INTRODUCTION:

Atorvastatin calcium is a synthetic pentasubstituted pyrrole heptanoic acid derivative antilipemic agent. It limits cholesterol formation by competitively inhibiting the conversion of HMG – CoA to mevalonate by HMG – CoA reductase.Chemically, atorvastatin calcium is the calcium salt of (β R, 8R) – 2 –(4 – fluorophenyl) – α,δ, - dihydroxy – 5 – (1 – methyl ethyl) – 3 – phenyl – 4 – [ (phenyl amino) carbonyl] – 1 H – pyrrole – 1 – heptanoic acid trihydrate.

Fig 1.Stucture of Atorvastatin Calcium

Clopidogrel bisulphate is a thieno pyridine drug that target P2Y₁₂, a key ADP receptor on platelets. Activity of clopidogrel dependent on hepatic transformation to an active metabolite. The principle metabolite found in human plasma (SR 26334) is inactive. Clopidogrel selectively inhibit ADH – induced platelet aggregation by irreversibly blocking P2Y₁₂. Chemically, Clopidogrel bisulphate is methyl (S) – α – (o –chlorophenyl) – 6,7 – dihydrothieno [3,2 C] pyridine – 5 – (4H) – acetate sulphate.^5,6,7

Fig 2. Stucture of Clopidogrel Bisulphate

Literature survey revealed that only one RP– HPLC^1,2,3,4method have been reported for the determination of Atorvastatin and Clopidogrel in combined dosage form. Hence the aim of the study was to develop a rapid, sensitive, simple and precise RP-HPLC method which can estimate both the drugs simultaneously.

MATERIALS AND METHODS^8,9:

Chemicals and reagents:

Atorvastatin Calcium and Clopidogrel Bisulphate was obtained as gift sample from Dr. Reddy’s Hyderabad. HPLC grade Acetonitrile, Water and Methanol were purchased from Nice Pharmaceuticals. Analytical Grade Ortho-Phosphoric acid was purchased from Merck chemicals, Mumbai. The capsule Clopitorva – 75 was purchased from local market.

Instrumentation:

The proposed work was carried out on a Waters HPLC , which posses PDA – 2998 detector, pump 515 HPLC and the column used was Spherisorb 5µsilica (4× 250mm I.D.) All weighing was done on electronic balance (Shimadzu ATY224).An ultrasonic cleaner (Equitron) was used for degassing the mobile phase.

Selection of solvents:

Fig 3. A typical Chromatogram of Atorvastatin and Clopidogrel

On the basis of solubility study methanol was selected as the solvent for dissolving atorvastatin calcium and Clopidogrel bi sulphate.

Preparation of standard stock solution:

Standard stock solutions for both Atorvastatin andClopidogrel were prepared separately in a 100ml volumetric flask by dissolving 100mg of the drugs in HPLC grade Methanol. The stock solutions were protected from light using aluminium foil and stored at room temperature.

Preparation of Resolution Mixture:

Resolution mixture is prepared by dilution of the standard stock solutions of Atorvastatin and Clopidogrel using mobile phase. It was so prepared that the drugs Atorvastatin and Clopidorgrel will be in the ratio same as that of the marketed formulation available (ie 10:75)

Preparation of Test solutions:

For the estimation of drugs in the commercial formulation, Capsule powder equivalent to about 10mg of Clopidogrel was transferred to 100ml volumetric flask and 25ml methanol was added and sonicated for 20min, volume was then made up to the mark with Methanol. The resulting solution was mixed and filtered through Whatmann no.1 filter paper and filtrate was diluted with mobile phase to get approximate concentration of 10µg/ml of Atorvastatin and 75µg/ml of Clopidogrel. The solution was filtered through 0.2µm filter before injection into the column.

RESULTS:

In this work an isocratic, simple, accurate and sensitive RP-HPLC method suitable for the simultaneous determination of Atorvastatin Calcium and Clopidogrel Bisulphate in capsule dosage form has been developed.Initially various mobile phases with different ratios were investigated in an attempt to obtain best resolution for Atorvastatin calcium and Clopidogrel Bisulphate.

Optimization of Chromatographic Conditions:^[8,9]

Equipment : Waters HPLC with 2998 PDA detector:

Stationary phase : Spherisorb, 5µ silica (4×250mm) column

Mobile Phase : Acetonitrile :0.1% orthophosphoric acid

Mobile Phase ratio : 65: 35% v/v

Detection wavelength :245nm

Flow rate : 1ml/min

Sample Volume : 20µl

Observation:

Using the optimized chromatographic conditions, resolved sharp peaks corresponding to Atorvastatin Calcium and Clopidogrel Bisulphate can be obtained at retention time of 2.007 and 5.977 min respectively.

VALIDATION:

The proposed HPLC assay for simultaneous determination of Atorvastatin Calcium and Clopidogrel Bisulphate was validated in terms of linearity, precision and accuracy¹¹.

Linearity:

Linearity was demonstration by analyzing 5 different concentrations of active compounds. The linearity was observed in the concentration of (6,8,10,12, 14)µg/ml for Atorvastatin and (45, 60, 75, 90, 105)µg/ml for Clopidogrel. The calibration plot wasconstructed by plotting peak area versus concentration of Atorvastatin and Clopidogrel. The slope, y-intercept and correlation coefficient were calculated.

Table 1: Linearity Results for Atorvastatin Calcium and Clopidogrel Bisulphate

Concentration µg/ml ATR: CLP	Peak Area
Concentration µg/ml ATR: CLP	Atorvastatin	Clopidogrel
6:45	317345	642414
8:60	421155	853852
10:75	527254	1068018
12:90	631486	1267355
14:105	723645	1484350
Regression Equation	Y=51148x+12695	y = 13983x + 14510
R²	R²=0.999	R²=0.999

Fig 4. Calibration Curve of Atorvastatin

Fig.5 Calibration Curve of Clopidogrel

Precision:

The precision of the method was demonstrated by intraday precision (repeatability) and intermediate precision studies. Repeatability of the method was ascertained for 5 samples of CLOPITORVA 75 capsules. It was evaluated by carrying out 5 independent assays of samples against qualified standard drugs. Intermediate precision of the method was evaluated on 3 consecutive days by carrying out the same assay procedure The average % RSD was found to be 0.3775 for ATR and 0.0411 for CLP. The standard deviation and % RSD shows that the method was precise.

Accuracy:

The accuracy of the method was evaluated in triplicate at three concentration levels of linearity range. The concentrations selected were Atorvastatin and clopidogrel in the ratios (8:60µg/ml, 10:75µg/ml and 12:90µg/ml).

Table:2 Intra Day Precision studies

	Atorvastatin	Clopidogrel
Conc µg/ml ATR:CLP	10	75
Mean% Assay	98.85	98.91
Std deviation	0.4686	0.1686
% RSD	0.4740	0.1704

Table 3: Intermediate Precision studies

	Atorvastatin (10 µg/ml)			Clopidogrel (75 µg/ml)
	Day 1	Day 2	Day 3	Day 1	Day 2	Day 3
Mean % Assay	98.798	98.934	98.51	98.882	98.85	98.824
Std deviation	0.3668	0.2825	0.4688	0.07259	0.02449	0.02510
% RSD	0.3712	0.2855	0.4759	0.0734	0.0247	0.02539
Avg % RSD	0.3775			0.0411

Table.4 Recovery study I

	Atorvastatin			Clopidogrel
	I	II	III	I	II	III
Conc (ATR:CLP)- µg/ml	8	10	12	60	75	90
Amt. of std added (µg/ml	4	5	6	30	37.5	45
Average % Recovery	98. 62	98. 55	98. 60	98. 46	98. 39	98. 48
Std deviation	0.15 53	0.087 37	0.14 19	0.15 28	0.19 43	0.10 00
%RSD	0.15 75	0.08 86	0.14 39	0.15 52	0.19 75	0.10 15

To determine the accuracy of the method, the standard solutions of the respective drugs were added to the test solution at a level of 50%. The peak areas of these solutions were compared with that of the standard of same concentration.The percentage recoveries were calculated.

Estimation of Atorvastatin and Clopidogrel in Combined Dosage Form^[10]

For the estimation of Atorvastatin and Clopidogrel in combined dosage form, a resolution mixture in the ratio 10:75 was prepared and the prepared solution was run for 5 times and average peak area was calculated .Test solution was prepared in the same concentration and run in the similar manner for the quantitative estimation of ATR and CLP in combined dosage form. The percentage of ATR and CLP were calculated.

Table 5. Assay of Clopitorva75 capsule

	Atorvastatin	Clopidogrel
Conc µg/ml	10	75
Average %Assay	98.83		98.724
Std.deviation	0.3334		0.3927
%RSD	0.3373		0.3978

DISCUSSION:

In this method the HPLC conditions were optimized to obtain, an adequate separation of eluted compounds. The system with acetonitrile: 0.1% orthophosphoric acid (65:35 % v/v) with 1 ml/min flow rate was quite good. The optimum wavelength for detection was 245nm at which better detector response for drugs were obtained. The retention times for Atorvastain calcium and Clopidogrel Bisulphate was found to be 2.007 and 5.977 respectively. To ascertain the effectiveness of the method various validation parameters were checked as per ICH guidelines.

CONCLUSION:

The newly developed RP-HPLC method for the simultaneous estimation of Atorvastatin Calcium and Clopidogrel Bisulphate was validated in terms of linearity, precision,and accuracy. R², standard deviation and %RSD were calculated. The results indicated that the developed method was simple, rapid, inexpensive, precise and accurate. This method can be used for the routine analysis of Atorvastatin Calcium and Clopidogrel Bisulphate in combined dosage forms.

ACKNOWLEDGEMENT:

The authors are thankful to Prof. R. Raju for his kind guidance extended throughout the work.We are also thankful to Dr. Reddy’s Pharmaceuticals for providing the gift samples.

REFERENCE:

1. S.D. Raut, A.R. Umarkar, D.R Chaple, A.V. Kasture. Development of RP-HPLC method for simultaneous estimation of atorvastatin calcium and clopidogrel bisulphate in their pharmaceutical preparation. Journal of Pharmacy Research. 2011; 4(7) :2307-09

2. S.V. Londhe, R.S. Deshmukh, S.V. Mulgund, K.S. Jain. Development and validation of a reversed-phase HPLC method for simultaneous determination of aspirin, atorvastatin calcium and clopidogrel bisulphate in capsules. Indian Journal Of Pharmaceutical Sciences.2011; 73 (1):23-9

3. H. O. Kaila, M. A. Ambasana and A. K. Shah. A simple and rapid ultra-performance liquid chromatographic assay method for the simultaneous determination of aspirin,clopidogrelbisulphate and atorvastatin calcium in capsule dosage form.International Journal Of Chemtech Research.2011; 3(1): 459-65

4. Devika G.S. , M. Sudhakar, J. Venkateshwara Rao.A new simple RP-HPLC method for simultaneous estimation of asprin, atorvastatin and clopidogrel in capsule dosage form. Asian Journal Of Research In Chemistry.2011; 04(05):795

5. Indian Pharmacopoeia. Vol-I. 6^thedition. Ghaziabad: The Indian Pharmacopoeia commission;2010.141,153, 274.

6. Mary J.M, RichardA.H.,PamelaC.C.Lippincott’s illustrated reviews;pharmacology.2^nd edition.448-49

7. The Merk Index, 13^thedition.NewJersy:Merk research laboratories.;1996.148,420

8. Michael W.Dong. Modern HPLC for practicing scientists.NewJersy: John Wiley and sons;2006.27-34,194-06

9. Snyder R.L, Kirkland.J.J, Giajch.L.J. Practical HPLC method Development. 2nd edition.USA:John Wiley and sons.3-19,653-57.

10. Anthony C Moffat, M David Osselton and Brian Widdop. Clark Analysis of drugs and poisons. Vol-1,3^rdedition; P_hP Publishers.508

11. https://www.ich.org/fileadmin/Public_Web.../Guidelines/./Q2_R1__Guideline.pdf

Received on 19.07.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(3):1227- 1230.

DOI: 10.5958/0974-360X.2020.00226.7