A Validated Ultra Performance Liquid Chromatography Method for Simultaneous Estimation of Diacerein and Aceclofenac in Bulk and Pharmaceutical Formulation

 

Bharatee P. Chaudhari, Kratika Daniel

Faculty of Pharmacy, Mandsaur University, Mandsaur, Madhya Pradesh, India.

*Corresponding Author E-mail: bharatichaware@gmail.com

 

ABSTRACT:

A simple, exceptionally cost-effective, extremely accurate, quite precise and highly reproducible analytical method was developed and validated for simultaneous estimation of Diacerein and Aceclofenac in bulk and Pharmaceutical formulation. The new Ultra Performance Liquid Chromatography technique was developed. Method- The separation has been done on column ACQUITY UPLC BEH Shield C18 (50 x 2.1mm), 1.7µm (40°C temperature). The mobile phase contained Buffer and Acetonitrile (Buffer: ACN) (55:45 V/V).. The flow rate was set at 0.4ml/min, and detected at 268nm with PDA detector. Mobile phase was sonicated for 15 min. before use. 5μl of samples of standard stock solution and tablet solution were injected. Different trials were performed to separate diacerein and aceclofenac. The total run time of the detection was 4 min. The developed method was validated against different validation parameters. Results - The retention times were obtained at 1.762min. and 2.891 min. for Diacerein and Aceclofenac respectively. Accuracy study was observed in the range of 100.33% to 101.67% with less than 2% RSD. LOQ was found to be 2.98μg/ml and 5.9041μg/ml. similarly LOD was found to be 0.9841μg/ml and 1.9467μg/ml. for Diacerein and Aceclofenac respectively. Precision %RSD for intraday and interday were found to be 0.19 and 0.18 for Diacerein , 0.44 and 0.16 for Aceclofenac respectively. Linearity was found in the range of 2.5-17.5μg/ml and 5-35μg/ml for Diacerein and Aceclofenac respectively. The method was found robust by deliberate changes in the flow rate, ratio of mobile phase and detection wavelength. Conclusion-The method was found to be satisfactory and can be used successfully for determination of Diacerein and Aceclofenac simultaneously in bulk and pharmaceutical dosage form.

 

KEYWORDS: UPLC, Diacerein, Aceclofenac, Anti-inflammatory, Method development.

 

 

INTRODUCTION:

Diacerein is 1,8-diacetoxy-3-carboxyanthraquinone, used to treat osteoarthritis. It is synthesised from glucopyranoside aloin. Diacerein is a selective inhibitor of interleukin-1 having protective effect on granuloma-induced cartilage breakdown by a reduction in the concentrations of proinflammatory cytokines1. It is metabolized into its active metabolite rhein. Diacerein and rhein both are anthraquinone compounds2,3.

 

Aceclofenac is (2-[(2,6-dichlorophenyl) amino] phenyl acetoxyacetic acid). It is used as an effective non-steroidal anti-inflammatory drug (NSAID) derived from the phenylacetic acid with pronounced anti-inflammatory, analgesic and antipyretic properties. It is use in painful conditions like rheumatoid arthritis, osteoarthritis and ankylosing spondylatis4,5,6,7.

 

A combined fixed dose of 50mg diacerein and 100mg aceclofenac tablet is available for the treatment of osteoarthritis. The two main aspects of drug product analysis that play an important role in shelf life determinations are assay of active drug and degradants generated during the stability study.

 

The literature survey revealed that few methods have been reported for the estimation of Diacerein and Aceclofenac8. Some spectroscopic9,10,11, HPLC 12,13,14,15and HPTLC16,17 methods were reported. However, most of reference HPLC methods are less sensitive. RP-UPLC method for the simultaneous determination of aceclofenac and diacerein in tablet dosage form is not available in any pharmacopeia. Hence, it was felt essential to develop and validate a sensitive, accurate UPLC method for the simultaneous determination of aceclofenac and diacerein in tablet dosage form. The chemical structures of diacerein and aceclofenac are shown in Figure 1.and 2.

 

Figure No.1 Structure of Diacerein

 

Figure No.2 Structure of Aceclofenac

 

MATERIAL AND METHOD:

For the present study all the materials were obtained from Merck India Ltd, Mumbai. All the material was of analytical grade. Diacerein and aceclofenac were obtained from Balaji Drugs Surat, Gujrat. Diacerien, aceclofenac tablets were purchase from local market.

 

Chromatographic Condition:

WATER ACQUITY UPLC H –CLASS SYSTEM consist of column ACQUITY UPLC BEH ShieldC18 (50x2.1)mm, 1.7µm particle size, equipped with EMPOWER software for processing data. Sonicator and pH meter also used. The mobile phase Buffer:Acetonitrile (55:45 V/V). was filter with 0.22μm membrane filter and sonicate for 15 minutes. Injecting 5μl of sample with 0.4ml/min flow rate and 25±20C column temperature. Sample was detected at 268nm with PDA detector. The total run time of the detection is 4 min.

 

Preparation of Standard Solution:

The standard stock solution was prepared by taking 50mg of diacerein and 100mg of Aceclofenac in 100ml volumetric flask with Methanol. 1ml of the solution was taken from the standard stock solution in 100ml volumetric flask and diluted with mobile phase to make the final concentration of 5μg/ml of Diacerein and 10μg/ml of Aceclofenac.

 

Preparation of sample solution:

Total of 20 tablets were weighted and triturated. An amount equivalent to 50mg of Diacerien and 100mg of Aceclofenac were transferred to 100ml volumetric flask. Diluted the solution with methanol and sonicate. Solution was filtered through 0.22μm membrane filter, 1 ml of the prepared solution was taken into 100ml volumetric flask and adjusts the volume with mobile phase. Analyze the solution with optimized chromatographic conditions.

 

Method development:

Different trials were done to study effect of some important factors by changing one parameter at a time and keeping other constant. Different chromatograms were observed to separate Diacerein and Aceclofenac properly.

 

Validation:

Developed method was validated by various parameters as per ICH guidelines.18,19,20

 

Linearity:

Linearity was determined at 2.5-17.5μg/ml range (i.e. 50%-350%) for Diacerein and 5-150μg/ml (i.e. 50%-350%) for aceclofenac standard solutions. Peak area for all the observations were recorded and graphs were constructed for both as Peak area Vs. concentration.

 

Accuracy:

Accuracy was checked by performing assays in triplicate at three concentration level 80%, 100% and 120% by standard addition method where known amount of standard mixture of both the drugs was added to pre-analyzed sample and subjected to analysis.

 

Precision:

Precision of method was carried out by six independent samples of preparations against standard for intraday precision and was determined by calculating %RSD. For interday precision a different analyst on different day in a same laboratory analyzed 6 test samples against standard and %RSD was determine.

 

Robustness:

To study robustness small but deliberate variations in chromatographic conditions were studied. The effect of change in flow rate, ratio of mobile phase and wavelength on system suitability parameter were studied.

 

Limit of detection and Limit of quantification:

LOD and LOQ of the method were determined by analyzing low concentrations of the standard solutions. LOD is the smallest concentration of analyte where signal to noise ratio 3:1 and LOQ is the smallest concentration of the analyte where signal to noise ratio 10:1.21-25

 

RESULT AND DISCUSSION:

In the present study wavelength was selected at 268nm by the overlay UV spectra of diacerein and aceclofenac. After various trials it was found that Diacerein and Aceclofenac were separated with adequate resolution with Buffer:Acetonitrile (55:45 V/V).as mobile phase at flow rate 0.4ml/min on Acquity UPLC BEH C18 (50x2.1) mm, 1.7µm column detected on PDA detector, when sample was run for 4 minutes. The retention time was found as 1.762 min. for diacerein and 2.891 min for aceclofenac. Fig. 3 represent chromatogram for Diacerein and Aceclofenac. Optimize mobile phase proportion was provide good resolution between Diacerein and Aceclofenac.

 

Figure No.3: Chromatogram for Diacerrine and Aceclofenac

 

Validation:

Linearity:

The response of the drug was found to be linear in the investigation concentration range 2.5μg/ml to 17.5μg/ml for Diacerine and 5μg/ml to 35μg/ml for Aceclofenac with correlation coefficient 0.9996 (Figure4) and for aceclofenac correlation coefficient 0.9999 (Figure5).

 

Figure No.4: Linearity study for Diacerein

 

Figure No.5: Linearity study for aceclofenac

 

Accuracy:

The mean recovery for Diacerein and aceclofenac (Table 1) indicate that the method was accurate.

Table No.1: Accuracy data for Diacerein and aceclofenac

Drug

Level %

Amount added(μg/ml)

Amount Found(μg/ml)

%Recovery

Mean Recovery %

SD

%RSD

 

 

 

 

Diacerein

80

4

4.14

103.50

 

101.67

 

0.08

0.07

 

4

3.96

99.00

4

4.10

102.50

100

5

4.98

99.60

 

101.47

1.36

 

1.34

 

5

5.10

102.00

5

5.14

102.80

120

6

6.10

101.60

101.31

1.24

 

1.22

 

6

6.16

102.67

6

5.98

99.67

 

 

 

 

Aceclofenac

80

8

8.12

101.50

101.46

0.25

 

0.25

 

8

8.14

101.75

8

8.09

101.13

100

10

10.10

101.00

100.70

0.65

 

0.65

 

10

9.98

99.80

10

10.13

101.30

120

12

12.16

101.33

100.33

0.72

0.72

12

11.96

99.67

12

12.00

100.00

 

Precision:

The RSD values for intraday precision study and interday precision study was < 2.0 % for Diacerein and Aceclofenac. It confirmed that the method was precise.

 

Robustness:

The result of robustness study showed no effect on actual value. System suitability parameters were found satisfactory. So the present UPLC method would be remarked as robust.

 

Table No.2: Precision data for Diacerein and aceclofenac

Assay

Set

Paracetamol

Etodolac

Interday

Intraday

Interday

Intraday

1

100.25

100.25

99.98

100.24

2

99.92

100.45

99.69

101.1

3

99.83

99.85

99.94

100.54

4

100.34

99.96

100.14

99.92

5

100.05

100.16

100.11

99.95

6

100.13

100.12

100.13

99.88

Mean

100.09

100.13

99.99

100.27

SD

0.18

0.19

0.16

0.44

%RSD

0.18

0.19

0.16

0.44

 

Table No.3: Robustness study for aceclofenac

Robust Condition

Assay %

System suitability Parameter

Theoretical Plate

Asymmetry

%RSD

Diacerein

Aceclofenac

Diacerein

Aceclofenac

Diacerein

Aceclofenac

Diacerein

Aceclofenac

Flow rate(+0.1)

99.65

100.06

3235

5043

1.20

1.16

0.26

0.24

Flow Rate

(-0.1)

100.01

99.12

3256

5154

1.25

1.13

0.24

0.29

Mobile Phase (56:44)

99.50

100.86

3486

5127

1.22

1.18

0.23

0.18

Mobile Phase (54:46)

100.06

100.39

3526

5016

1.24

1.16

0.28

0.19

Wavelength (+5)

100.04

99.96

3368

5138

1.23

1.19

0.19

0.26

Wavelength (-5)

99.78

100.35

3246

5024

1.22

1.17

0.21

0.28

 

LOD and LOQ:

The limit of detection and limit of quantitation were determined for Diacerein and aceclofenac as per ICH Q2R1 guideline. The LOD and LOQ for Diacerein and aceclofenac were estimated at a signal-to- noise ratio of 3:1 and 10:1, respectively by injecting a series of diluted solutions with known concentration. The limit of detection for Diacerein and Aceclofenac were 0.9841 μg/ml and 1.9467μg/ml. similarly LOQ for Diacerein and Aceclofenac were found as 2.98μg/ml and 5.9041μg/ml respectively.

 

CONCLUSION:

In the present research work a UPLC method was developed for the estimation of Diacerein and aceclofenac from bulk and dosage form. Present UPLC method was validated for precision, linearity, accuracy, ,Robustness and LOD and LOQ. Very simple method was used for the preparations, shorter run time was applied and low concentration of organic solvent was used. The retention time was found as 1.762 min. for diacerein and 2.891 min for aceclofenac. It indicates that both the drugs were separated with proper resolution. Hence the present method can be considered as simple, precise, accurate and easy to apply for the routine estimation of diacerein and aceclofenac.

 

AKNOWLEDGEMENT:

The authors are thankful to the management of Mandsour University, Mandsour for providing all the facilities and encouragement for the work.

 

REFERENCES:

1.      Tamura T, Ohmori K. Rhein an Active Metabolite of Diacerhein Suppresses the Interleukin-1 Induced proteoglycan degradation in cultured rabbit articular chondrocytes. Japanes Journal of Pharmacology. 2001; 85: 101–104. doi: 10.1254/jjp.85.101.

2.      Sweetman SC. Martindale: The Complete Drug Reference, 35th ed., London, UK: Pharmaceutical Press; 2007: 35-36.

3.      Government of India Ministry of Health & Family Welfare. Indian Pharmacopoeia. 5th Ed. Ghaziabad (INDIA): The Indian Pharmacopoeia Commission; 2007; 2. P. 63-64.

4.      Sharma MC, Sharma S. Stability and Reverse Phase HPLC Assay Method For Determination of Diacerein and Aceclofenac In Tablet Dosage Form - Application To Dissolution Studies. Asian Journal of Pharmaceutical and Clinical Research. 2011;4(1): 31-34. idosi.org/aejsr/6(3)11/5.

5.      Ojha A, Rathod R, Padh H. Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma. Journal of Chromatography B. 2009; 877:1145–1148. DOI: 10.1016/j.jchromb.2009.02.061

6.      Hinz B, Auge D, Rau T. Rietbrock S, Brune K, Werner U. Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography. Biomedical Chromatography. 2003;17:268–275. doi.org/10.1002/bmc.243

7.      Godse VP, Deodhar MN, Bhosale AV, Sonawane RA, Sakpal PS, Borkar DD, Bafana YS. Reverse Phase HPLC Method for Determination of Aceclofenac and Paracetamol in Tablet Dosage Form. Asian Journal of Research in Chemistry. 2009;2:37–40. DOI:10.4103/0250-474X.26672

8.      Hasan NY, Abdel-Elkawy M, Elzeany BE and Wagieh. Stability indicating methods for the determination of aceclofenac. IL Farmaco. 2003; 58: 91-99. doi.org/10.1016/S0014-827X(02)01271-5.

9.      Borgmann SHM, Parcianello L, Arend MZ, Bajerski L, Cardoso G. Development and Validation of a Dissolution Method with Spectrophotometric Analysis for Diacerhein Capsules. Scientia Pharmaceutica. 2008;76:541–554. doi.org/10.3797/scipharm.0804-17

10.   Nyola NK, Kalra N. Spectrophotometric determination of diacerin in bulk and pharmaceutical formulation. International journal of Pharma and Bio Sciences. 2010; 1(4):202-07.

11.   Dhaduk DM, Rathod BG, Patel PB. Estimation of Sildenafil Citrate and Diacerein Hydrochloride in their Combined Dosage Form by Validated UV Spectroscopic method. Inventi Rapid: Pharm Analysis & Quality Assuranc. 2012;5(10): 337.

12.   Bhinge JR, Kumar RV, Sinha VR. A simple and sensitive stability indicating RP-HPLC assay method for the determinationofaceclofenac.JournalofChromatographicScience.2008;46:440444. doi.org/10.1093/chromsci/46.5.440

13.   Momin MY, Yeole PG, Puranik MP, Wadher SJ. Reverse phase HPLC method for determination of aceclofenac and paracetamol in tablet dosage form. Indian Journal of Pharmaceutical Sciences. 2006;68:387–389. DOI: 10.4103/0250-474X.26672

14.   Karthikeyan V, Yuvaraj V, Nema RK. Simultaneous estimation of paracetamol, chlorzoxazone and aceclofenac in pharmaceutical formulation by HPLC method. International Journal of ChemTech Research. 2009;1:457–460.

15.   Kachhadia PK, Doshi AS, Ram VR, Joshi HS. Validated LC Method for Simultaneous Analysis of Tramadol HydrochlorideandAceclofenacinaCommercialTablet.Chromatographia.2008;68:997 52.doi: 10.3797/scipharm.1108-04

16.   Gandhi SP, Dewani MG, Borole TC, Damle MC. Development and Validation of Stability Indicating HPTLC Method for Determination of Diacerein and Aceclofenac as Bulk Drug and in Tablet Dosage Form. European journal of Advanced chemistry research. 2012; 9(4): 2023-28.

17.   Chaudhari B, Daniel K. A Validated RP-HPLC Method for Simultaneous Estimation of Tizanidine and Nimesulide in Bulk and Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 13(9): September 2020. 4207-4212. doi: 10.5958/0974-360X.2020.00743.

18.   The International Conference on Harmonization, Q2 (R1), Validation of Analytical Procedure: Text and Methodology: 2005.

19.   Validation of analytical procedures: text and methodology (2005) 254 In: ICH Harmonized Tripartite Guidelines Q2 (R1). ICH, Swtizerland.

20.   Padmaja V, Swapna G, Prasanthi M. Stability Indicating UPLC Method Development and Validation for the Determination of Reserpine in Pharmaceutical Dosage Forms. Research Journal of Pharmacy and Technology. 2018; 11(11): 5111-5114. doi: 10.5958/0974-360X.2018.00933.2

21.   Khan H, Ali M, Ahuja A, Ali J. Validated UPLC/Q-TOF-MS Method for Simultaneous Determination of Telmisartan and Hydrochlorothiazide in Human Plasma. Research Journal of Pharmacy and Technology. 2016; 9(9):1441-1446. DOI: 10.5958/0974-360X.2016.00278.X

22.   Eswarudu MM, Eswaraiah MC, Prasanna Kumar K, Sudhakar K. Ultra Performance Liquid Chromatography (UPLC): A Preeminent Technique in Pharmaceutical Analysis. Research Journal of Pharmacy and Technology. 2012; 5(12): 1484-1489.

23.   Swetha Sri R, Bhavya Sri K, Mounika. A Review on Comparative study of HPLC and UPLC. Research Journal of Pharmacy and Technology. 2020; 13(3):1570-1574. DOI: 10.5958/0974-360X.2020.00284.X

24.   Dhalape VM, Santosh T, Khadangale R, Pinjari V. RP-HPLC Method Validation for Quantitative Analysis of Pemetrexed Disodium Hemipentahydrate. Research Journal of Pharmacy and Technology. 2020; 13(8):3685-3689. DOI: 10.5958/0974-360X.2020.00652.6

 

 

 

 

 

Received on 23.02.2021            Modified on 26.05.2021

Accepted on 27.07.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1467-1471.

DOI: 10.52711/0974-360X.2022.00243