Development and Validation of UV Spectrophotometry and RP-HPLC Method for simultaneous determination of Rosuvastin and Clopidogrel in Tablet Dosage Form

 

Roshan Telrandhe

Kamla Nehru College of Pharmacy, Butibori, Nagpur 441108, Maharashtra, India

*Corresponding Author E-mail:

 

ABSTRACT:

The aimed of research the method development and validation by UV spectrophotometry and RP HPLC method of the Rosuvastatin calcium and Clopidogrel bisulphate. The method is a simple, accurate, specific, precise, reproducible and sensitive. The λ max of ROSU and CLOP was found to be 240nm. The coefficient correlation 0.999, the Beer’s Law limit 50-150 µg/ml, from the four trial of different concentration of mobile phase was selected Methanol:Water 80:20 v/v, pH 3.0 at 240nm, flow rate 1ml/min, sample inlet 20 µL, C 18 Prontosil, %RSD of ROSU 1.017 and CLOP 0.173, theoretical plates ROSU 7797.53 and ROSU 8257.53, Retention time of ROSU 3.483min and CLOP 4.983min, Tailing factor ROSU 1.1787 and CLOP 1.074, limits 2 NMT, Accuracy ROSU 0.37 % RSD, Recovery 99.59% and CLOP 0.18 %RSD, recovery 100.41% was show good efficacy and results. The methods indicate future scope in analysis quality control of the estimation of ROSU and CLOP for routine drug quality analysis investigation.

 

KEYWORDS: UV Spectrophotometry, RP HPLC, Rosuvastatin calcium, Clopidogrel bisulphate, Simultanious estimation, validation.

 

 


INTRODUCTION:

Rosuvastatin calcium (ROSU) is chemically 7-[4-(4-Fluorophenyl)-6-(1-methylethyl)-2-(methyl-methyl sulfonyl-amino)-pyrimidin-5-yl]-3,5-dihydroxy-hept-6-enoic acid calcium (Fig 1a). It is in a group of drug called hydroxymethylglutaryl coenzyme A (HMG CoA) reductase inhibitors, or statins. ROSU reduces levels of low density lipoprotein and triglycerides in blood, when increases level of high density liproprotein in management of hyperlipidaemias1.

 

If we manufacture nanoparticle attached to UV scattering substance like ZnO and TiO2 and specifically target these nanoparticles to skin cells with sunscreen on nanoscale2.

 

Clopidogrel bisulphate (CLOP) is chemically methyl (2S)-2-(2-chlorophenyl)-2-(4H, 5H, 6H, 7H-thieno[3,2 c]pyridine-5-yl) acetate sulfate (Fig 1b). It is a USP-NF enlist drug and a new thienopyridine derivative. CLOP is an anti-platelet agent, which directly inhibit the binding of adenosine diphosphate (ADP) to its platelet receptor and blocks the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex, so inhibiting platelet aggregation3. In the last few decades, the application of nanomaterials in the area of biology and medicine has revolutionized the field of drug delivery, theranostics, imaging, diagnosis, wound healing and medical devices with miscellaneous properties4.

 

Rosuvastatin calcium is determined alone also by UV-method5 and RP-HPLC method6. The gel stimulates cell growth and enhances the restoration of damaged skin7.Clopidogrel was also estimated using UV-method, derivative spectroscopy, HPLC, HPTLC and LCMS/MS8.The chewing sticks have been widely used in the Indian subcontinent, Middle East and Africa since ancient time period9.

 

The aim of current research to developed and validated UV spectrophotometry and RP HPLC for simultaneous estimation of rosuvastatin calcium and clopdogrel bisulphate. The accuracy, precision, %RSD and recovery study was indicated the reproducibility.

 

 

Fig 1a: Chemical structure of Rosuvastatin calcium

 

 

Fig 1b: Chemical structure of Clopidogrel bisulphate

 

MATERIALS AND METHOD:

Chemicals and Reagents:

·         Water (HPLC grade)

·         Methonol (HPLC grade)

·         OPA

All reagents and chemicals were used of HPLC grade.

Pure Sample:

Table 1: Pure drug information

Drugs

Supplier

Quantity

Purity

Rosuvastatin calcium

Zim Laboratory

10.0 g

99.98% w/w

Clopidogrel bisulphate

Zim Laboratory

10.0 g

99.99% w/w

 

The drugs used for the present investigation were donated as gift samples.

 

Marketed formulation available:

Table 2: Marketed formulation information

Brand Name

Mfg By

Content

Quantity

Rosloy CV 5mg/75mg Tab

Lloyed Healthcare Pvt Ltd

Rosuvastatin calcium

5 mg

Clopidogrel

Bisulphate

75 mg

 

The marketed formulation was purchased from local market.

 

Instrumentation:

Table 3: Required Instruments information

Name of Equipment

Make

Model

UV-Visible Spectrophotometer

Thermo Electron

Double beam carry-07 Bio

HPLC

Water

996 PDA Detector

600E EMPOWER Software with Autosampler

pH Meter

Systronics

pH meter 335

Balance

Citizen

CY 104 (Micro Analytical Balance)

Column

Prontosil (5µm)

C18 [4.6 x 250 mm(id)]

 

EXPERIMENTAL:

A. UV-Spectrophotometry Method10-12

Preparation of standard stock solution10

An accurate weighed quantity of Rosuvastatin calcium about 10 mg and Clopidogrel bisulphate 10 mg was transfer separately in two volumetric flasks of 10 ml and dissolved the water. The volume was made up to mark and the solutions make to produce concentration about 1000 mg/ml.

 

λ Max determination10

The aliquot portions of stock standard solutions were diluted appropriately with Diluents Water to obtained concentration 10 mg/ml of each drug. The solutions were separately scanned in the range 200-400 nm in 1 cm cell against blank. The UV absorbance spectrum of Rosuvastatin calcium (Fig 2a) and Clopidogrel bisulphate (Fig 2b), the overlain spectrum of both the drugs (Fig 2c) is also run.

 

Preparation of Calibration Curve11

Accurately weighed ROSU and CLOP separately about 10 mg and dissolved in methanol with volume made up to 10ml mark to obtain 1000 µg/ml. the stock standard solutions was diluted further with concentration range 50-150 µg/ml.

The (Fig 3a and Fig 3b) and (Table 4) shows the calibration curve and peak area data.

 

Assay of tablet formulation12

The twenty tablet of Rosley CV 5mg/75mg Tab content 5mg ROSU and 75mg CLOP were weighed accurately and finely powered separetly. Powder equivalent to 5mg rosuvastatin calcium and 75mg clopidogrel bisulphate was weighed and transferred to sintered glass crucible and drug extracted with three 20ml quantity of methanol and then final volume made up to 100ml to produce the 1000µg/ml of solution. Pipette out 1ml of solution in 10ml volumetric flask and make up the volume with diluents to make 50µg/ml ROSU and 750µg/ml CLOP.

 

 

B. HPLC Method13

Mobile phase:

From the various tried (Fig 4a,4b,4c,4d) , mobile phase containing, methanol: water with different concentration at pH 3.0 with column C 18 was selected since it gives sharp reproducible retention time for ROSU and CLOP (Fig 4d), chromatographic condition also determined. (Table 6)

 

Standard solution:

Accurately weighed separately about 5 mg ROSU and 75mg CLOP was dissolved into separate volumetric flask in methanol and volume was made up to mark 10 ml by same to obtain 500 mg/ml ROSU and 7500mg/ml stock solution.

 

Pipette out 1 ml from standard stock solutions and diluted it with 10ml methanol to obtain 50mg/ml ROSU and 750mg/ml CLOP.

 

From the stock solution were dilutions were made in the concentration 50, 80, 100, 120, 150 mg/ml of ROSU and CLOP. A 20mL of each sample was injected and chromatogram was recorded at 240nm. This above concentration range was found to be linear and obeys Beer’s law.

 

Analysis of marketed preparation:14

The twenty tablet of Rosloy CV 5mg/75mg Tab (Label claim: Rosuvastain calcium 5mg and Clopidogrel bisulphate 75mg) was weight to equivalent to label claim. Finely powder and prepare the solutions of ROSU and CLOP separately in 100 ml of volumetric flasks. Add 50ml methanol and disperse the powder completely. Then sonicated for 10min to it added 50ml of diluents to make up volume and sonicate 5min to dissolved with intermittent shaking. Allow the solutions to cool at room temperature. The stock of 1000mg/ml was prepared. From this stock solution pipette out 1ml supernatant liquid in 10ml volumetric flask and make up the volume with diluents to make 100ppm. To produce the 50mg/ml ROSU and 750mg/ml CLOP.

 

Method Validation:15-16

The method was developed and validated according to ICH guidelines.

 

Linearity:

Calibration graphs constructed by plotting peak area v/s concentration of ROSU and CLOP, the regression equation were calculated. The calibration range 50-150 mg/ml for both the drugs. Aliquots 20mL of each solution injected under the operating chromatographic condition. (Table 7)

 

 

System suitability test:

System suitability is a pharmacopoeial requirement and used to verify, whether the resolution and reproducibility of chromatographic system are adequate for analysis to be done. The tests were performed by collecting data from 5 replicate injections of standard solutions. (Table 8)

 

Accuracy:

The accuracy of the methods was established by recovery studies of ROSU (Table 9) and CLOP (Table 10)

 

Precision:

The intraday and interday precision of the proposed method was determined by analyzed the solution of ROSU and CLOP on the same and different days. (Fig 6) (Table 11 and 12)

 

Limit of detection and limit of quantitation (LOD and LOQ):

The LOD and LOQ of ROSU and CLOP were determined by calculating signal to noise (S/N) ratio , according to International Conference on Hormonization guidelines.

 

Robustness:

The robustness of the method was evaluated by assaying the sample solution after slight but deliberate changes in the analytical condition inject into HPLC system at -10% flow rate (0.9mL/min) and +10% flow rate (1.1mL/min).

The flow rate 1ml/min, 0.9ml/min and 1.1ml/min. (Fig 7, 8, 9) (Table 13)

 

Organic changes ie -10% and +10% Methanol also studied. (Fig 10, 11) (Table 14)

 

RESULTS AND DISCUSSION:

A. UV-Spectrophotometry method:

λ max determination:

 

 

Fig 2a: Spectrum of Rosuvastatin calcium

 

Fig 2b: Spectrum of Clopidogrel bisulphate

 

The λ max of ROSU and CLOP was found to be 240 nm.

 

Fig 2c: Overlain spectrum of ROSU and CLOP

 

Calibration curve:

Table 4: Calibration data

Sr. No

Concentration (µg/ml)

Peak area

ROSU

CLOP

1

50

79.91

390.65

2

80

127.85

635.04

3

100

159.82

781.3

4

120

191.78

935.53

5

150

235.76

1165.56

 

 

Fig 3a: Calibration curve of ROSU

 

Fig 3b: Calibration curve of CLOP

 

B. HPLC Method:

Mobile phase:

 

Fig 4a: Trial 1: chromatogram of ROSU and CLOP with mobile phase Methanol: water (100:00 v/v) 1ml/min at 270nm

 

 

Fig 4b: Trial 2: chromatogram of ROSU and CLOP with mobile phase Methanol: Water (80:20 v/v) pH 3.0

 

Fig 4c: Trial 3: chromatogram of ROSU and CLOP with mobile phase Methanol: Water (70:30 v/v) pH 3.0

 

 

Fig 4d: Trial 4: Chromatogram of ROSU and CLOP with mobile phase Methanol: water (80:20 v/v) pH 3.0

 

From the above four trial the observations are fallows

 

Table 5: Observations for trial of different concentration of mobile phase

Trial

Mobile phase

Peak characteristics

1

Methanol:Water (100:00 v/v) 1ml/min at 270nm

Peaks are not separated.

(Fig 4a)

2

Methanol:Water (80:20 v/v) [pH 3.0] 1ml/min at 270nm

Peaks were separated but asymmetry was out range.

(Fig 4b)

3

Methanol;Water (70:30 v/v) [pH 3.0] 1ml/min at 240nm

Peaks were separated but resolution are out of range.

(Fig 4c)

4

Methanol:Water (80:20 v/v) [pH 3.0] 1ml/min at 240nm

All parameters are in range as per USP.

(Fig 4d)

 

From the above four trials (Fig 4a,4b, 4c, 4d) and (Table 5) the selection of mobile phase done and chromatographic condition established.

 

Chromatographic conditions:

Table 6: Chromatographic condition

Column

Prontosil [4.6 x 250mm (id) ]

Particle size packing

10 mm

Stationaray phase

C 18 Prontosil (5mm)

Mobile phase

Methanol:Water (80:20 v/v) pH 3.0

Detection wavelength

240nm

Flow rate

1ml/min

Run time

08 min

Temperature

Ambient

Sample size

20 mL

Diluent

Methanol

 

Fig 5: Separation of two drug in selected mobile phase showing retention time ROSU 3.483 min and CLOP 4.983 min.

 

Method validation:

Linearity:

Table 7: Linearity observation

Parameters

ROSU

CLOP

λmax, nm

240

240

Beer’s Law limit (µg/ml)

50-150

50-150

Regression equation (Y*)

Y=1.564x+2.625

Y=7.716+9.972

Correlation coefficient (r2)

0.999

0.999

Slope (b)

1.564

7.716

Intercept (a)

2.62

9.97

 


 

Table 8: Observation of system suitability

Sr.No

Peak  area

Retention Time

Asymmetry

No .of theoretical Plates

Resolution

ROSU

CLOP

ROSU

CLOP

ROSU

CLOP

ROSU

CLOP

1

75.0602

408.35

 3.483

4.983

0.752

0.956

7849.1

8338.7

6.62

2

74.6202

408.85

3.450

4.950

0.763

0.954

7700.2

8142.8

6.42

3

76.1110

409.75

3.483

5.083

0.742

0.971

7843.3

8290.0

6.40

4

72.0124

406.82

3.466

5.066

0.766

0.956

7794.1

8067.3

6.42

5

74.1202

408.15

3.450

4.950

0.757

0.978

7681.5

8800.2

6.40

mean

75.26

408.98

3.460

4.994

0.752

0.960

7797.53

8257.53

6.48

 S.D

0.765

0.709

0.017

0.076

0.010

0.009

84.34

101.99

0.121

% R.S.D

1.017

0.173

0.513

1.537

1.39

0.967

1.081

1.235

1.877


 

System suitability: Accuracy:

Table 9: Accuracy of ROSU

 

Rosuvastatine calcium

Peak Area

Amount Taken(mg)

Amount recovered (mg)

% Recovery

Average recovery

% RSD

80%

120.10

1.6

3.63

98.61

100.66

0.27

120.59

1.6

3.65

99.63

120.32

1.6

3.64

99.06

100%

132.490

02

4.04

99.30

99.28

0.38

133.44

02

4.07

100.86

133.120

02

4.06

100.34

120%

145.13

2.4

4.46

100.10

99.44

0.46

146.020

2.4

4.49

101.32

146.270

2.4

4.50

101.67

Mean

99.59

0.37


 

Table 10: Accuracy of CLOP

Recovery level

Clopidogrel bisulphate

Area

Amount Taken(mg)

Amount recovered (mg)

% Recovery

Average recovery

% RSD

80%

601.21

24

53.95

99.54

99.41

0.22

602.780

24

54.13

99.32

600.890

24

53.91

99.38

 

100%

654.810

30

60.36

100.21

100.49

0.13

  655.090

30

60.47

100.56

656.090

30

60.51

100.72

120%

708.510

36

66.36

101.46

101.33

0.20

  707.160

36

66.62

100.91

 709.290

36

66.88

101.62

Mean

100.41

0.18

 


Precision:

Table 11: Observation of Precision

Sr. No.

Parameter

Observations

Limits

ROSU

CLOP

1

The % RSD of peak area response for three replicate injections of standard

1.017

0.173

NMT 2.0

2

Theoretical plates

7797.53

8257.53

NLT 2000

3

Tailing factor

1.1787

1.074

NMT 2.0

 

 

Fig 6: Chromatogram System precision showing Repeatability

 

 

 

 

Robustness:

Flow rate:

 

Fig 7: Chromatogram showing Flow rate 1ml/min

 

System Precision:

Table 12: Observation of System Precision

Injection No.

Area Response

ROSU

CLOP

1

133,72

676.566

2

134.77

667.67

3

133.92

676.44

Average

134.345

673.558

SD

0.601

5.100

% RSD

0.447

0.757

 

 

Fig 8: Chromatogram showing Flow rate 0.9ml/min

 

Fig 9: Chromatogram showing flow rate 1.1ml/min

 


Table 13: Observation of changing in flow rate

Sr. No.

System Suitability parameter

Observations for flow rate

Limits

Unchanged

0.9 ml

1.1 ml

1

The % RSD of peak area response for three replicate injections

ROSU

1.017

0.82

0.75

NMT 2.0

CLOP

0.173

0.20

0.05

2

Theoretical plates

ROSU

7797.53

6038.7

4556.9

NLT 2000

CLOP

8257.53

5965.7

5328.9

3

Tailing factor

ROSU

1.28

1.91

1.10

NMT 2.0

CLOP

1.06

0.950

1.00

4

Retention Time (Min)

ROSU

3.483

3.85

2.86

 

CLOP

4.983

5.46

4.13

 


Organic composition changes:

Fig 10: -10% Methanol

 

 

Fig 11: +10% Methanol

 


Table 14: Observaation of changes in organic composition (-10% and +10% Methanol)

Sr. No.

System Suitability parameter

Observations

Limits

Unchanged

- 10%

+ 10%

1

The % RSD of peak area response for three replicate injections

ROSU

1.017

0.655

0.046

NMT 2.0

CLOP

0.173

0.021

0.030

2

Theoretical plates

ROSU

7797.53

4896

6347.6

NLT 2000

CLOP

8257.53

8060

9386.6

3

Tailing factor

ROSU

1.28

1.166

1.08

NMT 2.0

CLOP

1.06

1.062

0.88

4

Retention Time (Min)

ROSU

3.483

3.46

3.38

 

CLOP

4.983

5.08

4.80


 

CONCLUSION:

The research indicate that UV spectrophotomerty and RP-HPLC method to be simple, accurate, specific, precise, reproducible, and sensitive. No interference of additives, matrix and good recovery and environmently friendly method. This implies that proposed UV and HPLC method can be used for routine quality control analysis of ROSU and CLOP in combination pharmaceutical dosage form.

 

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Received on 03.12.2017          Accepted on 11.01.2018        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1): 25-32.

DOI: 10.5958/2231-5675.2018.00005.4