DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF AMLODIPINE BESYLATE AND ATORVASTATIN CALCIUM IN COMBINED DOSAGE FORM

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About Authors: MALLIKARJUNA RAO N.
1.     Research scholar of Jawaharlal Nehru Technological University, Department of Pharmaceutical Analysis, College of Pharmacy, Kakinada, Andhra Pradesh, India.  

Reference ID: PHARMATUTOR-ART-1042

ABSTRACT
Objective:
This present study reports for the first time simultaneous quantitation of Amlodipine besylate and Atorvastatin calciumby HPTLC from a combined dosage form.
Methods: Chromatographic separation of the drugs were performed on aluminum plates precoated with silica gel 60 F254 used as stationary phase and the chromatogram was developed using Ethyl acetate: Methanol: Ammonia (7.5 : 2 : 0.5 %v/v/v) as mobile phase. Amlodipine besylate and Atorvastatin calcium showed Rf values 0.50 ±0.02 and 0.26 ±0.02 respectively. Densiometric analysis of both the drugs was carried out in the absorbance mode at 365 nm. The method has been successfully applied to tablets and was validated according to ICH Harmonized Tripartite guidelines.
Results: The linearity regression analysis for calibration showed 0.9983 (r2) and 0.9994 (r2) for amlodipine besylate and atorvastatin calcium with respect to peak area and height in the concentration range of 100-500ng/spot and 200-600ng/spot respectively.  The percentage recovery for amlodipine besylate was found to be 101.82 (at 50%), 99.12 (at 100%) and 101.41 (at 50%), 101.71 (at 100%) for atorvastatin calcium. The limit of detection was 30 ng/spot and    60 ng/spot for amlodipine besylate and atorvastatin calcium respectively. The limit of quantification was found to be 100 ng/spot and 200 ng/spot for amlodipine besylate and atorvastatin calcium respectively.
Conclusion: The developed TLC technique is precise, specific and accurate. It was concluded that the developed method offered several advantages such as rapid, cost effective, simple mobile phase and sample preparation steps and improved sensitivity made it specific, reliable and easily reproducible in any quality control set-up providing all the parameters are followed accurately for its intended use.

INTRODUCTION
Amlodipine is a white crystalline powder which is slightly soluble in water, sparingly soluble in ethanol and freely soluble in methanol. It is official in B.P. Chemically Amlodipine, (Fig 1.) is 3-Ethyl-5-methyl (±)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3, 5-pyridine dicarboxylatebenzenesulfonat[1]. Amlodipine is a dihydropyridine derivative with calcium antagonist activity[2]. It is used in the management of hypertension, chronic stable angina pectoris and prinzmetal variant angina[3]. Amlodipine acts by inhibiting the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle and also acts directly on vascular smooth muscle to cause a reduction in peripheral vascular resistance and reduction in blood pressure. Atorvastatin is a synthetic hydroxyl methyl glutaryl coenzyme A (HMG?CoA) reductase inhibitor that has been used as a lipid lowering agent[4].

Chemically, Atorvastatin (Fig 2.) is [R-(R*, R*)]-2-(4-flurophenyl)-B, B-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid[5]. Atorvastatin is a competitive inhibitor of HMG?CoA reductase. This enzyme catalyzes the reduction of 3?hydroxy?3?methylgultaryl?coenzyme?A to mevalonate, which is the rate?determining step in hepatic cholesterol synthesis. Because cholesterol synthesis decreases, hepatic cells increase the number of LDL receptors on the surface of the cells, which inturn increase the amount of LDL uptake by the hepatic cells, and decrease the amount of LDL in the blood[6-8].

HPLC methods are official in I.P[9] for the estimation of atorvastatin while in I.P[10], B.P[11], E.P[12] and USP[13] for the determination of amlodipie, but they do not involve simultaneous determination of atorvastatin and mlodipine. Detailed survey of literature for atorvastatin revealed several methods based on different techniques, viz. HPLC[14-16] and LC-MS[17-19] for its determination in plasma/serum; HPLC[20] for its determination in human serum and pharmaceutical formulations; HPLC[21-22]; Similarly, survey of literature for amlodipine revealed methods based on spectrophotometry[23], RP-HPLC[24] using fluorescence detection, HPLC-tandem mass spectrometry[25-26], RP-HPLC using UV detection[27-28], HPLC[29-33] in combination with other drugs, Flow injection analysis using UV-detection[34], stability indicating HPLC[35] and stability indicating HPLC[36] in combination with benazepril hydrochloride have been reported. Spectrophotometric[37], HPLC[38-39]  methods have been reported for simultaneous determination of atorvastatin and amlodipine. The reported HPLCmethods involve costly sophisticated instrumentation and time consuming process. Since no HPTLC method is reported for simultaneous estimation of Amlodipine and Atorvastatin calcium in combination therefore, in the present work, a successful attempt has been made to estimate both these drugs simultaneously. The proposed method was successfully applied for simultaneous determination of atorvastatin and amlodipine in combined dosage forms that are available in market.

MATERIALS AND METHODS

Reagents and chemicals

Atorvastatin calcium was obtained as gift sample from Micro labs and Amlodipine was obtained as gift sample from Cipla Pharmaceuticals. Purified water was prepared using a Millipore Milli?Q (Nanopure Diamond, Barnstead thermolyne, USA) water purification system. Acetonitrile, Methanol was purchased from Merck Ltd. (Mumbai, India)

Instrumentation

CAMAG HPTLC instrument was used in this method. CAMAG HPTLC is equipped with CAMAG TLC scanner-3, Linnomate V Automatic sample applicator controlled by WIN CATS software (1.4.3 version). Aluminum packed silica Gel 60 F254 HPTLC plates (20 X 10cm, layer thickness 0.2mm, E.MERCK).

Optimized chromatographic conditions

Stationary phase                      :           Silica gel 60GF254 precoated TLC plates                                      

 Mobile phase                           :             ethyl acetate: methanol: ammonia.

Mobile phase ratio                   :             7.5:2:0.5 %v/v/v.

Saturation time                        :             20 minutes.

Solvent front                           :             85 mm.

Band length                            :             6 mm.

Slit dimension                         :             5.00 x 0.45 mm.

Source of radiation                 :             Deuterium.

Scan wavelength                     :             365 nm.

Rf values

Atorvastatin calcium         :       0.26 ±0.02.

Amlodipine besylate         :       0.50 ±0.02.

Selection of detection wavelength

After chromatographic development, bands were scanned over the range of 200-400 nm and the overlain spectra were obtained. UV spectra of atorvastatin calcium and amlodipine besylate on precoated plate were recorded. The lmaxof atorvastatin calcium and amlodipine besylate was found to be 282 nm and 365 nm respectively.

The chromatogram scanned at 365 nm showed higher peak area and better peak shape for both atorvastatin calcium and amlodipine besylate than other wavelengths. So 365 nm was selected as the detection wavelength (fig.3).


 

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