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Research Article Pharmaceutical Sciences METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR
SIMULTANEOUS ESTIMATION OF DICYCLOMINE HYDROCHLORIDE AND
DICLOFENAC POTASSIUM IN TABLET DOSAGE FORMS
Vijjigiri Chaitanya, Daravath Bhaskar*, Kamarapu SK
Department of Pharmaceutical Analysis, Sri Shivani College Of Pharmacy,
Mulugu Road, Warangal, A.P, India, 506007
*Corresponding Author Email:
ABSTRACT
The study describes method development and subsequent validation of RP-HPLC method for simultaneous
estimation of Dicyclomine Hydrochloride, Diclofenac Potassium in combined tablet dosage forms.
Chromatographic separation was achieved on a Kromasil C18 (250 mm × 4.6 mm id, 5μm) column using a mobile
phase ratio consisting of (70:30) Methanol: Water at flow rate 1.0 ml/min. The detection wavelength is 263 nm.
The retention times of Dicyclomine Hydrochloride and Diclofenac Potassium were found to be 2.951 min and 4.195
min respectively. The developed method was validated as per ICH guidelines using the parameters such as
accuracy, precision, linearity, LOD, LOQ and robustness. The developed and validated method was successfully
used for the quantitative analysis of Dicyclomine Hydrochloride and Diclofenac Potassium in tablet dosage forms.
KEY WORDS
Dicyclomine Hydrochloride, Diclofenac Potassium, Cataspa tablet dosage forms, HPLC, Method development,
Method validation.
INTRODUCTION:
steroidal anti-inflammatory agent (NSAID) that is Diclofenac Potassium chemically, Potassium [2-[(2,6- widely used in pharmaceutical preparations for dichlorophenyl)amino]phenyl]acetate is a non- antipyretic and analgesic actions. Figure-1: Chemical structure of Diclofenac Potassium

Dicyclomine Hydrochloride is a gastrointestinal
achieved via a dual mechanism: (1) a specific antispasmodic antacid. The chemical name of the anticholinergic effect (antimuscarinic) acetylcholine-receptor sites and (2) a direct effect cyclohexylcyclohexane-1-carboxylate HCl. Its action is upon smooth muscle (musculotropic). International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar*et al
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Figure-2: Chemical structure of Dicyclomine Hydrochloride

Many UV, HPLC and HPTLC based methods have been
reported for estimation of these drugs alone as well as in combination with other drugs in pharmaceutical Standard Solution preparation:
dosage forms. But no method had yet been reported About 100 mg of pure samples of Dicyclomine for simultaneous estimation of these two drugs using Hydrochloride and Diclofenac Potassium were HPLC in bulk drug and pharmaceutical dosage forms. accurately weighed and transferred to a 100 ml Therefore, the present work was aimed to develop volumetric flask. Then they were dissolved in mobile and validate a new RP- HPLC method for phase and the solution was made up to volume with simultaneous estimation of Diclofenac Potassium and the same. Each ml of stock solution contained 1000 Dicyclomine Hydrochloride in pharmaceutical dosage g/ml. 10 ml of this stock solution was diluted to 100 ml with mobile phase to give 100 g/ml solution (Working Stock). EXPERIMENTAL: MATERIALS AND REAGENTS:
Dicyclomine Hydrochloride and Diclofenac Potassium Preparation of sample solution from dosage form:
were obtained from Active Pharma Labs, Hyderabad, Twenty tablets were weighed and crushed to fine India. A commercial preparation (Cataspa Tablet) powder. The tablet powder equivalent to 100 mg of used for analysis was procured from pharma market. Dicyclomine Hydrochloride and Diclofenac Potassium Each tablet contains 20mg Dicyclomine Hydrochloride was transferred to a 100 ml volumetric flask and and 50mg Diclofenac Potassium. HPLC grade dissolved in mobile phase and the content was made Methanol and water (Finar chemicals limited up to mark with mobile phase. Then the sample solution kept in sonicater for 15 min and the solution was filtered through 0.45µm filter paper. Instrumentation: RP-HPLC was performed using
Shimadzu HPLC system consisting of a pump LC-20AD plus, rheodyne sample injection port with 20 With the optimized chromatographic conditions microlitre loop , SPD-M20A Photo diode array mentioned early, a steady base line was recorded. detector (PDA), LC solutions software , column used After the stabilization of baseline, inject the sample was Kromasil C18 (250 x 4.6mm, 5μm). solution of a concentration 30 µg/ml of each Dicyclomine Hydrochloride and Diclofenac Potassium Chromatographic conditions:
respectively. Each solution was run an interval of 10 A reverse phase column [Kromasil C18 (250 x 4.6mm, minutes and the peak areas were found and amount 5μm particle size)], equilibrated with mobile phase of the drug and percentage of assay was calculated by [Methanol: Water] (70:30) was used. Mobile phase regression equations which were tabulated in Table-6
flow rate was maintained at 1ml/min and effluents and chromatograms were recorded and presented in were monitored at 263nm. The sample was injected Figure-3.
using 20 microlitre fixed loop rheodyne injector and Validation of HPLC method:
run time was 10 mins. The proposed RP-HPLC method was validated as per International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar* et al
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Linearity:
slope of calibration plot. Results are shown in the Hydrochloride and Diclofenac Potassium separately by plotting a Calibration curve of peak area against Robustness:
their respective concentration. From the calibration The robustness study was done by making small curve it was found that the curve was linear in the changes in the optimized method parameters like range of 20-80 µg/ml for Dicyclomine Hydrochloride ±1% change in pH and ±1% change in flow rate. There and Diclofenac Potassium. The regression equations was no significant impact on the retention time and for calibration curve was y=303.7x+0.428 (R2=1) for Dicyclomine Hydrochloride, y=1961.x+1.5 (R2=1) for Diclofenac Potassium. Results were shown in the RESULTS AND DISCUSSION
Table-1.
To develop a precise, accurate and suitable HPLC method for simultaneous estimation of Dicyclomine Precision:
Hydrochloride and Diclofenac Potassium in tablet Precision study was performed to find out intraday dosage form different mobile phases such as and interday variations. The intraday and interday Methanol and Water in different proportions were precision study of Dicyclomine Hydrochloride and used and finally Methanol: Water (70:30) was Diclofenac Potassium was carried out by estimating selected as an appropriate mobile phase, which give the correspondence response 3 times on the same good retention time and acceptable peak parameters day and on 3 different days for 3 different for Dicyclomine Hydrochloride and Diclofenac concentrations of Dicyclomine Hydrochloride and Potassium. The linear relationship was carried out Diclofenac Potassium and the results were reported between the peak area and concentration from a in terms of % relative standard deviation (%RSD). range of 20-80µg/ml for Dicyclomine Hydrochloride However, all results fall within acceptance limits (RSD and 20-80µg/ml for Diclofenac Potassium. The < 2), as shown in Table-4.
linearity can be expressed as correlation coefficient Accuracy:
0.998 and 0.999 for Dicyclomine Hydrochloride and The accuracy of the method was determined by calculating the recovery studies at three levels (50%, coefficient, y- intercept, slope of regression line is 100% and 150%) by standard addition method. shown in Table-1. Precision was determined as
intermediate precision as per ICH guidelines. It was Hydrochloride and Diclofenac Potassium were added assessed at 3 concentration levels %RSD obtained was to the pre quantified samples and they were less than 2% for both drugs. The results of precision subjected to proposed HPLC method. The recoveries are shown in Table-4. System suitability parameters
results of Dicyclomine Hydrochloride and Diclofenac for proposed method are shown in Table-8. Assay of
Potassium in pharmaceutical preparation are shown tablets Dicyclomine Hydrochloride and Diclofenac in the Table-3.
determinations were carried out on tablets. Limit of Detection (LOD) and Limit of Quantitation
Percentage purity was found to be 98.6% and 99.26%. Results of tablet analysis were shown in Table-2.
The LOD and LOQ for Dicyclomine Hydrochloride and Robustness studies were carried out after deliberate Diclofenac Potassium were separately determined by alterations of flow rate and mobile phase based on calculating the signal-to-noise ratio (s/n is compositions. It was observed that did not lead to 3.3 for LOD and 10 for LOQ) and from the calibration changes of retention times of peak of interest. curves the standard deviation of the y-intercepts and Percentage of recovery shows that method is free slope of the regression lines were used. σ is standard from interference of the excipients used in the deviation of response (y – intercept) and S is the formulation shown in Table-3.
International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar* et al
Int J Pharm Bio Sci
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Figure-3: Chromatogram of combined Tablet dosage form
Figure-4: Chromatogram of Dicyclomine Hydrochloride and Diclofenac Potassium for Recovery studies by HPLC
method (50%).
Figure-5: Chromatogram of Dicyclomine Hydrochloride and Diclofenac Potassium for Recovery studies by HPLC
method (100%).
International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar* et al
Int J Pharm Bio Sci
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Figure-6: Chromatogram of Dicyclomine Hydrochloride and Diclofenac Potassium for Recovery studies by HPLC
method (150%).
Figure-7: Chromatogram of Dicyclomine Hydrochloride and Diclofenac Potassium for Robustness by HPLC
method.
BRAND (Cataspa)
% Amount found ± SD
Hydrochloride 250mg + Diclofenac Potassium 4mg Diclofenac Potassium 99.26 ± 0.74 Table -1: Analysis of tablet formulation
Label claim Amount added Amount
% Recovery
Recovered
Table 2: Results of Recovery studies
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Theoretical
Intra-day concentration (µg/ml)
Inter-day concentration (µg/ml)
concentration
(µg/ml)
Hydrochloride 40 Table-3: Intra – day and Inter – day precision of Dicyclomine Hydrochloride and Diclofenac Potassium Standard
solutions

Parameters
Dicyclomine
Diclofenac Potassium
Limit of detection Limit of quantitation Table -4: Results of precision and LOD & LOQ
Flow rate
Mean (Peak Rt value
Overall %RSD
Table-5: Robustness-Effect of Flow rate (HPLC)
Parameters
Dicyclomine Hydrochloride
Diclofenac Potassium
Acceptance limits
Theoretical plates >2000
Asymmetry Factor Table-6: System suitability parameters
International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
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Concentration (g/ml)
Peak Area
Table-7: Linearity graph data of HPLC method for Dicyclomine Hydrochloride
Peak Area
(g /ml)
20
Table-8: Linearity graph data of HPLC method for Diclofenac Potassium
Figure-8: Linearity studies
Standard graph of Dicyclomine Hydrochloride
reaa 15000
y = 303.7x + 0.428 International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
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Standard garph of Diclofenac Potassium
reaa 100000

CONCLUSION
REFERENCES
The present paper describes proposed RP-HPLC 1. Skoog, D.A., West, D.M. and Holler, J.F., In; Analytical method for the simultaneous estimation of Chemistry-An Introduction, 6th Edn. Saunders College Dicyclomine Hydrochloride and Diclofenac Potassium Publishing, 1994, pp. in tablet dosage form is accurate, precise, linear, 2. Connors, K.A., In; A Text Book of Pharmaceutical Analysis, 3rd Edn. John Wiley and Sons, 2002, pp. 581. robust, simple and rapid. Acceptable regression 3. Sethi, P.D., In; Quantitative Analysis of drug in values, RSD % and standard deviations which make it versatile and valuable for simultaneous estimation of Publishers, 1997, pp. 8-9, 43-52. two drugs in tablet formulation. Acceptable values of 4. Beckett, A.H. and Stenlake, J.B., Eds., In; Practical precision and accuracy have been obtained as per Pharmaceutical Chemistry, 4th Edn. , Part two, CBS guidelines for assay validation. The run time is Publication, 2004, pp. 275, 276. relatively short i.e. within 10 mins. So, A large number 5. Scott, R.P.W., Eds., In; Liquid Chromatography for the of samples can be analysed in short period of time. Analyst, vol.67, Marcel Dekker, Inc., 1994, pp. 1. The results of this developed RP-HPLC method can be 6. Kasture, A.V., Mahadik, K.R., Wadodkar, S.G. and More, H.N., In; Pharmaceutical Analysis- Instrumental could be conveniently adopted for quality control Methods, Vol. II, NiraliPrakashan, 2001, pp. 6, 7. analysis of Dicyclomine Hydrochloride and Diclofenac 7. Willard, H.H., Merritt, L.L., Dean, J.A., and Settle, F.A., Potassium simultaneously from tablet dosage form. In; Instrumental Methods of Analysis, 7th Edn. CBS Publication, 1986,pp. 592-604. 8. Snyder, L.R., Kirkland, J.J. and Glajch, J.L., In; Practical The authors are thankful to Active Pharma Labs, HPLC Method Development, 2nd Edn. John Wiley and Hyderabad, India for providing sample of Dicyclomine Sons, Inc., 1997, pp. 1-2 Hydrochloride and Diclofenac Potassium for research. 9. ICH Harmonized Tripartite Guideline, Validation of Analytical Procedure: Methodology, Q2B, 1996, pp. 1- The authors are grateful to Department of Pharmaceutical Analysis, Sri Shivani College of 10. 10. Stephen, G., Schulman and Vogt, B.S., In; Munson, Pharmacy, Affiliated to Kakatiya University, Warangal, J.W., Eds, Pharmaceutical Analysis Modern Methods, Andhra pradesh, India, for providing all the facilities Part – II, Internationa Medical Book Distributors, to carry out the work. The authors express heartful Mumbai, India, 2001, pp. 15-10 thanks to Principal and Management of Sri Shivani 11. Connors Kenneth A. "A text book of Pharmaceutical college of Pharmacy for their best wishes and Analysis",Third edition, John Wiley and Sons, pp.373- constant support and encouragement during course 12. Willard - Hobart H, Lynne L. Merritt Jr, John A. Dean of project work. Frank A. Sttle Jr., "Instumental Methods of Analysis", International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
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Seventh edition, CBS Publishers and Distributors, New Delhi, pp.1-12,580-61,614-652. Acetaminophen And Clidinium Bromide In Solid Dosage 13. Chatwal Gurdeep R.,"Instrumental method of chemical Form; Bachani et al. Int J Pharm Pharm Sci, Vol 5, Suppl analysis" Himalaya publishing house,Delhi, pp.2.149- 2.184, 2.624-2.639 23. 22.Amey R. Keer, Shweta S. Havele, Kiran H. Gopani 14. USP NF 25 Page no 621,1065 and USP NF 20 Page No and Sunil R. Dhaneshwar* Application of High 2256 -2259,BP Appendix 3D 2004 software,IP 2007 performance thin layer chromatography-densitometry for the determination of Dicyclomine HCL in bulk drug 15. Sunil R Dhaneshwar*, Amey R Keer, Shweta S Havele, and injection formulation; Sunil R. Dhaneshwar et al Kiran H Gopani;Validated HPTLC Method for Der Pharma Chemica, 2011, 3 (1):549-556; Simultaneous Estimation of Diclofenac Potassium and 24. G. Rajalakshmi*, C.H. Vamsi, R. Balachandar, N. Dicyclomine Hcl in Tablet Formulation; October – Damodharan; Formulation and evaluation of diclofenac December 2011 RJPBCS Volume 2 Issue 4 Page No. potassium effervescent tablets; G. Rajalakshmi et al., Int J Pharm Biomed Res 2011, 2(4), 237-243. 16. Milindkumar P Rajput, Vishal V Bharekar, Savita S 25. 24.Sindhur Nag N1*, Gouthami B1, Madhuri L1, Yadav, Toufik S Mulla and Janhavi R Rao*Validated Krishnaveni N1, Meyyanathan S N1 and Suresh B2; Hptlc Method For Simultaneous Estimation Of Development and validation of a RP-HPLC method for Diclofenac Potassium And Metaxalone In Bulk Drug the simultaneous determination of paracetamol and And Formulation; Rao J R et al. / Pharmacie Globale diclofenac potassium on stainless steel surface of (IJCP) 2011, 12 (04) pharmaceutical manufacturing equipments; Sindhur 17. Roshni J. Hedpara*, Vibhuti Chhatarala and Ashwin Nag Net al J. Chem. Pharm. Res., 2012, 4(3):1670-1675. Agola;A Validated Rp- Hplc Method For The 26. 25.D.K.Mandloi1*, P.K.Tyagi2, V.K.Rai2, S. Dey2, R.K Simultaneous Estimation Of Paracetamol And Ashada1, P.Mohanraj1; Method development and validation of RP- HPLC in the application of invitro Formulations; Ijpsr (2013), Vol. 4, Issue 6 dissolution study of Lamivudine in bulk drug and tablet 18. K. Vinod kumar*1, M.sudhakar1, Y Padmanabha formulation; D.K.Mandloi et al Journal of Chemical and Reddy2, A. Ravindra2 Validated RP- HPLC Method for Pharmaceutical Research, 2009, 1(1): 286-296. Simultaneous Estimation of Metaxalone and Diclofenac 27. 26.Kantariya B*1, Agola A1, Roshani H1, Ghetia U1, Dr. potassium in Combined Dosage Form Vinodkumar et. S. Sai Shivam1 Development and Validation of a RP- al., Am. J. PharmTech Res. 2013; 3(4) ISSN: 2249-3387 HPLC Method for the Simultaneous Estimation of 19. Dipal Prajapati* And Dr. Hasumati Raj1. Simultaneous Dicyclominehydrochloride By Rp-Hplc Method; Int J IJPRS/V2/I2/00115, Pharm Bio Sci 2012 July; 3(3): (P) 611 – 625 Accepted On: 03/07/2013. 20. 20.J. Varshosaz1, J. Emami1, N. Tavakoli1, M. 28. 27.Chaitany A. Dave*, S. K. Tiwari, K. D. Brahmbhatt, P. Minaiyan2, N. Rahmani1, F. Ahmadi3,4 and F. M. Patel, S. B. Shah Development And Validation Of Dorkoosh5; Development and validation of a rapid Rp-Hplc Method For Estimation Of Omeprazole And HPLC method for simultaneous analysis of budesonide Dicyclomine Hydrochloride In Pharmaceutical Dosage and its novel synthesized hemiesters in colon specific Form Vol - 4, Issue - 3, Supl - 1 Apr-Jul 2013 ISSN: 0976- formulations; J. Varshosaz et al. / RPS 2011; 6(2): 107- 29. B.Gowramma*, S. Rajan, S. Muralidharan, S. N. 21. 20. Chintan R Patel1, Ritu V Kimbahune2, Prachi V Meyyanathan and B. Suresh A Validated Rp-Hplc Method For Simultaneous Estimation Of Paracetamol LVG1Spectrophotometric Estimation of Metaxalone and Diclofenac Potassium by Multicomponent Formulation; B.Gowramma et al /Int.J. ChemTech Analytical Method from Tablet Dosage Form; Patel et al., J Anal Bioanal Techniques 2012, 3:3 30. www.drugbank.com/dicyclomine hydrochloride cited 22. Manish Hiranand Bachani*, Dhaval Suresh Acharya, as on 06/08/2013 31. www.drugbank.com/diclofenac potassium cited as on Validation Of Hplc Method For Simultaneous International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar* et al
Int J Pharm Bio Sci
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IJPBS Volume 3 Issue 4 OCT-DEC 2013 255-264
*Corresponding Author:
Daravath Bhaskar *
Department of Pharmaceutical Analysis,
Sri Shivani College Of Pharmacy,
Mulugu Road, Warangal,
Andhra Pradesh, India, 506001.
International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)
Daravath Bhaskar* et al
Int J Pharm Bio Sci
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