & IBU for alteration in mobile phase composition was within the limits which are reported in table 6-C & 6-D.

Table 6-A: Data for effect of alteration in rate of flow for THIO

Table 6-B: Data for effect of alteration in rate of flow for IBU

Table 6-C: Data for effect of alteration in mobile phase composition for THIO

Table 6-D: Data for effect of alteration in mobile phase composition for IBU

6.6 Ruggedness:

To check ruggedness, we made system-system variability as a system 1 to system 2 and study has performed on different HPLC systems for THIO & IBU under same circumstances at different times. Samples of six were made for drugs and each was studies as per method of test. Results of differentiation for both the obtained on 2 dissimilar systems of HPLC, proves that the given method for test assay are robust for variability of system-system for this combination. % RSD was within the limit for the systems. For result of system 1 refer table 4 and result of system 2 are given in table 7.

Table 7: Data of system to system variability (sample) System-2 for THIO and IBU

6.7 Forced Degradation Studies:

Analysis of statistics proved that the given method for THIO and IBU gives result values as per guidelines of ICH. All the stability studies results are shown in table 2 and Fig. 5.

Figure 5-A: Chromatograms of Acid degradation

Figure 5-B: Chromatograms of Base degradation

Figure 5-C: Chromatograms of Peroxide degradation

Figure 5-D: Chromatograms of Thermal degradation

Figure 5-E: Chromatograms of Photostability at 256 nm

Figure 5-F: Chromatograms of Photostability at 228 nm

7. CONCLUSION:

In this a novel RP- HPLC (Stability- indicating) method has developed successfully & certified for the analysis of THIO and IBU in tablet dosage first time as stated by guidelines of ICH. This performed method was satisfyingly separated all the two compounds (drug) with degradants, estimate the active contents in forced degradation which is significant part of drug development stage and the pharmaceutical industry has a lots of attraction in this area.

CONFLICT OF INTEREST: 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

REFERENCES:

1. Jadhav AP, Datar PA, Kedar TR, Kardile DP, Shete RV, Analytical method for determination of Thiocolchicoside in marketed pharmaceutical preparation: A Review, International Journal of Technology, 2019; 9(2): 45-53 https://doi.org/10.5958/2231-3915.2019.00011.7

2. Arvind R, Umarkar, Niki S, Rewatkar, Manoj S, Charde, Ritu MC, Simultaneous estimation of Thiocolchicoside and Diclofenac Potassium by UV-Spectrophotometer using multicomponent method, International Journal of ChemTech Research CODEN, 2011; 3(2):944-947

3. Revankumar DN, Ashutosh DT, Sunil BC, Dr. Anil V, Simultaneous Estimation of Paracetamol, Thiocolchicoside and Aceclofenac by UV-Spectrophotometer using multicomponent mode method, Journal of Pharma Research, 2011; 4(7):2297-2299

4. Sunil RD, Kanchan OR, Vidhya KB,Validated HPLC method for simultaneous estimation of Paracetamol, Aceclofenac and Thiocolchicoside in bulk drug and formulation, Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2011; 2(2):435-445

5. Rupali SJ, Nilima SP, Sameer SK, Devendra BZ, Amol TS, Development and validation of UV-Spectrophotometric methods for simultaneous estimation of Paracetamol and Ibuprofen in pure and tablet dosage form, Der Pharmacia Sinica, 2011; 2(3):164-171

6. Rupali K, Chhotaram S, Pallavi S, Kiran P, Prof. Anita K, Prof. Pandurang D, Validated Spectroscopic method for estimation of Ibuprofen from tablet formulation, Arch Pharm Science Research, 2010; 2(1):271-273

7. Hapse SA, Kadaskar PT, Shirsath AS, Difference spectrophotometric estimation and validation of Ibuprofen from bulk and tablet dosage form, Der Pharmacia Letter, 2011; 3(6):18-23

8. Chaudhari B, Trivedi JB, Simultaneous spectrophotometric estimation of Thiocolchicoside and Dexketoprofen trometamol in pharmaceutical dosage form, International Journal of Biomedical and Advance Research, 2012; 03(03):179-183

9. Chatragadda N, Avula PR, Sunkara B, Chandra BS., Determination of ACE inhibitor, Perindopril erbumine in bulk and tablet dosage form with HPLC, A Chemical Sciences Journal, 2012; 2(4):161-176 https://doi.org/10.9734/ACSJ/2012/2045

10. Saurabh KB, Nitin MV, Simultaneous estimation of Amlodipine and Rosuvastatin in combined bulk forms by RP-HPLC using ultraviolet detection, Bull Pharma Research, 2013; 3(1):29-33

11. Varma P, Rao A, Dinda SC, Validated stability indicating reverse phase HPLC method for the simultaneous estimation of Perindopril and Indapamide in pharmaceutical dosage forms, International Journal of Pharmacy, 2013; 3(1):277-289 https://doi.org/10.1155/2013/747060

12. Sunil R, Kanchan O, Vidhya K, Validated HPLC method for simultaneous estimation of Paracetamol, Aceclofenac and Thiocolchicoside in bulk drug and formulation, Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2011; 2(2):435-445

13. Ramchandra P, Pravin OP, Sanjay BB, Dinesh MD, Simultaneous estimation of Etodolac and Thiocolchicoside in bulk and in tablet formulation by UV-Spectrophotometry, The Chemical Industry & Chemical Engineering Quarterly, 2014; 20(1):9-17 https://doi.org/10.2298/CICEQ120114098P

14. Rele RV, Mali RN, Advance simultaneous determination of Paracetamol, Thiocolchicoside and Aceclofenac in tablets by reverse phase high performance liquid chromatography, Der Pharmacia Sinica, 2014; 5(1):34-39

15. Ravisankar P, Aswini M, Srinivasa BP, Devadasu C, Sai AR, Prathima B, Bala GG, Development and validation of RP-HPLC method for simultaneous determination of Metoprolol and Amlodipine in tablet dosage form, Journal of Chemical and Pharmaceutical Sciences, 2013; 7(2):113-121

16. Chinna RP, Nirmala JP, Pallavi P, Rafath Md, Kiran KG, New spectrophotometric methods for the determination of Thiocolchicoside in bulk and pharmaceutical formulations, World Journal of Pharmacy and Pharmaceutical Sciences, 2015; 4(5):1158-1167

17. Ankita B, Toral J, Kartik V, Arvind, Development and validation of UV-Visible spectrophotometric method for simultaneous estimation of Ketoprofen and Thiocolchicoside in solid oral dosage form, International Research Journal of Pharmacy, 2016; 7(5):53-58 https://doi.org/10.7897/2230-8407.07552

18. Wani YB, Patil D, A Stability indicating RP-HPLC method for simultaneous determination of Ibuprofen and Famotidine, Analytical Chemistry: An Indian Journal, 2016; 16(13):1-16

19. Venkatakishore T, Dr. Prasadrao M, Thulasi B, Ambica K, Mohan SS, Jaswanth G, RP-HPLC method for simultaneous determination of ibuprofen and famotidine in pharmaceutical dosage form, World Journal of Pharmacy and Pharmaceutical Sciences, 2016; 5(12):1017-1025

20. Bhagat DM, Vaibhav R, Rakesh KJ, Varsha K, UV-Spectrophotometric and Stability Indicating RP-HPLC method for the simultaneous estimation of Amlodipine Besylate and Indapamide, Analytical Chemistry Letters, 2016; 6(4):354-370 https://doi.org/10.1080/22297928.2016.1217787

21. Soujanya S, Method development and validation of simultaneous estimation of Perindopril and Indapamide in tablet by RP-HPLC method, Indo American Journal of Pharmaceutical Research, 2017; 7(9):390-396

22. Ashutosh K, Manidipa D, Seshagiri R, Gowri S, A new RP-HPLC Stability indicating method development and validation for simultaneous estimation of Ibuprofen & Famotidine in bulk as well in pharmaceutical dosages form by using PDA detector, International Journal of Pharmaceutical Sciences and Research, 2014; 5(9):3829-3839

23. Kalpana N, Shanmukha KJ, Ramachandran D, Ganji R, Ganta S, Method development and validation of stability indicating RP-HPLC method for simultaneous estimation of Perindopril erbumine and Amlodipine besylate in bulk and its pharmaceutical formulations, Asian Journal of Agriculture and Development, 2014; 2(5):672-685

24. Santhoshi GR, Annapurna N, Santosh T, Durga B, Raziya SK, Evaluation of a new stability indicating method for the determination of Aceclofenac and Thiocolchicoside in Pharmaceutical dosage form by RP-HPLC, Oriental Journal of Chemistry, 2017; 33(3):1337-1346 https://doi.org/10.13005/ojc/330334