WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Ashraf et al.

World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 5.210

Volume 4, Issue 11, 310-329

Research Article

ISSN 2278 – 4357

NEW SIMPLE SPECTROPHOTOMETRIC AND DENSITOMETRIC METHODS FOR DETERMINATION OF ATORVASTATIN IN PRESENCE OF ASPIRIN IN PURE FORM AND PHARMACEUTICAL DOSAGE FORM. Khalid A. M. Attia, Mohamed W.I. Nassar and Ashraf Abdel-Fattah* Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. ABSTRACT Article Received on 05 Sep 2015, Revised on 27 Sep 2015, Accepted on 18 Oct 2015

Four, simple, accurate, selective and sensitive spectrophotometric and densitometric methods were developed for the determination of atorvastatin (ATV) in presence of Aspirin (ASP) without previous separation. Method A, Bivariate method using optimum wavelengths

*Correspondence for

(240 and 250 nm) which formed by Kaiser‘s method, with mean

Author

percentage recovery of 100.10±0.711. Method B, Area under curve

Ashraf Abdel-Fattah

method using two wavelength regions (2 5 225 nm) and (2 5 245

Pharmaceutical Analytical

nm) with mean percentage recovery of 100.54±0.318. Method C, Q-

Chemistry Department, Faculty of Pharmacy, Al-

analysis method where 277 nm is the iso-absorptive point for both the

Azhar University, Cairo,

drugs and 246 nm which is λ max of atorvastatin with mean percentage

Egypt.

recovery of 99.31±0.945. Method D, TLC-densitometry, separation was carried out on silica gel plates using methanol and chloroform (6:4 v/v). ATV and ASP were resolved with Rf values of 0.73 and 0.32 respectively. Determination was carried out at 246nm with mean percentage recovery of 100.04±0.782. Results were statistically compared to a reported method and no significant difference was noticed regarding accuracy and precision. KEYWORD: Bivariate; Area under curve; Q-analysis; Densitometric methods. 1. INTRODUCTION The combination of many active compounds in the same commercial preparation has been used in order to increase pharmacologic activity and one of the main challenges facing

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analytical chemists is the spectrophotometric determination of two compounds in the same sample without preliminary separation. The manuscript deals with pharmaceutical preparation containing atorvastatin (ATV) and Aspirin (ASP) combination. Atorvastatin (ATV)

is

(βR,δR)-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-

[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid as the calcium salt belongs to the group of statins (Fig. 1).

(Fig. 1) Chemical structure of atorvastatin. Aspirin is a cyclo-oxygenase inhibitor and commonly used as an analgesic, anti-inflammatory and antipyretic. Additionally, it also has an antiplatelet effect, hence, it also used for the prevention of heart attacks. ASP is chemically 2-(acetyloxy) benzoic acid (Fig.2).

(Fig. 2) chemical structure of aspirin. All statins, including ATV, induce the competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, thus reduce the cholesterol content in hepatocytes. ATV is a lipid regulating drug used to reduce LDL-cholesterol, apo-lipoprotein B and triglyceride and to increase HDL-cholestrolin

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in the treatment of hyperlipidemias. It is also used for prophylaxis of cardiovascular events in patients with multiple risk factors including diabetes mellitus.[1-3] In literature, several methods have been described for the quantitative determination of atorvastatin by HPLC in different pharmaceutical preparation[4-25], HPTLC[26-31], electrochemical[32,33], capillary electrophoresis[34], spectrofluorimetric[35], various spectrophotometric methods have been reported for the determination of atorvastatin[6,12,36-51] and LC-MS[52,53]. Reviewing the literature on the determination of atorvastatin in presence of aspirin revealed the lack of any stability indicating spectrophotometric methods. The aim of this work is to develop a simple, economic, rapid, sensitive, accurate and precise stability indicating methods for determination of ATV in presence of ASP without sophisticated instruments or any separation steps. 2. Theory 2.1.

Theory of Bivariate calibration method

This method is based on a simple mathematic algorithm, proposed for the resolution of binary mixture, in which the data used derives from four linear regression calibration equations, two calibration for each component (ATV and ASP) at two selected wavelengths (λ1, λ2), these selected wavelengths were selected using Kaiser’s method[54], which assured the best sensitivity for the quantitative determination of cited drug. The linear calibration regression equation for the spectrophotometric determination of an analyte A at a selected λ1 is given by AA1 = mA1. CA + eA1 Where, mA1 is the slope of linear regression, CA is the concentration of analyte A and eA1 is the intercept value. If the measurements of the binary mixture (A, B) are performed at two selected wavelengths (AAB1 = mA1CA + mB1CB + eAB1

(1)

AAB2 = mA2CA + mB2CB + eAB2

(2)



The resolution of such equations set allows the evaluation of C A and CB values:

CA = (AAB1 – eAB1– mB1CB)

(3)

CB = [mA2 (AAB1 – eAB1) + mA1 (eAB2 – AAB2)]/mA2mB1 – mA1mB2

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Where: - CA, CB are the concentrations of component A, component B. - mA1, mA2 are the slope values of component A at λ1, λ2. - mB1, mB2 are the slope values of component B at λ1, λ2. - AAB1, AAB2 are the absorbance values of the binary mixture at λ1, λ2. - eAB1, eAB2 are the sum of the intercepts of components A, B at λ1, λ2. 2.2.

Theory of area under curve method

When zero-order absorption spectra of the two components (ATV ‗ASP) showing sever overlapping, we can determine two areas (ATVA1, ATVA2) and (ASPA1, ASPA2) at the same wavelength ranges (λ1 λ2) and (λ3 λ4) under the spectra of each component (ATV, ASP) respectively and by measuring the absorptivity of each component according to each area under the curve with corresponding concentration of each one, we can measure the concentration of the proposed component by using equation: Cx = (A1 b2 A2 b1)/(a1 b2 a2 b1) Where, A1 is the area under curve of mixture of (ATV and ASP) at (λ1 λ2) range. A2 is the area under curve of mixture of (ATV and ASP) at (λ3 λ4) range. a1, a2 are the absorptivities of ATV at (λ1 λ2) and (λ3 λ4) respectively. b1, b2 are the absorptivities of ASP at (λ1 λ2) and (λ3 λ4) respectively. 2.3.

Theory of Q- Analysis method

When zero-order absorption spectra of the two components (ATV, ASP) showing sever overlapping with intersecting at iso-bestic point (λiso), use the absorption at two selected wavelengths. One is at λiso and other being the λmax of ATV, concentration of sample were obtained by using the equation: Cx = [(Qm – Qy)/(Qx – Qy)] × (Aiso/a) Where, CX is the concentration of ATV. Qm is the absorbance ratio of the mixture at λiso and the λmax of ATV. QX is the ratio of mean absorptivity of ATV at λiso and λmax. Qy is the ratio of mean absorptivity of ASP at λiso and λmax. Aiso is the absorbance of mixture at λiso.

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a is mean of the absorptivity of ATV or ASP at λiso. 2.4.

Theory of TLC- densitometric chromatographic method

This technique offers a simple way to quantify separated drugs directly on TLC plate by measuring the optical density of the separated bands. The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition.[55] 3. EXPERIMENTAL 3.1. Instruments 

Spectrophotometer: A double beam UV-Visible spectrophotometer (Shimadzu 1800, Japan) and it is connected to IBM compatible computer. The software UV-Probe Ver. 2.43.



TLC-densitometer: Precoated TLC-plates, silica gel 60 F254 (20 cm×20 cm, 0.25 mm), E. Merck (Darmstadt-Germany). Camag TLC scanner 3 S/N 130319 with winCATS software. Camag linomat 5 autosampler (Switzerland). Camag microsyring (100 ul).



UV lamp with short wavelength (246 nm.) (Desega-Germany).



Chromatographic tank (25 × 25 × 9 cm).

3.2. MATERIALS A. Atorvastatin calcium was kindly provided by ―Egyptian international pharmaceutical industries company‖. E.I.P.I.CO. 0th of Ramadan City, Egypt. B. Aspirin was kindly provided by Sigma Pharmaceutical Company, Cairo, Egypt. C. Methanol was obtained from El-Naser Pharmaceutical Company. D. Ecosprin-AV® capsule: manufactured in India by Accent Pharma; labeled to contain 10mg of atorvastatin and 150 mg of aspirin per capsule. 3.3. Standard solutions Stock solution of each compound was separately prepared, stock solution of 100 ug mL-1 for ATV was prepared by dissolving 10 mg of ATV in 50 mL of pure methanol and complete to the volume with the same solvent and stock solution of 1000 ug mL-1 for ASP was prepared by dissolving 100 mg of ASP in 500 mL of pure methanol and complete to the volume with same solvent, different sets of working solution at various concentrations were prepared by appropriate dilution of the stock solution.

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3.4. Laboratory prepared mixture 3.4.1. For spectrophotometric methods Accurate aliquots equivalent to (50 500 ug) of ATV into series of 10 ml volumetric flasks from its stock solution (100 ug mL-1) and portion equivalent to (750–7500 ug) of ASP from its stock solution (1000 ug mL-1) were added to the same flasks and volumes were completed to mark with pure methanol and mixed well. 3.4.1. For TLC-densitometric method A laboratory prepared mixtures of ATV and ASP (1:15 ratio of ATV and ASP respectively) within range of (2 6 ug ml-1) and (30 90 ug ml-1) of ATV and ASP respectively. 3.5. Pharmaceutical formulation Content of ten capsules were accurately weighed, crushed and mixed. An amount equivalent to 10 mg of ATV which will be simultaneously equal to 150 mg of ASP was weighed and dissolved in pure methanol then filtered through Whatman filter paper no 41, into 100 ml volumetric flask and the volume was completed to the mark with pure methanol. Tablet solution was diluted to the working calibration ranges. 4. Procedures 4.1. Construction of calibration curves 4.1.1. For spectrophotometric methods Accurately measured aliquots equivalent to (50 500 ug) of ATV from stock solution (100 ug mL-1) were, separately, transferred into a series of 10 ml volumetric flasks and Accurately measured aliquots equivalent to (750 7500 ug) of ASP from stock solution (1000 ug mL-1) were, separately, transferred into the other series of 10 ml volumetric flasks, each flask was completed to the mark with pure methanol, yielding concentration range of (5 50 ug mL-1) and concentration range of (75 750 ug mL-1) of ATV and ASP respectively (Table 1). 4.1.1.1. For determination of ATV in presence of ASP using bivariate technique Record the zero-order absorption spectra which obtained from calibration solution of ATV and ASP in concentration range of (5 50 ug mL-1) and (75 750 ug mL-1), respectively, which were separately prepared from above stock solution and from the obtained absorption spectra, select the signals of ATV and ASP at seven wavelengths (240, 250, 260, 270, 280, 290 and 300 nm). The calibration curve equations and their respective linear regression coefficients are obtained directly with the aim of ensuring the linearity between the signals and the

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corresponding concentrations. Absorbance values were measured for both ATV and ASP at the optimum wavelengths (240 and 250 nm) which found by Kaiser’s method (Table 4), the obtained slope and intercept values were used for calculating the concentration of each component using equations (3) & (4) as mentioned under section 2.1. 4.1.1.2. For determination of ATV in presence of ASP using area under curve technique (AUC) At the stored spectra of zero-order absorption spectra of both ATV and ASP, we select two wavelength regions (2 5 225 nm) and (2 5 245 nm) and construct the area under each curve of both ATV and ASP, calculate the absorptivity for each area under the curve of each wavelength region with its corresponding concentration of both ATV and ASP within linearity range of (10 50 ug mL-1) and (150 750 ug mL-1) respectively. 4.1.1.3. For determination of ATV in presence of A

n

ana

t

n

From the overlain zero order absorption spectra of both ATV and ASP, two wavelengths were selected one at 277 nm which is the iso-absorptive point for both the drugs and the other at 246 nm which is λ max of ATV. The stock solution of both drugs were further diluted separately with methanol to get a series working solution of (10 50 ug mL-1) and (150 750 ug mL-1) of ATV and ASP respectively.

he absorbance of the sample solutions were

measured and the absorbance ratio values for both the drugs at selected wavelengths were also calculated, the method employs Q

alues and the concentration of ATV in sample

solution were determined by using equation which mentioned before in (section 2.3). 4.1.2. For densitometric methods For preparation of a calibration plot, 2, 3, 4, 5, 6 uL of standard Working solution of ATV (100 ug mL-1) which equal to (0.2, 0.3, 0.4, 0.5 and 0.6 ug mL-1) were spotted as bands of 5mm width on TLC plates (20 ×10 cm). Bands were applied at 15 mm interval and 15mm from the bottom and sides edges of the plate. Linear ascending development was performed in a chromatographic tank which previously saturated with methanol and chloroform (6:4 v/v) for 30 minutes at room temperature. ATV was scanned at 246 nm. A calibration curve relating the optical density of each spot to the corresponding concentration of ATV was constructed. The regression equation was then computed for the studied drug and used for determination of unknown samples containing it (Fig.3).

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(Fig. 3): Thin layer chromatogram of separated peaks of atorvastatin at various concentrations with Rf value (0.73±0.01) at 246 nm. 4.2. Accuracy Accuracy was assured by carrying out the previously mentioned procedures under (section 4.1.1.) for each technique, for the determination of different concentration of pure ATV. The concentrations were calculated from the corresponding equations of each technique. 4.3. Precision 4.3.1. Intra-day precision (Repeatability) Three concentrations of ATV were analyzed three times intraday using the previously mentioned procedures for each technique. The percentage of recoveries of each concentration of ATV and its relative standard deviation were calculated using the suggested methods (Table 2). 4.3.2. Intermediate precision Three concentrations of ATV were analyzed on three successive days using the previously mentioned procedures for each technique. The percentage of recoveries of each concentration of ATV and its relative standard deviation were calculated using the suggested methods (Table 2). 4.4. Limit of detection (LOD) and limit of quantification (LOQ) The LOD and LOQ parameters were determined from regression equation, LOD = 3.3 Sy/a LOQ = 10 Sy/a

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where (Sy) is a standard error of the calibration curve and (a) is the slope of the corresponding calibration curve (Table 1). Since, Bivariate method depends upon two wavelengths found by (Kaiser‘s method) as mentioned in section 4.1.1.1., area under curve method depends upon two wavelength areas as mentioned in section 4.1.1.2. and Q-analysis method depends upon two wavelengths (isosbestic point and λmax) as mentioned in section 4.1.1.3. So that, LOD and LOQ should be applied at the two wavelengths for each method. 4.5. Application to laboratory prepared mixtures Laboratory prepared mixtures containing different concentration of ATV were prepared, keeping the ratio between ATV and ASP within their calibration ranges. The spectra of these mixtures were recorded and the procedures under construction of calibration curves were then followed. Recovery was calculated as previously mentioned in accuracy 4.6. Application to pharmaceutical formulation Different concentrations within calibration range of each method (Bivariate, area under curve, Q analysis and TLC-densitometric methods) were prepared from the solution of the pharmaceutical preparation, the spectra and optical density of these prepared concentrations were recorded and procedures under construction of calibration curves were followed using the recorded spectra and optical density of the pharmaceutical formulation prepared solution. 5. RESULT AND DISCUSSION Simple spectrophotometric and TLC-densitometric methods were developed for the determination of ATV in presence of ASP without previous separation. 5.1. The bivariate method[56] Bivariate calibration spectrophotometric method is a direct method which has been proposed for the resolution of binary mixture. The principle of bivariate calibration is the measurement of two components (A and B) at two selected wavelengths (λ

and λ2) to obtain two

equations as mentioned before in (section 2.4.). The calibration curve equation and their respective linear regression coefficient are obtained directly with the aim of ensuring the linearity between the signal and the concentrations. The slope values of the linear regression were estimated for both ATV and ASP at the selected wavelengths and used for determination of the sensitivity matrics K, proposed by Kaiser ’s method.[53] A series of

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sensitivity matrices K, were created for ATV and ASP and for every pair of pre-selected wavelengths.

Where mA1,2 and mB1,2 are the sensitivity parameters (Slope) of the regression equations of A and B at the two selected wavelengths (λ1 and λ2). The determination of these matrics were calculated as shown in (Table 4). The wavelength set was selected for which the highest matrix determinant value was obtained. For bivariate determination of ATV in presence of ASP, wavelengths 240 and 250 nm were found to give the maximum value of K and thus can be used for the analysis, using the following linear regression calibration equations: For ATV

For ASP

A = 0.2018 x + 0.0024

r2 = 0.9999

at λ1 = 240 nm

A = 0.2068 x + 0.0027

r2 = 0.9999

at λ2 = 250 nm

A= 0.0259 x + 0.0027

r2 = 0.9999

at

λ1 = 240 nm

A = 0.0052 x + 0.0022

r2 = 0.9999

at

λ2 = 250 nm

Where A is the absorbance at the selected wavelength, X is the concentration in ug mL -1 and r is the correlation coefficient. 5.2. The area under curve method[57] Suitable dilution of standard stock solution of ATV and ASP were prepared separately in methanol in the concentration range of (10 50 ug mL-1) and (150 750 ug mL-1 ) respectively. he solutions of drugs were scanned in the range of 200 400 nm. or area under curve(A C) method, sampling wavelength ranges which selected for estimation of ATV in presence of ASP were 2 5 225 nm (λ λ2) and 2 5 245 nm (λ3 λ4), areas under the curves were integrated between these selected wavelength ranges for both drugs (Fig.4) and (Fig.5), which show linear response with increasing concentration. The same wavelength ranges were used for preparation of calibration curve and estimation of tablet formulation by measuring the absorptivity of each component according to each area under the curve with corresponding concentration of each one, we can measure the concentration of the proposed component by using equation which mentioned before in (section 2.5.). www.wjpps.com

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(Fig. 4): Area under the curve spectrum of ATV (30 ug mL-1) in methanol at wavelen t an

(

n ) and (

n )

(Fig. 5): Area under the curve spectrum of ASP (450 ug mL-1) n a

n t

an

(

n ) and (

t an

at

n )

5.3. The Q-analysis method[58,59] In quantitative estimation of a component in binary mixture by Q-analysis method, absorbance was measured at the iso-bestic wavelength and maximum absorption wavelength of one of the two drugs. From overlain spectra of ATV and ASP (Fig.6), absorbance was measured at the selected wavelengths 277 nm (isoabsorptive point) and at 246 nm (the maximum absorption of ATV). The absorptivity coefficients of each drug at both wavelengths were determined. The concentration of ATV in laboratory mixture and tablet formulation was determined by substituting the absorbance and absorptivity coefficients in the previous equation mentioned in (section 2.6.).

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(Fig. 6): Zero-order absorption spectra of (20 ug ml-1) of ATV, (300 ug ml-1) of ASP and mixture of ATV with ASP (10 + 150 ug m-1). 5.4. TLC-densitometric[56,60,61] To improve separation of bands, it was necessary to investigate the effect of different variables. Studying the optimium parameters for maximum separation was carried out as following. 5.4.1. Mobile phase Different developing systems of different composition and ratios were tried for separation, e.g. hexane-ethyl acetate (1:1, v/v), hexane-ethyl acetate (2:1, v/v), hexane-ethyl acetateacetic acid (8:2; 0.1, v/v/v), chloroform-hexane (5:5, v/v) and hexane-methanol (9:1, v/v). The best mobile phase was methanol-chloroform (6:4, v/v). This selected mobile phase allows the determination of ATV without interference from ASP without tailing of the separated bands. 5.4.2. Band dimension Different band dimension were tested in order to obtain sharp and symmetrical separated peaks. The optimum band width chosen was 5 mm and the inter-space between bands was 15 mm. 5.4.3. Scanning wavelength Different scanning wavelength (251, 246, 240, 235 and 224 nm) were tried. Peaks at 246 nm were more sharp, symmetrical and minimum noise was obtained.

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5.4.4. Slit dimension of scanning light beam The slit dimension of scanning light beam should ensure complete coverage of band dimensions on the scanned track without interference of adjacent bands. Different slit dimension were tried, where 5 mm × 0.2 mm proved to be the slit dimension of choice which provides highest sensitivity. This method is based on the difference in the Rf values of ATV (Rf = 0.73±0.01) and ASP (Rf = 0.32 ± 0.02) (Fig.7).

(Fig. 7): Thin layer chromatogram of separat d a

n n an

(

) and (

a )

t

t

t

he calibration curve was constructed by plotting the integrated pea corresponding concentration in the range of (0.2 0.6 ug m

a tat n and

-1

) and (

area versus the

9 ug m

-1

) for ATV

and ASP respectively. The regression equation was calculated and found to be Y = 0.8395 × 104 X – 0.326 × 103

r = 0.9996

Where Y is the integrated peak area, X is the concentration of ATV in ug/band and r is the correlation coefficient. As the method could effectively separate both ATV and ASP from each other, it can be employed as stability indicating one. 5.5. Accuracy and Precision According to the ICH guideline, three replicate determination of three different concentration of the studied drug in pure form within their linearity ranges were performed in the same day

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(intra-day) and in three successive days (inter-day) for each method. Concentrations of (10, 30 and 50 ug mL-1) were used in the first derivative methods, since concentrations of (20, 30 and 40 ug mL-1) were used in bivariate and area under curve methods, while concentrations of (20, 30 and 50 ug mL-1) were used in Q-analysis method and the concentration of (2, 4 and 6 ug mL-1) were used in densitometric method. Accuracy as recovery percent (R%) and precision as percentage relative standard deviation (RSD%) were calculated and results were listed in (Table 2). 5.6. Specificity The specificity of the proposed methods was assured by applying the laboratory prepared mixtures of ATV and ASP as mentioned before in (section 4.5.) and specificity of TLCdensitometric method was previously demonstrated in (Fig.7). The results were listed in (Table 3). 8. Pharmaceutical Applications The proposed methods were applied to the determination of the studied drug in Ecospirin

®

capsules. The statistical comparison between the results obtained by applying the proposed methods and those obtained by applying the reported method [62], showed less calculated t and F values revealing no significant difference in accuracy and precision, (Table 5). Table (1): Spectral data for determination of atorvastatin in presence of aspirin by proposed methods. Parameters Wavelength (nm) Linearity range (µg ml-1) LOD (µg ml-1) LOQ (µg ml-1) Regression equation* Slope (b) Intercept (a) Regression coefficient (r2)

Bivariate 240

250

5 ― 50

Area under curve 215-225

235-245

0 ― 50

Q-analysis 246

277

0 ― 05

TLCdensitometry 246 2

6

0.038 0.114

0.014 0.041

0.074 0.224

0.037 0.113

0.022 0.066

0.211 0.638

0.004 0.011

0.2018 0.0024

0.2068 0.0027

1.79 0.0011

2.0062 -0.0012

0.2099 -0.0001

0.1284 0.0008

0.8415×104 -0.366×102

0.9999

0.9999

0.9999

0.9999

0.9998

0.9998

0.9998

*y= a + bx where y is the response and x is the concentration.

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Table (2): Intraday and interday accuracy and precision for the determination of

TLC Area under Q - analysis Bivariate Method densitometry curve

atorvastatin by the proposed methods.

20

Intraday Found Accuracy Conc. + SD (R%) 20.076±0.008 100.38

30

30.168±0.005

100.56

0.170

30.126±0.017

100.42

0.572

40

39.852±0.010

99.63

0.251

39.772±0.018

99.43

0.453

20

19.986±0.014

99.93

0.690

19.996±0.016

99.98

0.777

30

30.151±0.003

100.50

0.097

30.414±0.033

101.38

1.082

40

40.096±0.008

100.24

0.202

40.201±0.020

100.50

0.489

20

20.050±0.012

100.25

0.607

20.016±0.015

100.08

0.736

30

29.659±0.012

98.83

0.410

29.514±0.008

98.38

0.276

Conc g.ml-1

Precision (RSD%) 0.380

Interday Found Accuracy Conc. + SD (R%) 19.950±0.015 99.75

Precision (RSD%) 0.738

50

50.080± 0.024

100.15

0.474

50.135±0.016

100.27

0.321

2

1.993±0.002

99.65

0.766

1.994±0.003

99.70

0.757

4

4.017±0.003

100.43

0.627

4.007±0.002

100.17

0.381

100.72

0.816

6.047±0.006

100.78

0.911

6

6.043±0.005

Table (3): Determination of atorvastatin in presence of aspirin in their laboratory mixtures by the proposed methods.

Q-analysis Area under curve

Bivariate

methods

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ATV (µg ml-1) 5 10 20 30 40 50 Mean ± RSD% 10 20 30 40 50 Mean ± RSD% 10 20 30 40

ASP (µg ml-1) 75 150 300 450 600 750

ATV found (µg ml-1) 4.930 9.950 20.140 30.241 39.941 49.922

150 300 450 600 750

10.070 20.148 29.559 39.988 49.920

150 300 450 600

9.883 20.008 29.406 39.860

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Recovery % of ATV 98.60 99.50 100.70 100.80 99.85 99.84 99.88 ± 0.814 100.70 100.74 98.53 99.97 99.84 99.96 ± 0.896 98.83 100.04 98.02 99.65

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TLC- densitometry

50 Mean ± RSD% 2 3 4 5 6 Mean ± RSD%

750

49.870

30 45 60 75 90

1.996 2.980 4.040 4.960 6.032

99.74 99.26 ± 0.828 99.79 99.33 101.00 99.14 100.45 99.94 ± 0.777

Table (4): Values of the sensitivity matrix determinants calculated according to kaiser's method(53) ( k x 106) for the mixture of atorvastatin and aspirin by the bivariate method. λλ 300 290 280 270 260 250 340

300 0

290 1512 0

280 413.65 631.03 0

270 77.5 1244.8 699.63 0

260 3132.6 1798.34 1208.53 475.66 0

250 3371 2050.92 1339.04 465.08 111.8 0

240 357.1 193.66 1198.67 2570.06 3786.92 4306.76 0

Table (5): Statistical comparison between the results obtained by applying the proposed (spectrophotometric and densitometric) and reported methods for determination of atorvastatin in Ecosprin® capsules. Bivariate N* mean SD Variance t-test F-value

5 100.10 0.711 0.711 0.453 (2.306) 1.591 (6.388)

Area under curve 5 100.54 0.460 0.457 0.659 (2.306) 3.802 (6.388)

Q-analysis 5 100.60 0.798 0.793 0.852 (2.306) 1.103 (6.388)

TLCdensitometry 5 100.04 0.782 0.781 0.539 (2.306) 1.325 (6.388)

Reported method[60] 5 100.33 0.897 0.894 —— ——

*No. of experimental. t –test and F-value at (p= 0.05). 9. CONCLUSION The present work is concerned with the determination of ATV in presence of ASP. Reviewing the literature on the determination of Atorvastatin in presence of Aspirin (Binary mixture) revealed the lack of any stability indicating spectrophotometric or densitometric methods for the determination of Atorvastatin.

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Bivariate, area under curve and Q - analysis spectrophotometric methods are well-established techniques that are able to enhance the resolution of overlapping bands. The advantages of TLC-densitometric method is that several samples can be run simultaneously using a small volume of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis and provide high sensitivity and selectivity. Analysis of authentic samples containing ATV and ASP showed no interference from the common additives and excipient. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. It can be easily and conveniently adopted for routine quality control analysis. The suggested methods are found to be simple, accurate, selective and equally sensitive with no significant difference of the precision compared with the reported method of analysis.[60] 10. ACKNOWLEDGMENT I am deeply thankful to ALLAH, by the grace of whom this work was realized. I wish to express my indebtedness and gratitude to staff members of Analytical Chemistry Department for their valuable supervision, continuous guidance, and encouragement throughout the whole work. 11. REFERENCES 1. S. c. Sweetman, thirty-sixth ed., Martindale-The Complete Drug Reference, The Pharmaceutical Press, London, UK, 2009; 1: 1218-1219, 1238-1239, 1284-1285, 1307-1310. 2. The Merck index, 13th ed., Merck and Co., Inc., White House Station, NJ, 1997; 868. 3. Y. Shitara, Y. Sugiyama; Pharmacokinetic and pharmacodynamics alteration of 3hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: drug-drug interaction and iter-individual differences in transporter and metabolic enzyme functions. Pharmacol Ther., 2006; 112: 71-105. 4. T. G. Altuntas, N. Erk; J. Liq. Chromatogr. Relat. Technol., 2004; 27: 83-93. 5. M. K. Pasha, S. Muzeeb, S. J. Basha, D Shashikumar, R. Mullangi, et al. Biomed. Chromatogr., 2006; 209: 282-293. 6. S. S. Sonawane, A. A. Shirkhedkar, R. A. Fursule, S. J. Surana; Eurasian J. Anal. Chem., 2006; 1: 31-41.

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