A simple and selective LC method is described for the determination of Rosuvastatin and Aspirin tablet dosage forms. Chromatographic separation was achieved on a C18 column using mobile phase consisting of A mixture of 60 volumes of 20mM Phosphate buffer pH 3.5: 20 volumes of Acetonitrile and 20 voulmes of Methanol. With detection of 232 nm. Linearity was observed in the range 12-28 µg /ml for Rosuvastatin (r2 =0.9977) and 90-210 µg /ml for Aspirin (r2 =0.9953) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing % RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.

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Available online at www.icjpir.com ISSN: 2349-5448
Intercontinental journal of pharmaceutical
Investigations and Research
ICJPIR |Volume 3 | Issue 1 | Jan – Mar- 2016 Research Article
New RP HPLC method for the simultaneous estimation of rosuvastatin
and aspirin in pharmaceutical dosage form
M. Sambasiva Rao, A. Sunil Kumar Reddy, A. Ashok Kumar
Professor & HOD OF Vijaya college of pharmacy, Munaganur (village), Hayathnagar (Mandal),
Ranga redy (District), Pin-501511
Corresponding Author: A. Ashok Kumar
Email: ashok576@gmail.com
ABSTRACT
A simple and selective LC method is described for the determination of Rosuvastatin and Aspirin tablet dosage
forms. Chromatographic separation was achieved on a C18 column using mobile phase consisting of A mixture
of 60 volumes of 20mM Phosphate buffer pH 3.5: 20 volumes of Acetonitrile and 20 voulmes of Methanol.
With detection of 232 nm. Linearity was observed in the range 12-28 µg /ml for Rosuvastatin (r2
=0.9977) and
90-210 µg /ml for Aspirin (r2
=0.9953) for the amount of drugs estimated by the proposed methods was in
good agreement with the label claim. The proposed methods were validated. The accuracy of the methods was
assessed by recovery studies at three different levels. Recovery experiments indicated the absence of
interference from commonly encountered pharmaceutical additives. The method was found to be precise as
indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the
methods and can be used for routine analysis of pharmaceutical dosage form.
Keywords: 60 volumes of 20mM Phosphate buffer pH 3.5: 20 volumes of Acetonitrile and 20 voulmes of
Methanol, Rosuvastatin and Aspirin.
INTRODUCTION
Pharmaceutical analysis is a branch of
chemistry involving a process of identification,
determination, quantification, purification and
separation of components in a mixture or
determination of chemical structure of compounds.
There are two main types of analysis – Qualitative
and Quantitative analysis1
.
Qualitative analysis is performed to establish
composition of a substance. It is done to determine
the presence of a compound or substance in a
given sample or not. The various qualitative tests
are detection of evolved gas, limit tests, color
change reactions, determination of melting point
and boiling point, mass spectroscopy,
determination of nuclear half life etc.

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Quantitative analysis techniques are mainly
used to determine the amount or concentration of
analyte in a sample and expressed as a numerical
value in appropriate units. These techniques are
based on suitable chemical reaction and either
measuring the amount of reagent added to
complete the reaction or measuring the amount of
reaction product obtained the characteristic
movement of a substance through a defined
medium under controlled conditions, electrical
measurement or measurement of spectroscopic
properties of the compound. 2
METHODOLOGY
Mobile Phase
A mixture of 60 volumes of 20mM Phosphate
buffer pH 3.5:20volumes of Acetonitrile and 20
voulmes of Methanol. The mobile phase was
sonicated for 10min to remove gases.
Preparation of Phosphate buffer (20mM)
2.721 gm. of potassium di hydrogen phosphate
(KH2PO4) was weighed and dissolved in 100ml of
water and volume was made up to 1000ml with
water. Adjust the pH to 3.5 using ortho phosphoric
acid. The buffer was filtered through 0.45µ filters
to remove all fine particles and gases.
Determination of Working Wavelength
(λmax)
In simultaneous estimation of two drugs
isobestic wavelength is used. Isobestic point is the
wavelength where the molar absorptivity is the
same for two substances that are interconvertible.
So this wavelength is used in simultaneous
estimation to estimate both drugs accurately.
Preparation of standard stock solution of
Rosuvastatin
25 mg of Rosuvastatin was weighed and
transferred in to 250ml volumetric flask and
dissolved in methanol and then make up to the
mark with methanol and prepare 10 µg /ml of
solution by diluting 1ml to 10ml with methanol.
Preparation of standard stock solution of
Aspirin:
25mg of Aspirin was weighed in to 250ml
volumetric flask and dissolved in Methanol and
then dilute up to the mark with methanol and
prepare 10 µg /ml of solution by diluting 1ml to
10ml with methanol.
RESULTS AND DISCUSSION
Solubility Studies
These studies are carried out at 25 0
C
Rosuvastatin
Sparingly soluble in methanol, Freely Soluble
in Water.
Aspirin
It is freely soluble in water,
sparingly soluble in methanol, very
slightly soluble in ethanol
METHOD DEVELOPMENT OF
ROSUVASTATIN AND ASPIRIN
Trial-5: (Optimised)
Preparation of mixed standard stock solution
Weigh accurately 20 mg of Rosuvastatin nand
150 mg of Aspirin in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase.
Then 1mL of the above solution was
transferred to 10mL volumetric flask and diluted to
volume with mobile phase.(This solution contains
20 µg/ml of Rosuvastatinand 150 µg/ml of
Aspirin). This solution is used for recording
chromatogram.

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Fig. 4: Chromatogram of Rosuvastatin and Aspirin by using mobile phase
Table 1: Result for ROSUVASTATINand ASPIRINby using mobile phase
S.No. Name Rt (min) Peak Area Asymmetry Efficiency Resolution
1 ASPIRIN 3.393 4341.612 1.526 2591 -
2 ROSUVASTATIN 4.407 296.410 1.133 3447 3.508
Observation
 There is good resolution (>2) between the
peaks.
 USP Tailing is within the limit (Not more than
2).
 USP plate count is > 2000, hence passed as per
ICH guidelines.
 The details are given in the table 1and figure
4, hence this method was taken for optimized.
Table 2: Optimized chromatographic conditions
Mobile phase KH2PO4 Buffer(pH 3.5):ACN:Methanol
pH 3.5
Column Inertsil ODS, 3V(250×4.6× 5µ)
Flow rate 1.0 ml/min
Column temperature Room temperature(20-25o
C)
Sample temperature Room temperature(20-25o
C)
Wavelength 232 nm
Injection volume 20 µl
Run time 6.0 min
Retention time About 3.393min for Aspirinand 4.407min for Rosuvastatin
Assay
Preparation of samples for Assay
Standard sample
Weigh accurately 20 mg of Rosuvastatin and
150 mg of Aspirin in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase.
Then 1mL of the above solution was
transferred to 10mL volumetric flask and diluted to
volume with mobile phase.(This solution contains
20 µg/ml of Rosuvastatinand 150 µg/ml of
Aspirin). This solution is used for recording
chromatogram.
Tablet sample
10tablets (each tablet contains 75 mg of
Aspirinand 10mg of Rosuvastatin) were weighed

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and taken into a mortar uniformly mixed. Test
stock solutions of Aspirin (150μg/ml) and
Rosuvastatin (20μg/ml) were prepared by
dissolving weight equivalent to 75 mg of
Aspirinand 10 mg of Rosuvastatin and dissolved in
sufficient mobile phase. After that filtered the
solution using 0.45-micron syringe filter and
Sonicated for 5 min and dilute to 100ml with
mobile phase. Further dilutions are prepared in 5
replicates of 50μg/ml of Aspirinand 100μg/ml of
Rosuvastatinwas made by adding 1 ml of stock
solution to 10 ml of mobile phase.
Fig.5: Chromatogram of Assay standard preparation-1
Fig. 6: Chromatogram of Assay standard preparation-2
Fig.7: Chromatogram of Assay standard preparation-3

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Fig.8: Chromatogram of Assay standard preparation-4
Fig.9: Chromatogram of Assay standard preparation-5
Fig. 10: Chromatogram of Assay sample preparation-1

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Fig.11: Chromatogram of Assay sample preparation-2
Fig. 12: Chromatogram of Assay sample preparation-3
Fig. 13: Chromatogram of Assay sample preparation-4
Fig. 14: Chromatogram of Assay sample preparation-5
Table No.3: Assay Results
ASPIRIN ROSUVASTATIN
Standard Area Sample Area Standard Area Sample Area
Injection-1 4392.560 4341.612 297.311 296.410
Injection-2 4431.255 4418.182 315.227 310.445
Injection-3 4412.074 4397.958 307.059 316.873
Injection-4 4367.707 4287.403 322.334 320.296
Injection-5 4345.146 4401.250 319.463 300.312

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Average Area 4389.748 4369.281 312.2788 308.8672
Tablet average weight 150 150
Standard weight 150 20
Sample weight 300 300
Label amount 75 10
std. purity 99.2 99.3
Amount found in mg 74.05 9.82
Assay(%purity) 98.74 98.22
Observation
The amount of Aspirin and Rosuvastatin
present in the taken dosage form was found to be
98.74 % and 98.22% respectively.
VALIDATIONS
System suitability
Standard solutions were prepared as per the test
method and injected into the chromatographic
system. The system suitability parameters like
theoretical plates, resolution and asymmetric factor
were evaluated.
Table 4: Results for system suitability of Aspirin
Injection Retention time (min) Peak area Theoretical plates (TP) Tailing factor (TF)
1 3.393 4399.321 2296 1.553
2 3.397 4411.932 2301 1.553
3 3.407 4401.157 2410 1.553
4 3.410 4446.486 2415 1.595
5 3.407 4406.215 2410 1.553
6 3.397 4345.829 2396 1.568
Mean 3.4018 4401.823 - -
SD 0.0070 32.429 - -
%RSD 0.21 0.74 - -
Table 5: Results for system suitability of Rosuvastatin
Injection Retention time (min) Peak area Theoretical plates Tailing factor Resolu
tion
1 4.407 301.0
17
3447 1.156 3.473
2 4.400 307.2
85
3310 1.239 3.406
3 4.403 304.6
78
3315 1.130 3.416
4 4.397 301.6
28
3431 1.205 3.415
5 4.403 301.3
85
3315 1.174 3.416
6 4.400 308.4
65
3310 1.295 3.439
Mean 4.402 304.0
76
- - -
SD 0.003 3.241 - - -

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%RSD 0.08 1.07 - - -
Acceptance criteria
1. The % RSD for the retention times of Aspirin
and Rosuvastatin Peaks from 6 replicate
injections of each Standard solution should be
not more than 2.0 %
2. The % RSD for the peak area responses of
Aspirin and Rosuvastatin peaks from 6
replicate injections of each standard solution
should be not more than 2.0%.
3. The number of theoretical plates (N) for the
Aspirin and Rosuvastatin peaks is not less than
2000.
4. The Tailing factor (T) for the Aspirin and
Rosuvastatin peak is not more than 2.0.
Observation
The % RSD for the retention times and peak
area of Aspirin and Rosuvastatin were found to be
less than 2%. The plate count and tailing factor
results were found to be satisfactory and are found
to be within the limit.
Specificity by Direct comparison method
There is no interference of mobile phase,
solvent and placebo with the analyte peak and also
the peak purity of analyte peak which indicate that
the method is specific for the analysis of analytes
in their dosage form.
Preparation of samples for Assay
Standard sample
Weigh accurately 20 mg of Rosuvastatin and
150 mg of Aspirin in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase.
Then 1mL of the above solution was
transferred to 10mL volumetric flask and diluted to
volume with mobile phase.(This solution contains
20 µg/ml of Rosuvastatin and 150 µg/ml of
Aspirin). This solution is used for recording
chromatogram.
Tablet sample
10tablets (each tablet contains 75 mg of Aspirin
and 10 mg of Rosuvastatin) were weighed and
taken into a mortar uniformly mixed. Test stock
solutions of Aspirin (150μg/ml) and Rosuvastatin
(20μg/ml) were prepared by dissolving weight
equivalent to 75 mg of Aspirin and 10 mg of
Rosuvastatin and dissolved in sufficient mobile
phase. After that filtered the solution using 0.45-
micron syringe filter and Sonicated for 5 min and
dilute to 100ml with mobile phase. Further
dilutions are prepared in 5 replicates of 50μg/ml of
Aspirin and 100 μg/ml of Rosuvastatin was made
by adding 1 ml of stock solution to 10 ml of
mobile phase.
Fig. 15: Blank chromatogram for specificity by using mobile phase

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Fig. 16: Chromatogram for specificity of Aspirin and Rosuvastatin standard.
Fig. 17: Chromatogram for Specificity of Aspirin and Rosuvastatin sample
Observation
It is observed from the above data, diluent or
excipient peaks are not interfering with the Aspirin
and Rosuvastatin peaks.
LINEARITY AND RANGE
Preparation of mixed standard stock solution
Weigh accurately 20 mg of Rosuvastatin and
150 mg of Aspirin in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase.
Then 1mL of the above solution was
transferred to 10mL volumetric flask and diluted to
volume with mobile phase.(This solution contains
20 µg/ml of Rosuvastatin and 150 µg/ml of
Aspirin). This solution is used for recording
chromatogram and further dilutions were given in
the table No 6
Table 6: Linearity Preparations
Preparations Volume from standard
stock transferred in ml
Volume made up in ml
(with mobile phase)
Concentration of solution(µg
/ml)
ASPIRIN ROSUVASTATIN
Preparation 1 0.6 10 90 12
Preparation 2 0.8 10 120 16
Preparation 3 1.0 10 150 20
Preparation 4 1.2 10 180 24
Preparation 5 1.4 10 210 28

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Fig. 18: Chromatogram of Aspirin and Rosuvastatinpreparation-1
Fig. 19: Chromatogram of Aspirin and Rosuvastatin preparation-2
Fig.20: Chromatogram of Aspirin and Rosuvastatin preparation-3
Fig.21: Chromatogram of Aspirin and Rosuvastatin preparation-4

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Fig. 22: Chromatogram of Aspirin and Rosuvastatinfor preparation-5
Table 7: linearity of ASPIRIN
S.No. Conc.(µg/ml ) Area
1 90 2593.335
2 120 3352.327
3 150 4455.003
4 180 5363.931
5 210 6312.861
Table 8: linearity of ROSUVASTATIN
S.No. Conc.(µg/ml ) Area
1 12 176.327
2 16 249.969
3 20 306.279
4 24 361.598
5 28 442.401
Fig. 23: Linearity graph of Aspirin and rosuvastatin
Linearity of Aspirin y = 31.502x - 309.84
R2
= 0.9977
0
1000
2000
3000
4000
5000
6000
7000
0 50 100 150 200 250
Conc
Area
Linearity of Rosuvastatin y = 16.094x - 14.574
R2
= 0.9953
0
50
100
150
200
250
300
350
400
450
500
0 5 10 15 20 25 30
Conc
Area

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Acceptance criteria
The relationship between the concentration of
Aspirin and Rosuvastatin and area of Aspirin and
Rosuvastatin should be linear in the specified range
and the correlation should not be less than 0.99.
Observation
The correlation coefficient for linear curve
obtained between concentration vs. Area for
standard preparations of Aspirin and Rosuvastatin
is 0.9977 and 0.9953. The relationship between the
concentration of Aspirin and Rosuvastatin and area
of Aspirin and Rosuvastatin is linear in the range
examined since all points lie in a straight line and
the correlation coefficient is well within limits.
ACCURACY
Accuracy of the method was determined by
Recovery studies. To the formulation (pre analyzed
sample), the reference standards of the drugs were
added at the level of 80%, 100%, 120%. The recovery
studies were carried out three times and the
percentage recovery and percentage mean recovery
were calculated for drug is shown in table. To check
the accuracy of the method, recovery studies were
carried out by addition of standard drug solution to
pre-analyzed sample solution at three different levels
80%, 100%, 120%.
Fig. 24: Chromatogram of 80% recovery (injection 1)
Fig. 25: Chromatogram of 80% recovery (injection 2)

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Fig. 26: Chromatogram of 80% recovery (injection 3)
Fig. 27: Chromatogram of 100% recovery (injection 1)
Fig. 28: Chromatogram of 100% recovery (injection 2)
Fig. 29: Chromatogram of 100% recovery (injection 3)

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Fig. 30: Chromatogram of 120% recovery (injection 1)
Fig. 31: Chromatogram of 120% recovery (injection 2)
Fig. 32: Chromatogram of 120% recovery(injection 3)
Acceptance criteria
The % recovery of Aspirin and Rosuvastatin should lie between 98% and 102%.
Table 9: Recovery results for Aspirin
Recovery Accuracy ASPIRIN Average%

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Table 10 : Recovery results for Rosuvastatin
Observation
The percentage mean recovery of Aspirin and
Rosuvastatin is99.83% and 99.92% respectively.
PRECISION
Method precision
Prepared sample preparations of Aspirin and
Rosuvastatin as per test method and injected 6
times in to the column.
Acceptance criteria
The % Relative standard deviation of Assay
preparations of Aspirin and Rosuvastatin should be
not more than 2.0%.
level Amount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery Recovery
80% 150 4039.822 4164.483 149.07 99.38
99.83%
150 4432.307
150 4021.321
100% 180 5402.156 5429.798 182.82 101.57
180 5460.411
180 5426.827
120% 210 6249.806 6167.095 206.95 98.55
210 6024.208
210 6227.270
Recovery
level
Accuracy ROSUVASTATIN Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 20 300.213 308.539 19.75 98.74
99.92 %
20 311.768
20 313.636
100% 24 365.486 368.172 24.04 100.17
24 379.319
24 359.712
120% 28 433.330 425.524 28.24 100.87
28 417.930
28 425.311

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Fig. 33: Chromatogram of precision injection 1
Fig. 34: Chromatogram of precision injection 2
Fig. 35: Chromatogram of precision injection 3
Fig. 36: Chromatogram of precision injection 4

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Fig. 37: Chromatogram of precision injection 5
Fig. 38: Chromatogram of precision injection 6
Table 11: Results for Method precision of Aspirin and Rosuvastatin
ASPIRIN ROSUVASTATIN
S.No. Rt Area S.No. Rt Area
1 3.393 4399.321 1 4.407 301.017
2 3.397 4411.932 2 4.400 307.285
3 3.407 4401.157 3 4.403 304.678
4 3.410 4446.486 4 4.397 301.628
5 3.407 4406.215 5 4.403 301.385
6 3.397 4345.829 6 4.400 308.465
Avg 3.4018 4401.823 Avg 4.402 304.076
Stdev 0.0070 32.429 Stdev 0.003 3.241
%RSD 0.21 0.74 %RSD 0.08 1.07
Observation
Test results for Aspirin and Rosuvastatin are
showing that the %RSD of Assay results are within
limits. The results were shown in table Table9.5.1.
LIMIT OF DETECTION
Where, σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte.

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Fig. 40: Calibration graphs of ASPIRIN and ROSUVASTATIN
Table No 12: Results for calibration graph
S.No.
ASPIRIN ROSUVASTATIN
Concentration
µg/ml
Peak Area Concentration
µg/ml
Peak Area
1
90 2593.335 12 176.327
2 120 3352.327 16 249.969
3
150 4455.003 20 306.279
4 180 5363.931 24 361.598
5
210 6312.861 28 442.401
S.D. 47.4 1496 6.3246 102
Slope 31.50 16.09
Observation
The LOD for this method was found to be
4.97µg/ml & area 156.72for ASPIRIN and 1.30
µg/ml & area 20.93 for ROSUVASTATIN.
LIMIT OF QUANTIFICATION
Where,
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte. The LOQ for this method was
found to be 15.04 µg/ml & area 474.91 for
ASPIRINand 3.93 µg/ml & area 63.41 for
ROSUVASTATIN.
ROBUSTNESS
Chromatographic conditions variation
To demonstrate the robustness of the method,
prepared solution as per test method and injected at
different variable conditions like using different
conditions like Temperature and wavelength.
System suitability parameters were compared with
that of method precision.
Acceptance criteria
The system suitability should pass as per the
test method at variable conditions.
Linearity of Aspirin
y = 31.502x - 309.84
R2
= 0.9977
0
1000
2000
3000
4000
5000
6000
7000
0 50 100 150 200 250
Conc
Area
Linearity of Rosuvastatin y = 16.094x - 14.574
R2
= 0.9953
0
50
100
150
200
250
300
350
400
450
500
0 5 10 15 20 25 30
Conc
Area

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Fig. 41: Chromatogram of Aspirin and Rosuvastatin Robustness (Flow: 0.8 ml/min)
Fig. 42: Chromatogram of Aspirin and Rosuvastatin Robustness (Flow: 1.2 ml/min)
Fig. 43: Chromatogram of Aspirin and Rosuvastatin for Robustness (230nm)
Fig. 44: Chromatogram of Aspirin and Rosuvastatin for Robustness (234nm)

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Table 13: Result of Robustness study
Parameter
ASPIRIN ROSUVASTATIN
Retention time(min) Tailing factor Retention time(min) Tailing factor
Flow
0.8ml/min
1.0ml/min
1.2ml/min
4.283
3.393
2.887
1.5
1.5
1.5
5.480
4.407
3.693
1.2
1.2
1.2
Wavelength
230nm
232nm
234nm
3.413
3.393
3.423
1.4
1.5
1.5
4.383
4.407
4.390
1.1
1.1
1.1
Observation
From the observation it was found that the
system suitability parameters were within limit at
all variable conditions.
RUGGEDNESS
The ruggedness of the method was studied by
the determining the analyst to analyst variation by
performing the Assay by two different analysts
Acceptance criteria
The % Relative standard deviation of Assay
values between two analysts should be not more
than 2.0%.
Fig. 45: Chromatogram of Analyst 01 standard preparation
Fig. 46: Chromatogram of Analyst 01 sample preparation

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Fig. 47: Chromatogram of Analyst 02 standard preparation
Fig. 48: Chromatogram of Analyst 02 sample preparation
Table 14: Results for Ruggedness
ASPIRIN %Assay ROSUVASTATIN %Assay
Analyst 01 98.33 Analyst 01 96.29
Analyst 02 100.37 Analyst 02 102.44
%RSD 0.24 %RSD 0.10
Observation
From the observation the %RSD between two
analysts Assay values not greater than 2.0%, hence
the method was rugged.
CONCLUSION
From the above experimental results and
parameters it was concluded that, this newly
developed method for the simultaneous estimation
of Rosuvastatin and Aspirin was found to be
simple, precise, accurate and high resolution and
shorter retention time makes this method more
acceptable and cost effective and it can be
effectively applied for routine analysis in research
institutions, quality control department in meant in
industries, approved testing laboratories, bio-
pharmaceutical and bio-equivalence studies and in
clinical pharmacokinetic studies in near future.
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