Simultaneous Estimation of Olmesartan and Atorvastatin by RP-HPLC in Tablet Dosage Form

 

Ajay Kumar D*, R Anusha Naik

 

ABSTRACT:

A new, precise, rapid, accurate RP-HPLC method was developed for the Simultaneous Estimation of Olmesartan and Atorvastatin in tablet dosage form. After optimization the good chromatographic separation was achieved by Isocratic mode with a mixture of Acetonitrile: Phosphate Buffer pH 5.8 in the ratio of 60:40 v/v as the mobile phase with ZODIAC C₁₈ (150 × 4.6 mm I.D) 5μm, column as stationary phase at flow rate of 1 mL/min and detection wavelength of 212 nm. The retention times Olmesartan and Atorvastatin found to be 2.67 3 min and 3.717 min respectively. The linearity of this method was found in the concentration range of 60μg/mL to 140 μg/mL for Olmesartanand 30μg/mL to 70μg/mL for Atorvastatin. The correlation coefficient R² value is found to be 0.9956for Olmesartan and 0.9973for Atorvastatin. The LOD and LOQ for Olmesartan were found to be 15.935ppm and 48.2864ppm respectively. The LOD and LOQ for Atorvastatin were found to be 12.16ppm and 36.86 ppmrespectively. This method was found to be good percentage recovery Olmesartan and Atorvastatin were found to be 100.67 and 99.54 respectively indicates that the proposed method is highly accurate.

 

 

 

INTRODUCTION:

High Performance Liquid Chromatography:

High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. Advances in column technology, high-pressure pumping system and sensitive detectors have transformed liquid column chromatography into high speed, efficient, accurate and highly resolved method of separation.

 

 

MATERIALS AND METHODS:

METHOD DEVELOPMENT:

The objective of this experiment was to optimize the assay method for simultaneous estimation of Olmesartan and Atorvastatin based on the literature survey made and the methods given in official pharmacopoeias. Trials done for optimization are as follows

 

Solubility Studies

These studies are carried out at 25⁰C

Solvent Name	Olmesartan	Atorvastatin
Water	insoluble	insoluble
Ethanol	Slightly Soluble	Sparingly soluble
Methanol	Sparingly Soluble	Slightly Soluble
Acetonitrile	Freely Soluble	Very Slightly Soluble

 

Determination of Working Wavelength (λmax):

In simultaneous estimation of two drugs isobestic wavelength is used. Isobestic point is the wavelength where the molar absorptivity is the same for two substances that are inter convertible. So this wavelength is used in simultaneous estimation to estimate both drugs accurately.

 

Preparation of Standard solution:

About 100 mg of olmesartan and 50 mg of atorvastatine were weighed into a 100 mL volumetric flask, to this 50 mL of mobile phase was added, sonicated and the volume was made up to mark with the mobile phase.

 

STEPS INVOLVED IN METHOD DEVELOPMENT:

Method development in HPLC is a complex process that involves a number of steps, which are as follows

 

Step 1: Method selection:

When developing a HPLC method first step is always to consult the literature to ascertain whether separation has been previously performed if so, under what conditions this will save time doing unnecessary experimental work. This information from literature should contain

 

Solubility profile – which includes solubility of drug substance in different solvents and at different Ph Conditions?

 

Analytical profile –which includes analytical profile of the drug substance, impurity and degradation products?

 

Stability profile –which includes stability profile of the drug substance with respect to storage conditions?

 

Step 2: Selection of initial conditions:

This step determines the optimum conditions to adequately retain all analytes i.e., ensures no analytes has capacity factor of less than 0.5 (Poor retention could result in peak overlapping) and no analyte has capacity factor 10 –15 (excessive retention leads to long analysis time and broad peaks with poor detectability). Selection of the following is then required.

 

Step-3: Selectivity optimization:

The aim of this step is to achieve adequate selectivity (peak spacing).The mobile phase and stationary phase compositions need to be taken into account. To minimize the number of trail chromatograms involved, only the parameters that are likely to have a significant effect on selectivity in the optimization must be examined. To select these, the nature of the analytes must be considered. The optimization of mobile phase parameters is always considered first as this is much easier and convenient than stationary phase optimization.

 

Step-4: System Optimization:

This is used to find the desired balance between resolution and analysis time after satisfactory selectivity has been achieved. The parameters involved included column dimensions, column-packing particle size and flow rate. These parameters may be changed without affecting capacity factors or selectivity.

 

Step-5: Method Validation:

Proper validation of analytical methods is important for pharmaceutical analysis when ensuring of the continuing efficacy and safety of each batch manufactured relies solely in the determination of quality. Method Validation is generally a one-time process performed after the method has been developed to demonstrate that the method is scientifically sound and that it serves the intended analytical purpose.

 

RESULTS AND DISCUSSION:

PRECISION OF OLMESARTAN and ATORVASTATIN:

 

 

Fig no.1 Precision Chromatogram Of Olmesartan and Atorvastatin

 

Fig no.2 Precision Chromatogram 1 Of Olmesartan and Atorvastatin

 

 

Figure no.3 Precision Chromatogram 2 Of Olmesartan and Atorvastatin

 

 

Fig no.4 Precision Chromatogram 3 Of Olmesartan and Atorvastatin

 

 

Fig no.5 Precision Chromatogram 4 Of Olmesartan and Atorvastatin

 

Fig no.6 Precision Chromatogram 5  Of Olmesartan and Atorvastatin

 

Table No: 1 Precision Results

S.No	OLMESARTAN Area	ATORVASTATIN Area
1	810.419	286.026
2	820.419	276.026
3	811.688	282.016
4	812.647	288.483
5	831.524	285.746
6	828.437	286.026
Avg.Assay	819.189	284.054
%RSD	1.11	1.57

 

ACCURACY:

100% level:

 

Fig No.7 Chromatogram Of 100% Accuracy OfOlmesartan and Atorvastatin

 

 

Fig no.8 Chromatogram Of 100% 1 Accuracy of Olmesartan and Atorvastatin

 

Fig no.9 Chromatogram Of 100% 2 Accuracy Of Olmesartan and Atorvastatin

 

120% level:

 

Fig no.10 Chromatogram of 120% Accuracy ofOlmesartan and Atorvastatin

 

Fig no.11 Chromatogram Of 120% 2 Accuracy Of Olmesartan and Atorvastatin

 

 

Fig no.12 Chromatogram Of 140% 1 Accuracy of Olmesartan and Atorvastatin

 

Table No.2 100% Accuracy Results

Inj.Sample	Spike level	Area	Amount added	Amount recovered	%  recovered	Mean recovery
OLMESARTAN	100%-1	801.032	99.94	99.94	100	99.97
	100%-2	816.586	99.58	99.54	99.95
	100%-3	820.921	99.97	99.96	99.99
ATORVASTATIN	100%-1	284.882	49.91	49.90	99.97	99.97
	100%-2	287.502	49.85	49.84	99.97
	100%-3	287.785	49.86	49.83	99.9

Table No.3  120% Accuracy Results

Inj.Sample	Spike level	Area	Amount added	Amount recovered	%  recovered	Mean recovery
OLMESARTAN	120%-1	911.538	119.89	119.87	99.98	99.97
	120%-2	911.492	119.75	119.72	99.97
	120%-3	916.756	119.97	119.93	99.96
ATORVASTATIN	120%-1	331.628	59.65	59.50	99.74	99.82
	120%-2	311.036	59.21	59.15	99.89
	120%-3	334.897	59.85	59.75	99.83

 

140% level:

 

Fig no.13 Chromatogram of 140% Accuracy ofOlmesartan and Atorvastatin

 

 

Fig no.14 Chromatogram Of 140% 1 Accuracy Of Olmesartan and Atorvastatin

Fig no.15 Chromatogram Of 140% 2 Accuracy Of Olmesartan and Atorvastatin

 

Table No. 4 140% Accuracy Results

LINEARITY OF OLMESARTAN and ATORVASTATIN:

Inj.Sample	Spike level	Area	Amount added	Amount recovered	%  recovered	Mean recovery
OLMESARTAN	140%-1	1000.092	138.90	138.70	99.85	99.92
	140%-2	1014.597	139.97	139.92	99.96
	140%-3	1020.027	139.85	139.81	99.97
ATORVASTATIN	70%-1	356.491	69.83	69.74	99.87	99.88
	70%-2	366.526	69.39	69.35	99.94
	70%-3	368.706	69.12	69.02	99.85

 

Linearity-60%:

 

Fig no.16 Linearity 60% Chromatogram of Olmesartan and Atorvastatin

 

Linearity-80%

 

Fig No.17 Linearity-80% Chromatogram Of Olmesartan and Atorvastatin

Linearity-100%:

 

FigNo.18 Linearity-100% Chromatogram Of Olmesartan and Atorvastatin

 

Linearity-120%:

 

Fig No.19 Linearity-120% Chromatogram Of Olmesartan and Atorvastatin

Linearity-140%:

 

Fig No.20 Linearity-140% Chromatogram Of Olmesartan and Atorvastatin

 

Table No.5 Linearity Results

OLMESARTAN		ATORVASTATIN
%	Area	%	Area
60	470.702	60	179.350
80	639.263	80	228.733
100	751.231	100	269.831
120	879.442	120	306.058
140	1005.606	140	355.197
Correlation Coefficient	0.9956	Correlation Coefficient	0.9973

 

ROBUSTNESS:

Flow rate (1):

 

Fig No.21 Chromatogram of Robustness with More Flow ofOlmesartan and Atorvastatin

 

 

Flow rate (2):

Fig No.22 Chromatogram of Robustness with Less Flow ofOlmesartan and Atorvastatin

 

Wavelength– (1):

 

Fig No.23 Chromatogramof Robustness withfewer WavelengthsofOlmesartan and Atorvastatin

 

Wavelength- (2):

 

Fig No.24 Chromatogram of Robustness with More WavelengthsofOlmesartan and Atorvastatin

 

Table No.6 Robustness Results

Inj.Sample	Flow Rate (ml/min)	USP Plate Count	USP Tailing	Wavelength (nm)	USP Plate Count	USP Tailing
OLMESARTAN	0.8	3906	1.333	210	3363	1.241
	1.2	4288	1.391	212	3620	1.286
ATORVASTATIN	0.8	5117	1.171	210	4659	1.111
	1.2	5577	1.167	212	4700	1.111

 

RUGGEDNESS:

Analyst-01:

 

Fig No.25 Chromatogram of Ruggedness of Analyst 1 of Olmesartan and Atorvastatin

 

Analyst-02:

 

Fig No.26 Chromatogram of Ruggedness of Analyst 2 Olmesartan and Atorvastatin

 

table no. 7 Ruggedness results

Parameters	OLMESARTAN	ATORVASTATIN
Tailing factor (T)	1.259	1.143
Number of theoretical plate(n)	3708	4770
Retention time (R	2.673	3.717
%RSD	0.48	0.37

 

Assay of OLMESARTAN and ATORVASTATIN:

Chromatogram of standard:

 

Fig no.27 Chromatogram of standard Olmesartan and Atorvastatin

Table No: 8 System Suitability Parameters

	Compound	Tailing factor	Number Theoretical Plates
Analyst-1	OLMESARTAN	1.250	3506
	ATORVASTATIN	1.333	3594
Analyst-2	OLMESARTAN	1.176	4813
	ATORVASTATIN	1.114	4916

 

Chromatogram of sample:

 

Fig no.28 chromatogram of sample Olmesartan and Atorvastatin

 

Table no. 9 ASSAY RESULT

 

CONCLUSION:

The findings of the present investigation are summarized as follows

1.   A suitable chromatographic method was developed through optimization by changing various parameters such as the mobile phase, injection volume, flow rate etc.

2.   In the present method a ^{Zodiac C}₁₈^{, 250X 4.6 mm, 5µ,} column has been used for Olmesartan and Atorvastatin drugs respectively.

^{3. pH 5.8 with dilute phosphoric acid: Acetonitrile 40:60 mobile phases used} for Olmesartan and Atorvastatin drugs respectively, Retention of Olmesartan and Atorvastatin have more dependence on the mobile phase.

4.   The separation of the two peaks was also dependent on the buffer and the percentage of mobile phases. Olmesartan and Atorvastatin were eluted at acceptable retention times and got good resolution.

5.   Several assay methods has been developed for the determination of Olmesartan and Atorvastatin in formulations and biological fluids but this method is most economic and accurate so this method is very useful for the determination of Olmesartan and Atorvastatin in tablet formulations. This method was validated as per ICH-Q2 (R1) guidelines and met the regulatory requirements for selectivity, accuracy and stability. Considering the obtained data, it was possible to affirm that the proposed method was fast, simple and suitable for the accurate determination of drug Olmesartan and Atorvastatin in tablet formulation.

 

REFERENCES:

1.     Beckett H, Stenlake JB. Practical Pharmaceutical Chemistry. 4th Edition. New Delhi (Ind): C.B.S Publications; 2002. Page.1.

2.     Willard HH , Merritt LL ,Jr.Dean JA ,Frank A S.Instrumental methods of Analysis ,7^th Edition,CBS Publishers and Distributors: New Delhi ,1986 ; page.1-5

3.     Bassett J, Denny RC, Jeffry GH, Mandham J. Vogel’s Text book of quantitative inorganic analysis. 4th ed. England: Longman group; 1986.

4.     Mecek, K. Pharmaceutical applications of thin-layer and paper chromatography. Amsterdam: Elsevier Publishing Company; 1972.

5.     High Performance Liquid Chromatography (HPLC) in the Pharmaceutical Analysis by Shulamit Levin, Medtechnica Feb 2010

6.     Tips on Liquid chromatography (online). Available from: URL:http://www.waters.com

7.     International conference of Harmonization Guidelines on validation of Analytical Procedures Definitions and Terminology Federal Register; (March 1, 1995).

8.     “Validation of compendia methods” USP23<1225>USPC Rockville Maryland USA 1994.

9.     International Journal of Current Pharmaceutical Research Vol 2, Issue 2, 2010 Received 21Dec 2009, revised andAccepted 08 Jan 2010.

10.   Indian J Pharm Sci. 2008 Nov;70(6):754-60. doi: 10.4103/0250-474X.49117.

11.   Indian J Pharm Sci. 2008 Mar-Apr; 70(2):263-5. doi: 10.4103/0250-474X.41473.

12.   J Chromatogr Sci. 2012 Sep;50(8):680-7. doi: 10.1093/chromsci/bms067. Epub 2012 May 16.

13.   International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304 Vol. 3, No.2, pp 668-675, April-June 2011 ISSN: 2320 – 3471(Online)

14.   Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology Volume 1(2) March-April 2013

15.   June 2011, Journal of Pharmacy Research;Jun2011, Vol. 4 Issue 6, p1711, Academic Journal Article

 

 

 

 

 

 

 

Accepted on 16.05.2018            © A&V Publications All right reserved