VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ATORVASTATIN CALCIUM AND OLMESARTAN MEDOXOMIL IN TABLET DOSAGE FORM

About Author:
D. J. Kalena^*, C. N. Patel
Department of Quality Assurance,
Shri Sarvajanik Pharmacy College,
Near arvind baug, Mehsana - 384 001, Gujarat, India

ABSTRACT
Combination therapy of Atorvastatin calcium (AT) and Olmesartan medoxomil (OLM) is used for the treatment of coexisting essential hypertension and hyperlipidemia in adult persons. In the present study a simple, precise, rapid, efficient and reproducible reversed?phase high performance liquid chromatography (RP?HPLC) method has been developed for the simultaneous estimation of AT and OLM present in its tablet dosage forms. Chromatographic separations were carried out isocratically at 30°C ± 0.5°C on a Kromasil C18 Column (5 μm, 250mm x 4.60mm) with a mobile phase composed of Methanol: Acetonitrile: Water (pH 3.65) in the ratio of 50:27:23 % v/v at a flow rate of 1.0 ml/min. Detection is carried out using a UV detector at 260 nm. The retention times for AT and OLM were 5.3 ± 0.5 min and 3.4 ± 0.5 min respectively. The linearity range for AT and OLM were found to be 10?60 μg/ml and 20?120μg/ml with correlation coefficient of 0.996 and 0.999 respectively. The %recovery of the proposed method was found in the range of 98.36-100.91 for AT and 99.27-100.99 for OLM. The relative standard deviations for three replicate measurements in three concentrations of standard solution were always less than 2%. The results of the study showed that the proposed RP?HPLC method is simple, rapid, precise and accurate, which may be useful for the routine estimation of AT and OM in bulk drug and in its pharmaceutical dosage form.

Reference Id: PHARMATUTOR-ART-1159

INRODUCTION
Atorvastatin calcium (AT), [R- (R*, R*)]-2-(4-fluorophenyl)-β, δ-dihydroxy-5-(1 Methyl ethyl)-3-phenyl-4-[(phenyl amino) carbonyl] 1Hpyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate, is a white crystalline powder, slightly soluble in water and ethanol and freely soluble in methanol. AT is a HMG-CoA reductase inhibitor. HMG-CoA reductase catalyzes the reduction of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) to mevalonate, which is the rate-limiting step in hepatic cholesterol biosynthesis^{[1, 2]}. It is official in Indian pharmacopoeia^[3]. Olmesartan medoxomil (OLM), [2, 3?dihydroxy?2?butenyl 4?(1?hydroxy?1?methyl ethyl) ?2?propyl? 1?[p?(o?1H?tetrazol?5?yl phenyl) benzyl] imidazole?5?carboxylate, cyclic 2, 3?carbonate] is a white to yellow white crystalline powder, insoluble in water and sparingly soluble in methanol. OLM is an angiotensin II receptor blocker (ARB).

It blocks the vasoconstrictor effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in vascular smooth muscle^{[2, 4, 5]}. Several analytical methods that have been reported for the estimation of AT in biological fluids and/or pharmaceutical formulations include spectrophotometric ^{[6, 7]}, high performance liquid chromatography ^[8-10]], while OLM determinations have been reported by UV?Vis spectrophotometry ^{[13, 14, 15]}, HPLC ^[16-19] and HPTLC ^[20]. However there is no method available for the simultaneous determination of AT and OLM in combined fixed dosage form. Therefore, an attempt was made to develop a rapid, economic and sensitive method for the simultaneous determination of AT and OLM. To access the reproducibility and wide applicability of the developed method, it was validated as per ICH norm, which is mandatory, also^[21].

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MATERIAL AND METHODS
AT and OLM of pharmaceutical grade were kindly supplied as gift samples by Sun pharmaceutical Ltd. Baroda, India. Methanol, acetonitrile and water used were of HPLC grade (Finar chemicals Ltd.). Tablet containing 10 mg of AT and 20 mg of OLM (OLMESAR AV, Macleod pharma, Mumbai) was procured from local market and used for analysis of marketed formulation. The liquid chromatographic system was of LC-2010 CHT (Schimadzu, Japan), with SPD-20A prominence diode array detector (Schimadzu, Japan). The chromatographic analysis was performed using LC solution software on a Kromasil-RP-18 column (250×4.6 mm, 5 μm particle size). In addition, an electronic analytical balance (Acculab, model ALC-210), a pH meter (Chemi line, model CL-180), a sonicator, were used in this study.

Chromatographic condition:
The isocratic mobile phase consisted of methanol: acetonitrile: water in the ratio of 50:27:23 v/v/v, pH 3.65 adjusted by ortho phosphoric acid, flowing through the column at a constant flow rate of 1.0 ml/min. A Kromasil (C?18) Column (5 μm, 250mm x 4.60mm i.d) was used as the stationary phase and 260 nm was selected as the detection wavelength for UV?PDA detector.

Standard stock solution preparation:
Standard stock solutions of 1000 μg/ml ofAT and OLM were prepared in methanol.Working standard solutions were prepared in mobile phase by taking dilutions ranging from 10?60 and 20-120 μg/ml for AT and OLM, respectively.

Sample preparation:
Twenty tablets of OLMESAR AV (Macleod Pharma, Mumbai) containing AT and OLM in ratio of 10 mg: 20mg respectively was weighed and crushed to fine powder. Powder equivalent to 10 mg AT and 20 mg of OLM was weighed and dissolved in 100 ml of methanol, sonicated for 10 min and filtered through Whatmann filter paper No. 42. Appropriate volume of the aliquot was transferred to a 10 ml volumetric flask and the volume was made upto the mark with mobile phase to obtain a solution containing 20 μg/ml of AT and 40 μg/ml of OLM. A 20 μl volume of above sample solution was injected into HPLC and peak areas were measured under optimized chromatographic conditions.

Calibration curves for AT and OLM:
Appropriate aliquots of ATand OLM stock solutions were taken in different 10 ml volumetric flasks and diluted up to themark with mobile phase to obtain final concentrations of 10-60 μg/ml of AT and 20-120 μg/ml of OLM. The solutions were injected andchromatograms were recorded. Calibration curves were constructed by plotting average peak areasvs.concentrations and regression equations were computed for both the drugs.

Method Validation:
The method of analysis was validated as per the recommendations of ICH and USP for the parameters like accuracy, linearity, precision, detection limit, quantitation limit and robustness. The accuracy of the method was determined by calculating percentage recovery of AT and OLM. For both the drugs, recovery studies were carried out by applying the method to drug sample to which known amount of AT and OLM corresponding to 50, 100 and 150% of label claim had been added (standard addition method). At each level of the amount three determinations were performed and the results obtained were compared.

Precision was measured by analysis of working standard solution at three different concentrations level. The precision of the method, as intraday precision (%RSD) was determined by analysis of AT and OLM standard solutions in the range 10-60 µg/ml and 20-120 µg/ml three times on the same day. Inter-day precision (%RSD) was assessed by analysis of the same solution on three different days over a period of one week. The results from study of precision are shown in Table 1.

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3*σ/S and LOQ= 10* σ/S, where σ =standard deviation of response (peak area) and S= average of the slope of the calibration curve.

System suitability tests are an integral part of chromatographic method which is used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the standard drug solution at the concentration level 30 μg/ml and 60 μg/ml for AT and OLM, respectively to check the reproducibility of the system and the results are shown in Table 1

RESULTS AND DISCUSSION
The mobile phase consisting of methanol: acetonitrile: water (50:27:23, v/v/v) pH 3.65 adjusted with orthophosphoric acid, at 1ml/min flow rate was optimized which gave two sharp, well-resolved peaks with minimum tailing factor for AT and OLM (fig. 1). The retention times for AT and OLM were 5.3±0.2 min and 4.3±0.2 min, respectively. UV overlain spectra of both AT and OLM showed that both drugs absorbed appreciably at 260 nm, so this wavelength was selected as the detection wavelength. The calibration curve for AT and OLM was found to be linear over the range of 10-60 μg/ml and 20-120 μg/ml, respectively. The developed method was also found to be specific, since it was able to separate other excipients present in tablet from the two drugs, also the good resolved separated peak show the specificity of the method. The LOD for AT and OLM were found to be 0.14 μg/ml and 0.03 μg/ml, respectively, while LOQ were 0.44 μg/ml and 0.1 μg/ml, respectively. The results for validation and system suitability test parameters are summarized in Table 1. Assay results for combined dosage form using proposed method showed 99.69±1.40 % of AT and 100.95±0.66 % of OLM. (Table 2)

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CONCLUSION
In the proposed study, RP-HPLC method was developed for the simultaneous determination of AT and OLM and validated as per ICH guidelines. Statistical analysis proved that method was accurate, precise, and repeatable. The developed method was found to be simple, sensitive and selective for analysis of AT and OLM in combination without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation.

TABLE 1: SUMMARY OF SYSTEM SUITABILITY AND LINEARITY DATA

TABLE 2: ANALYSIS OF MARKETED FORMULATION