INTRODUCTION:

Atrovastatin Calcium (fig.1a) chemically it is 2-(4-flurorophenyl) β, δ-dihydroxy-5-isopropyl-3-phenyl-4-(phenylamino) carbonyl-1H-Pyrrole-1-heptanoic acid trihydrate calcium salt[1,2,3]. It is a synthetic lipid lowering agent and an inhibitor of 3-hydroxy-3-methylglutaryl-coenyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an early and rate limiting stage in cholesterol biosynthesis.

Clopidogrel Bisulphate (fig.1b) chemically it is Methyl (2-chlorophenyl) (4, 5, 6, 7-tetrahydrothieno [3, 2 c] pyridin-5(4H)-yl) acetate bisulphate.[4,5]it belonging to the category Anti-platelet Drug. Due to opening of the thiophene ring, the metabolite chemical structure now has three sites of chirality, making a total of eight possible isomers. These are; a stereocentre at C4 (attached to the —SH thiol group), a stereobond at C3—C16 double bound and the original stereocentre at C7. Only one of the eight structures is an active antiplatelet drug. As the thiol group is too reactive, work with the active metabolite of the related drug Prasugrel suggests that the (R)-configuration of the C4 group is critical for P2Y₁₂ and platelet-inhibitory activities.

Literature survey reveals that Spectrophotometric HPLC, RP-HPLC methods are available for determination of Atrovastatin Calcium from pharmaceutical preparations and biological formulation More ever, the literature survey revealed that so far, no method has been reported for estimation of ATOR and CLOP in combined dosage form, hence U V Spectrophotometric methods have been developed for simultaneous estimation of ATOR and CLOP in capsule dosage form by Multicomponent method.

MATERIALS AND METHODS:

Materials:

All the chemicals and solvents used were of AR. The pure drugs ATOR (99.91 %) and CLOP (99.50%) were gift sample from vama pharmaceutical Pvt. Ltd. Nagpur, and were used as reference standards. The capsule formulation was purchased from local market and it was coded by the symbol C and used for analysis.

Methods:

The sampling wavelengths 219,225,232,237,246 nm were selected on trial and error basis. The concentrations of individual drug (i.e. ATOR and CLOP) in the respective six mixed standard solutions (Table 10) were feed to multicomponent mode of the instrument. All the six mixed standards were scanned in the range of 200 nm to290 nm

The basic necessity for the application of the proposed method is that at all the selected sampling wavelengths the mixed standard solutions must follow the Beer-Lambert’s law. The study shows that at all the selected sampling wavelengths, the mixed standard solutions obey the Beer-Lambert’s law.

(fig.1a) Atrovastatin Calcium

(fig.1b) Clopidogrel Bisulphate

Analysis of Pharmaceutical Formulation:

For the simultaneous estimation of commercial formulation twenty capsule of ATOR (Labail claim: Atrovastatin Calcium and Clopidogrel Bisulphate) was taken. The average weight of capsule was determined. powder equivalent to 10 mg ATOR was accurately weighed, transferred to a 10 ml volumetric flask, dissolved in 10ml solvent for 20 minutes with vigorous shaking. Finally the volume was made-up to the mark with solvent. The solution was filtered through Whatman filter paper-41, first few drops were rejected. The filtrate was then appropriately diluted to get final concentration of 2 _g/ml of and 5 _g/ml of ATOR. Absorbance of this solution was measured at appropriate wavelengths and values were substituted in the respective formula to obtain concentration. The result of analysis was mentioned in Table 2.

Table 1:- Range for mixture

Validation of the proposed method:

a) Accuracy: - Accuracy of the proposed method was ascertained on the basis of recovery studies performed by Standard addition method. The procedure for mixed standard solution is same as given in Table4.

b) Precision: - It is expressed as ±SD and % RSD of any measurements. Precision of estimation of ATOR and CLOP by proposed method was ascertained by replicate analysis of homogenous samples of tablet powder. The results are also shown in Table 3.

c) Linearity and range: - The study was performed over the series of concentrations ranging from 2-10 µg/mL for mixture. The graphs of concentration against absorbance (Fig.5) found to be straight line over the concentration range of these range.

d) Ruggedness: - The studies were carried out for different parameters i.e. different elapsed times (intraday and interday) different analysts.

Fig 1. Linearity graph of mixture at five selected wavelength

Table 2:- Analysis of standard laboratory mixture

S. No.	Amount of drugs taken (µg/mL)		Amount of drug estimated (µg/mL )		% of drugs estimated.
S. No.	ATOR	CLOP	ATOR	CLOP	ATOR	CLOP
1	2	4	1.99	3.98	99.5	99
2	3	6	2.97	5.99	99.25	99.75
3	4	8	3.96	7.89	99.33	98.16
4	5	10	4.98	9.99	99.75	99.87
5	6	12	5.94	11.96	99.4	99.6

Statistics

Drug	Mean	± SD	%RSD
ATOR	99.44	0.193	0.194
CLOP	99.27	0.707	0.712

Table 3. Analysis Data of Tablet Formulation

S. No.	Amount of capsule powder taken (µg/mL)	Amount of drug estimated ( µg/mL)		% of labeled claim
S. No.	Amount of capsule powder taken (µg/mL)	ATOR	CLOP	ATOR	CLOP
1	10	10.02	10.12	100.01	100.23
2	10	9.86	9.84	99.24	99.20
3	10	9.90	10.02	98.67	100.01
4	10	10.15	10.00	100.08	100.00

Statistics

Drug	Mean	± SD	%RSD
ATOR	99.49	51.87	1.912
CLOP	99.86	52.048	1.913

Recovery Study:

To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiment was carried out by standard addition method. From the total amount of drug found, the percentage recovery was calculated. The results are reported in Table 5.

Table 4- Results of ruggedness studies

Drugs	Parameter	Method precision	Intermediate Precision
Drugs	Parameter		Interday	Intraday	Different Analysts
ATOR	Mean ± SD	99.18 ±1.2945	98.15 ± 1.124	99.91 ± 1.350	100.5 ± 0.912
ATOR	% RSD	1.2857	1.145	1.364	0.907
CLOP	Mean ± SD	99.91 ±0.5635	99.83 ± 1.004	98.74 1.675	99.78 ± 0.473
CLOP	% RSD	0.5639	1.005	1.696	0.474

Table 5. Recovery Studies

S.N.	Amount of drugs added (µg/mL)		Amount of drugs recovered (µg/mL)		% of drugs recovered
	ATOR	CLOP	ATOR	CLOP	ATOR	CLOP
1	2	2	2.00	2.00	100	100
2	2	2	1.97	1.99	98.5	99.5
3	2	2	1.98	1.96	99	98
4	2	2	1.99	1.98	99.5	99

Statistic

Drug	Mean	± SD	% RSD
ATOR	99.25	0.6454	0.650
CLOP	99.21	0.8539	0.860

* Recovery is mean of five estimation. Method Multicompanant method while Method 2 is simultaneous equation method and R.S.D is the relative standard deviation.

Name of drug

Concentration in (µg/mL)