Development and Validation of HPTLC 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

A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the simultaneous estimation of atorvastatin calcium and olmesartan medoxomil in combined dosage forms. The stationary phase used was TLC aluminium plate precoated with silica gel 60F₂₅₄. The mobile phase used was a mixture of acetonitrile: chloroform: methanol: 10% glacial acetic acid (7: 2: 1.5: 0.1 v/v/v/v). The system was found to give compact spot for both atorvastatin and olmesartan (R_f value 0.5±0.01 and 0.76±0.02 respectively). The densitometric analysis of spot was carried out in reflectance mode at 253 nm. The method was validated in terms of linearity, specificity, precision, robustness and accuracy. The calibration curve was found to be linear in the range of 200 to 800 ng/spot for atorvastatin and 400 to 1600 ng/spot for olmesartan. The limit of detection and the limit of quantification for the atorvastatin were found to be 178.239 and 540.11 ng/spot and for olmesartan 40.10 and 121.51 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of marketed tablet formulation.

REFERENCE ID: PHARMATUTOR-ART-1143

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 [Figure 1], 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-methylglutaryl-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] [Figure 2] 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^{[4, 5]}.

Tablet dosage forms containing AT and OLM in ratio of 10mg and 20 mg is newly approved combination by Central Drug Standard Control Organization (CDSCO) for marketing in India for the treatment of coexisting essential hypertension and hyperlipidemia in adult persons in October 2009. 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] and high-performance thin layer chromatography^[11], while OLM determinations have been reported by UV?Vis spectrophotometry ^[12-14], HPLC ^[15-18] and HPTLC ^{[19, 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, 22]}.

MATERIAL AND METHODS
Materials:
Working standards of AT and OLM were procured as gift sample from Sun pharmaceuticals Ltd. Baroda. Silica gel 60F₂₅₄ TLC plate (20×20 cm, layer thickness 0.2 mm, Merck, Mumbai) was used as a stationary phase. All chemicals and reagent used were of analytical grade. Tablet containing AT (10mg) and OM (20mg) were procured from local pharmacy store (Olmesar-AV, Macleod pharma).

Instrumentation:
Chromatographic separation of drugs was performed on Merck TLC plates precoated with silica gel 60 F254 (20×20 cm, with 0.2 mm layer thickness). The samples were applied onto the plates as a band with 5 mm width using Desaga 100 μl sample syringe (Hamilton, Switzerland) with an AS-30 sample applicator (Desaga, Switzerland). Linear ascending development was carried out in a twin trough glass chamber (for 10x10 cm). Densitometric scanning was performed using Densitometer CD-60 TLC scanner (Desaga) in the range of 200- 400 nm and operated by proquant software (Desaga).

Standard preparation:
Standard stock solution of 1000 µg/ml of AT and OLM were prepared in methanol. Mix Working standard were prepared by taking dilution ranging from 20-80 µg/ml of AT and 40-160 µg/ml of OLM. This solution were used for linearity range

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Sample preparation:
Weight accurately twenty tablets of OLMESAR AV (Macleod Pharma) containing AT and OLM in the ratio of 10mg and 20mg respectively and crushed to fine powder. Powder equivalent to 5mg of AT and 10mg of OLM were transferred to 100 ml volumetric flask, sonicated for 15 min and filtered through Whatmann filter paper No 41 and make up to mark with methanol.

Selection of Detection Wavelength:
After chromatographic development bands were scanned over the range of 200-400 nm and the spectra were overlain. It was observed that both the drug showed considerable absorbance at 253 nm. So, 253 nm was selected as the wavelength for detection.

Method development:
Method development for resolution of AT and OLM in combination was started with development of densitogram with neat solvent and combinations of Chloroform,Toluene, Methanol, Acetone, acetonitrile, Ethyl acetate and Benzene. Finally acetonitrile, chloroform, and methanol used in the ratio of 7: 2: 1.5, v/v/v as mobile phase, resulted in AT and OLM getting separated, but spot shape was still unsatisfactory. An attempt to improve spot shape was made by adding 10% glacial acetic acid (GAA) to the mobile phase. The presence of GAA in mobile phase resulted in excellent overall chromatography with an appropriate compact spot. Eventually, a mobile phase of acetonitrile: chloroform: methanol: 10% GAA (7: 2: 1.5: 0.1, v/v/v/v) provided the best chromatographic response and was used for further studies.

Chromatographic condition:
Chromatography was performed on 20 cm × 10 cm aluminum pre-coated plates with 0.2 mm layer of silica gel 60 GF₂₅₄. Standard and sample solution of AT and OLM (10 μl) were applied to the plates as 5-mm bands, 20 mm apart from the x-coordinate and 15 mm from the y-coordinate on the plate, by use of DESAGA sample applicator fitted with 100-μl syringe. The constant application rate was 5sμl^-1 and a nitrogen aspirator was used. Plates were developed with acetonitrile: chloroform: methanol: 10% GAA (7:2:1.5:0.1, v/v/v/v) as mobile phase. Liner ascending development was performed in 20 cm × 10 cm twin through glass chamber previously equilibrated with mobile phase vapor for 30 min at room temp (the optimum chamber-saturation conditions). The development distance was 8.0 cm. Approximately 20 ml mobile phase (10 ml in trough containing plate and 10 ml in other trough) was used for each development, which required 10 min. After development the plates were dried in an oven at 50 ?C for 5 min. Densitometric scanning was performed with a DESAGA TLC scanner in reflectance mode at the wavelengths of 253 nm for AT and OLM. The slit dimensions were height 0.02 mm and width 0.40 mm, smoothing value 15 and the scanning rate was 20 mm/s. The source of radiation used was the deuterium lamp. Evaluation was achieved by linear regression of peak area response against amount of drug by using Desaga Proquant software for peak-area measurement and data processing.

Validation parameter:
AT and OLM were separated at R_f 0.50 and 0.76 respectively. The method was validated for assay of AT and OLM in accordance with ICH guidelines.

1) Linearity:
Amounts of working standard solution of AT equivalent to 200, 300, 400, 500, 600, 700 and 800 ng/spot and 400, 600, 800, 1000, 1200, 1400 and 1600 ng/spot of OLM were applied to a HPTLC plate. The plate was developed, dried and scanned as represented above. A calibration plots were constructed by plotting peak areas against amounts of AT and OLM. The linearity of the response to AT and OLM were assessed in the concentration ranges 200-800 and 400-1600 ng/band with correlation coefficient of 0.995 and 0.996, respectively as shown in Table 1.

2) Sensitivity:
The sensitivity of measurement of AT and OLM using the proposed method was estimated as the limit of quantification (LOQ) and the lowest concentration detected under these chromatographic condition as the limit of detection (LOD). The LOD and LOQ were calculated by using the equations LOD= 3.3*σ/S and LOQ= 10*σ/S, where σ is the standard deviation of y-intercept and S is the slops of the corresponding calibration plot. The results were shown in Table 1.

3) Precision:
Precision was measured by analysis of sample 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 200-800 ng/spot and 400-1600 ng/spot 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.

4) Accuracy:
The accuracy of the method was determined by analysis of standard addition at three levels, i.e. multiple-level recovery studies. Reference standard at three different concentrations (50. 100, 150%) were added to a fixed amount of pre-analyzed test sample and the amounts of drug were analyzed by the proposed method. Results from the recovery studies are given in Table 2.

5) Specificity:
The mobile phase used enable good resolution of AT and OLM (R_f 0.50 and 0.76 when using wavelength 253 nm). To confirm the specificity of the proposed method, the solution of the formulation was spotted on the TLC plate, developed and scanned. It was observed that the excipients present in the formulation did not interfere with the peaks of AT and OLM.

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6) Robustness and Ruggedness:
There were no significant difference between results obtained by applying the analytical condition established for the method and those obtained in experiments in which some of the conditions were varied slightly. Thus the method was shown to be robust to changes in acetonitrile proportion from 12-18 %. Plates were developed 5 min after spotting and scanned 15 min after development. The ruggedness of the method was checked by using different analysts and days. The relative standard deviation of the results obtained by different analysts using instruments was <2.0%.

7) Solution stability:
To check the stability of the drug by use of the proposed method, freshly prepared solutions of analytes were applied to the plates and developed after intervals of 2 h, 6 h and 12 h. Decomposition of the drugs were not observed during chromatogram development and no change in the peak area of the drugs were observed during solution stability studies.

8) Assay:
The proposed method was also evaluated by the assay of test sample of commercially availabletablets of AT and OLM. The % assay (Mean ± S.D) was found to be 99.89 ± 0.14 for AT and102.2 ± 0.11 for OLM

RESULTS AND DISCUSSION
The mobile phase consisting of acetonitrile: chloroform: methanol: 10% GAA (7:2:1.5:0.1, v/v/v/v) gave R_f value of 0.50±0.01 and 0.76±0.02 for AT and OLM, respectively. The linear regression data (n=5, Table 1) showed a good linear relationship over a concentration range of 200-800 ng/spot for AT and 400-1600 ng/spot for OLM, respectively. The limit of detection and limit of quantification for AT was found to be 178.23 and 540.11 ng/spot and for OLM 40.1 and 121.51ng/spot, respectively.  The intra-day and inter-day coefficients of variation were less than 2 for both AT and OLM. Repeatability of sample application was assessed by spotting 5µl of drug solution 5 times on a TLC plate followed by development of plate and recording the peak area for 5 spots. The %RSD for peak area values of AT and OLM were found to be 1.09 and 1.06, respectively. To confirm the specificity of the proposed method, the solution of the formulation was spotted on the TLC plate, developed and scanned. It was observed that the excipients present in the formulation did not interfere with the peaks of drugs. Multiple recovery study was carried out for accuracy parameter. % recovery was found to be within the limit as listed in Table 2. The assay value for marketed formulation was found to be within the limits as listed in Table 3.

The developed HPTLC technique is simple, precise, specific and accurate and the statistical analysis proved that method is reproducible and selective for the analysis of olmesartan medoxomil and atorvastatin calcium in bulk drug and tablet formulation.

ACKNOWLEDGMENT
The authors wish to express their gratitude to Sun Pharmaceutical Ltd. Baroda (India), for providing gift sample of AT and OLM. The authors are also thankful to the Principal and Management, Shri Sarvajanik Pharmacy College for providing necessary facilities.

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TABLE 1: METHOD VALIDATION PARAMETER OF THE PROPOSED METHOD

Table 2: RECOVERY DATA FOR THE METHOD

TABLE 3: ANALYSIS OF MARKETED FORMULATION

*Average of three estimations

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