Single RP-HPLC Method for Quantification of 3 Drugs

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490

ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries An International peer
Review Journal for Science

Research Article Available online through www.ijsidonline.info

SINGLE RP-HPLC METHOD FOR THE QUANTIFICATION OF ACECLOFENAC, PARACETAMOL AND
CHLOROZOXAZONE IN FORMULATIONS

1Dept. of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India; 2Department of Chemistry,
Hari kishan Reddy Ganthi1*, Hanimi Reddy Bapatu2, Maram Ravi Kumar3, Useni Reddy Mallu1,

JNT University, Kukatpally, Hyderabad, AP, India-500072.; 3 AR&D, Custom Pharmaceutical Services,
Dr. Reddy’s Laboratories Ltd, Bachupally, Hyd-72, India.

Received: 14-08-2012

Accepted: 17-10-2012 ABSTRACT

Objectives: To develop a single RP-HPLC method for determination of Aceclofenac,
Paracetamol and Chlorozoxazone contents in formulation.
*Corresponding Author

Methods: Chromatographic separation was achieved on Inertsil ODS 3V, 150 x4.6mm, 5µ
column. Mobile phase composed of phosphate b u f f e r of pH 6.0 and
a c e t o n i t r i l e i n t h e r a t i o n 6 7 : 3 3 v / v . 1.0ml per min flow rate and detection
was at 275 nm.
Results: High resolution was achieved with the simple mobile phase composition and
retention time of Paracetamol, Chlorozoxazone, and Aceclofenac are about 2.1min, 8.8min
and 20.7min, respectively. The area of all ingredient peaks were a linear function of
concentration in the range 150.4 to 752.3 ppm for Paracetamol, 120.2 to 761.4 ppm for
Chlorozoxazone and 29.9 to 159.9 ppm for Aceclofenac and the correlation co-efficient
Address:

value of all activeINTRODUCTION limit (0.999).
ingredients within the
Name:

Conclusion: Proposed HPLC method was validated with specificity, linearity, accuracy,
Hari Kishan Reddy Ganthi
Place:

reproducibility and ruggedness and it is applicable for regular analysis.
Sri Krishnadevaraya University

Keywords: Aceclofenac, Paracetamol, Chlorozoxazone and RP-HPLC method.
Anantapur, AP, India
E-mail:
kishangan05ster@gmail.com

INTRODUCTION

471
International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012


Paracetamol (acetaminophen) is one of the most popular over-the-counter (OTC) analgesic and antipyretic drugs.
INTRODUCTION

Paracetamol (1-8) is available in different dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories.
Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID). It is used for the relief of pain and inflammation in
rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The dose is 100 mg twice daily. It should not be given to
people with porphyria or breast-feeding mothers, and is not recommended for children.
Chlorzoxazone is used to relieve pain and stiffness caused by muscle strains and sprains. It is used in combination with
physical therapy, analgesics (such as aspirin or acetaminophen), and rest. The side effects are upset stomach, drowsiness,
dizziness, lightheadedness, weakness, skin rash or itching, yellowing of the skin or eyes and stomach pain.
Chemical structures of all ingredients were represented in figure-1.All three ingredients are available in liquid pharmaceutical
dosage forms.
Paracetamol Chlorozoxazone Aceclofenac

Figure-1: Chemical structure of all ingredients

All ingredients have reported methods for individual and other combination products, but the objective of the present
study is to develop a single RP-HPLC method for the estimation of Paracetamol, Aceclofenac and Chlorozoxazone in
formulations and the developed and validated method is simple, novel, rugged and linear.
MATERIALS AND METHODS

Various buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization of
Selection of mobile phase:

mobile phase. Finally well shaped and high resolution was achieved with pH 6.0 phosphate buffer and acetonitrile at 67:33 v/v
ratio.

Ortho phosphoric acid, triethyl amine (AR Grade) was procured from S.D fine chemicals. High pure (NLT 98.5%) standards
Chemicals and reagents:

(Aceclofenac, Paracetamol and Chlorozoxazone) were used for this study. HPLC grade acetonitrile were procured from
Spectrochem Pvt. Ltd. Water is prepared by mili Q system (Milli-pore).
Buffer preparation: Diluted 2.0mL of ortho phosphoric acid to 1000 mL of water. Adjusted to pH 6.0 with triethyl amine
Filtered through 0.45µm membrane filter and degassed.
Mobile Phase: Mixed the buffer and acetonitrile in the ration 67:33 v/v.
Diluent: Mobile phase.

Column : Inertsil ODS 3V (150X4.6mm, 5μm)
HPLC conditions:

Wavelength : 275nm
Injection volume: 20 μL

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Flow rate : 1.0 mL/min
Temperature : 30°C
Run time : 30min
Mobile phase : Buffer and Acetonitrile 67:33 v/v

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about
Preparation of standard solution: For 100/ 500 / 500 mg Tablets:

30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration of
Aceclofenac is about 2000µg/mL)
Accurately weigh and transfer 100 mg of Paracetamol working standard, and 100 mg of Chlorzoxazone working standard
in to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up
to the mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration of
Paracetamol is about 500µg/mL and concentration of Chlorzoxazone is about 500µg/mL ).

Preparation of standard solution: For 100/ 325/ 250 mg Tablets:

Accurately weigh and transfer 65 mg of Paracetamol working standard, and 50 mg of Chlorzoxazone working standard in
to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to
the mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration of
Paracetamol is about 325µg/mL and concentration of Chlorzoxazone is about 250µg/mL).

Preparation of standard solution: For 100/ 500 mg Tablets:

Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10
mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.
(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).

Preparation of standard solution: For 100/ 500/15 mg Tablets:

Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10
mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.
(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50.0 mL volumetric flask, add to it
Preparation of standard solution: For 100mg Tablets:

about 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration
of Aceclofenac is about 2000µg/mL)

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Dilute the 10 mL of above Aceclofenac stock solution to 200 mL with mobile phase and mix well. (Concentration of
Aceclofenac is about 100 µg/mL).

Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weigh
Preparation of test solution: For 100/ 500 / 500 mg Tablets:

accurately the powder equivalent to 500 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml of
acetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minutes
with intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.
Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL with
diluent.


diluent.


diluent.
Prepare the test solutions in duplicate.

Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Inject blank, standard and
Procedure:

sample preparation filter through 0.45µ nylon filter. Inject blank (diluent) in single, standard preparation in five
replicates, and each test preparation into the chromatographic system and record the chromatograms. Inject standard
preparation as a bracketing after every six injections of test preparations. Evaluate the system suitability parameters from
the standard chromatograms. The order of elution is Paracetamol, Chlorzoxazone and Aceclofenac.
% content= Test solution area x Std. Dilution factor x Std. Potency
Standard solution area x Test dilution factor
Calculation:

RESULTS AND DISCUSSION

Standard solution was prepared as per the proposed test method and injected into the HPLC system. The results of the
System suitability:

system suitability assessment for initial validation study parameters were tabulated in Table 1.

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Table 1: System suitability
Sr. Observations
System Suitability parameter Limits

The % RSD of peak area response for
No. Aceclofenac Paracetamol Chlorzoxazone

0.3 0.1 0.2 NMT 2.0
five replicate injections of standard
1

Theoretical plates 2963 1506 3986 NLT 1000
Tailing factor 2.0 1.8 1.9 NMT 2.0
2
3
Precision Studies:

Standard solution of working standard was prepared as per the proposed test procedure for repeatability studies. Five
System Precision:

replicate injections were injected into the HPLC system. % RSD for the peak responses as the peak area was calculated,
Results are tabulated in Table 2.
Table 2: System Precision
Area Response

3481243 9580728 14147918
Injection No.
Aceclofenac Paracetamol Chlorzoxazone

3480070 9596079 14192275
1

3474685 9605600 14200613
2

3456683 9587599 14148073
3

3462315 9597724 14154756
4

3470999 9593546 14168727
5

10967.78 9602.586 25622.336
Average

0.3 0.1 0.2
SD
% RSD

Six test preparations were prepared as per the proposed test method for individual test preparation. All individual test
Method Precision:

preparations were injected into the HPLC system as per the test method. The %assay results were calculated for each
individual test sample, along with average assay and % RSD for the six preparations. The results are tabulated in Table 3.

Table 3: Method Precision Studies
% Assay

98.4 95.8 103.4
Sr. No

98.1 95.9 103.4
1

98.7 95.8 103.5
2

98.6 96.1 103.9
3

98.2 95.9 103.5
4

98.8 95.6 103.2
5

98.5 95.9 103.5
6

0.2805 0.1643 0.2317
Average

0.3 0.2 0.2
SD
% RSD

Six test preparations were prepared as per the proposed test method for individual test preparation and injected into the
Ruggedness (Intermediate precision):

different HPLC system by the different analyst using different make of HPLC Column at different day.

The %assay results were calculated for each of the sample for each of the variability. Average assay values and % RSD for
the six preparations were calculated. The results are tabulated in Table 4.

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Table 4: Ruggedness
% Assay

98.8 95.8 103.8
Sr. No

98.4 95.7 103.7
1

98.6 95.8 103.8
2

98.8 96.1 104.0
3

98.9 96.0 103.9
4

98.9 96.0 103.9
5

98.7 95.9 103.9
6

0.1966 0.1549 0.1049
Average

0.2 0.2 0.1
SD
% RSD
Table 5: Ruggedness data evaluation
% Assay

98.4 98.8 95.8 95.8 103.4 103.8
Sr. No

98.1 98.4 95.9 95.7 103.4 103.7
1

98.7 98.6 95.8 95.8 103.5 103.8
2

98.6 98.8 96.1 96.1 103.9 104.0
3

98.2 98.9 95.9 96.0 103.5 103.9
4

98.8 98.9 95.6 96.0 103.2 103.9
5
6
Overall
98.6 95.9 103.7
Average
Overall SD 0.2697 0.1545 0.2570
Overall
0.3 0.2 0.2
% RSD
Linearity and Range:

The linearity studies of detector response for analytes were evaluated in the concentration range from about 50% of lower
Linearity of Detector Response:

strength to 150% of the higher strength of the targeted concentration. The diluted standard solutions were prepared from
stock solution in the above range and analysed using proposed analytical method by injecting each level in to the system.
The Linearity graph of average area response verses concentration was plotted and the correlation coefficient was
calculated. The results are tabulated in Table 6.
Table6: Linearity of Detector Response:
Sr. No Concentration Area Concentration Area Concentration Area

29.9976 1051778 150.4635 2644943 120.2281 3603632
(g/mL) response (g/mL) response (g/mL) response

59.9951 2105682 200.6180 3519271 200.3802 6047772
1

75.9938 2451169 401.2361 7010633 320.6083 9611741
2

99.9919 3503313 501.5451 8745948 500.9505 14946798
3

109.9911 3849570 551.6996 9595498 601.1406 17884943
4

119.9903 4187013 626.9314 10901381 721.3687 21323730
5

159.9870 5600698 752.3177 13044334 761.4448 22494050
6

0.999 1.000 1.000
7
Correlation

35288.4907 17291.5590 29427.4152
Coefficient

56878.5049 55950.5535 139465.4305
Slope
Intercept

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The results obtained in accuracy study were further interpreted for the evaluation of linearity of the test method. The
Linearity of Test Method (Inferred from Accuracy):

results of average of mg added at each spiked level was plotted against average mg recovered at each accuracy level and the
correlation coefficient for set of data was evaluated. The results are tabulated in Table 7.
Table 7: Linearity of Test method (Inferred from accuracy)

For 100/500/500 mg Tablets:

Aceclofenac Paracetomol Chlorzoxazone
Recovery level Amount Amount Amount Amount Amount
Amount
Recovered Added Recovered Added Recovered

79.4 81.0 397.2 401.0 397.1 403.0
Added (mg)
(mg) (mg) (mg) (mg) (mg)

99.6 101.0 495.8 498.0 495.6 502.0
80%

119.6 121.0 594.8 590.0 595.0 594.0
100%

1.000 0.9999 0.9997
120%
Correlation

0.995 0.9565 0.9651
Coefficient

1.9627 21.9933 21.0867
Slope
Intercept

Precision at lower and higher extreme range concentration for Aceclofenac (29.9976 µg/ml and 159.9870 µg/ml),
Precision at Lower and Higher extreme concentrations:

Paracetamol (150.4635 µg/ml and 752.3177 µg/ml), and Chlorzoxazone (120.2281 µg/ml and 761.4448 µg/ml) of
Linearity levels were determined as per proposed method by injecting six replicate injections of standards for both the
levels. % RSD for peak responses for both the level was evaluated. The results are tabulated in Table 8.
Table 8: Precision at Lower and Higher extreme Levels of linearity
Area Responses
Area Responses Aceclofenac Area Responses Paracetamol
Chlorzoxazone
Injection Number
Lower level Higher level Lower level Higher level Lower level Higher level

1051198 5593151 2654481 13053921 3607531 22487458
(25%) (150%) (25%) (150%) (25%) (150%)

1053062 5599304 2655088 13029744 3605991 22475748
1

1052477 5593984 2646499 13073632 3602871 22554993
2

1051003 5598529 2637079 13028568 3600717 22472092
3

1052376 5602959 2635339 13056944 3598395 22506195
4

1050552 5616261 2641174 13023193 3606288 22467814
5

1051778 5600698 2644943 13044334 3603632 22494050
6

993.4749 8438.0317 8539.324 20088.1667 3586.7515 32895.1346
Average

0.1 0.2 0.3 0.2 0.1 0.1
SD
% RSD

An accuracy study was conducted by spiking the known amount of analytes in the equivalent weight of placebo. Accuracy
Accuracy:

study was conducted in triplicate at three different levels, (80%, 100% and 120% of target concentration). The samples
were analyzed as per the proposed test procedure and the % recovery for each spike level was calculated. The precision at
each spike level was also established.
The results are tabulated in Table 9, 10 and 11.

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Table 9: Accuracy of Aceclofenac

Aceclofenac
Recovery
Level Amount Amount Average

79.4 80.5 101.4
% Recovery % RSD
Added (mg) Recovered (mg) Recovery (%)

79.4 80.9 101.9 101.5 0.4
79.5 80.4 101.1
80%

99.6 101.7 102.1
99.6 101.1 101.5 101.7 0.3
99.6 101.2 101.6
100%

119.6 119.8 100.2
119.6 121.3 101.4 101.3 1.0
119.6 122.4 102.3
120%

Table 10: Accuracy Paracetamol
Overall 101.5 0.6

Paracetamol
Recovery
Amount Amount Average

397.1 399.9 100.7
Level % Recovery % RSD

397.1 402.6 101.4 101.0 0.4
397.3 400.3 100.8
80%

495.8 500.1 100.9
495.8 495.9 100.0 100.3 0.5
495.9 496.5 100.1
100%

594.8 582.6 97.9
594.8 590.9 99.3 99.2 1.3
594.8 597.3 100.4
120%

Table 11: Accuracy of Chlorzoxazone
Overall 100.2 1.0

Chlorzoxazone
Recovery
Amount Amount Average

397.1 403.6 101.6
Level % Recovery % RSD

397.1 405.6 102.1 101.6 0.5
397.2 401.1 101.0
80%

495.6 502.6 101.4
495.7 502.0 101.3 101.4 0.1
495.6 502.4 101.4
100%

595.0 588.2 98.9
595.0 591.4 99.4 99.8 1.2
595.0 601.3 101.1
120%

Overall 100.9 1.0
Robustness:

Mobile phase Flow rate as per proposed analytical method is 1.0 ml/min. Change in flow rate by –10% = 0.9 ml/min. Change in
Effect of variation in Flow rate of mobile phase (±10%):

flow rate by +10% = 1.1 ml/min. The effect due to change in flow rate on the system suitability parameters are compared.
Results are tabulated in Table 12.

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Table 12: System suitability of change in Flow Rate
Sr. Observations

The % RSD of Aceclofenac 0.1 0.1 0.2
No. As Such - 10% + 10%

peak area Paracetamol 0.1 0.1 0.2
response for five NMT 2.0
replicate Chlorzoxazone 0.1 0.2 0.3
1

injections
Aceclofenac 6209 6301 6158
Theoretical plates Paracetamol 2559 2697 2413 NLT 1000
Chlorzoxazone 10476 10660 9988
2

Aceclofenac 2.0 2.0 2.0
Tailing factor Paracetamol 1.4 1.4 1.3 NMT 2.0
Chlorzoxazone 1.1 1.1 1.1
3

The Column Oven temperature as per proposed analytical method is 30°C. Change in Column oven Temperature by –5°C =
Effect of variation in Column oven temperature (±5°C):

25°C. Change in Column oven Temperature by +5°C = 35°C. The effect due to change in Column oven temperature on the
system suitability parameters are compared. Results are tabulated in Table 13.
Table 13: System suitability of change in Column Oven temperature

Sr. Observations

No.

The % RSD of peak
As Such -5°C + 5°C

area response for five Paracetamol 0.1 0.0 0.1 NMT 2.0
replicate injections Chlorzoxazone 0.1 0.0 0.1
1

2

3

The Organic phase ratio of mobile phase as per proposed analytical method is 0.2% v/v Orthophosphoric acid: Acetonitrile is
Effect of variation in organic phase composition (± 10%):

67:33 v/v. Change in Organic phase ratio of mobile phase by +10% and -10% of 0.2% v/v Orthophosphoric acid:
Acetonitrile was performed. The effect due to change in organic phase composition on the system suitability parameters are
compared. Results are tabulated in Table 14.
Table 14: System suitability of change in organic phase composition

Sr. Observations

The % RSD of peak Aceclofenac 0.1 0.2 0.1
No. As Such -5% + 5%

area response for five Paracetamol 0.2 0.1 0.2 NMT 2.0
replicate injections Chlorzoxazone 0.4 0.1 0.1
1

2

3

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The pH of mobile phase A as per proposed analytical method is 6.0. Change in pH of mobile phase A by – 0.2 units is 5.8
Effect of variation in Mobile phase pH (± 0.2 Units):

Change in pH of mobile phase A by + 0.2 units is 6.2 The effect due to change in pH of mobile phase A on the system
suitability parameters are compared. Results are tabulated in Table 15.
Table 15: System suitability of change in mobile phase pH

Sr. Observations

The % RSD of Aceclofenac 0.2 0.1 0.0
No. As Such - 0.2 units + 0.2units

peak area Paracetamol 0.1 0.1 0.0
response for five NMT 2.0
replicate Chlorzoxazone 0.2 0.1 0.1
1

injections
2

3

Standard solutions and sample solution were prepared. Some portion of above solutions was filtered through 0.45
Filter Interference studies:

membrane filter. Both the samples further diluted as per proposed method and injected in to the HPLC system. For
comparison the % area difference was calculated between unfiltered and filtered solutions. The data tabulated in Table 16,

Table 16: Filter paper details
17, 18 and 19.

Nylon MDI Syringe 0.45µ 25 mm SN0850
Type Make Micron size Diameter Lot No

Table 17: Filter paper interference study Aceclofenac
Aceclofenac
Average Standard Area Average Test Area
Prep. No. % %
STD-1 % Diff STD-2 Test-1 % Diff Test-2

3551905 NA 3524335 NA 3595532 NA 3591554 NA
Diff Diff

3548229 0.1 3563626 1.1 3588334 0.2 3581025 0.3
Unfiltered
0.45µFilter

Table 18: Filter paper interference study Paracetamol
Paracetamol
Prep. No. % %

9556575 NA 9525387 NA 9730520 NA 9753048 NA
STD-1 STD-2 % Diff Test-1 % Diff Test-2
Diff Diff

9512533 0.5 9564122 0.4 9720541 0.1 9713146 0.4
Unfiltered
0.45µFilter

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Table 19: Filter paper interference study Chlorzoxazone
Chlorzoxazone
Prep. No. % %
STD-1 % Diff STD-2 Test-1 % Diff Test-2

14171478 NA 14113560 NA 15017843 NA 15040877 NA
Diff Diff

14143527 0.2 14114024 0.0 14979503 0.3 14994880 0.3
Unfiltered
0.45µFilter

Test preparation was prepared as per the test method and injected into HPLC system. The peak purity result was
Peak Purity results of Sample As such:

evaluated by Photo Diode Array Detector.
The results are tabulated in Table 20 and 21.
Table 20: Sample As such Higher Strength 100/500/500 mg tablets
Peak purity results
Parameter Aceclofenac Chlorzoxazone Paracetamol
Purity-1 angle 0.071 0.271 2.092
Purity-1 threshold 0.251 0.282 6.619
Purity Flag No No No

Table 21: Sample As such Lower Strength 100/500/15 mg tablets
Peak purity results
Parameter Aceclofenac Paracetamol
Purity-1 angle 0.057 2.171
Purity-1 threshold 0.232 6.775
Purity Flag No No

The stress degradation study was carried out on the sample preparations (100/500/500 mg and 100/500/15 mg strength)
Forced Degradation studies:

of Tablet, and the degradation was evaluated by calculating the % degradation of in comparison with unstressed sample
preparation. The degradation of 10-30% was tried by following stress conditions to prove the stability indicating
characteristics of the method. The stress conditions and results were compiled in Table 22 and 23.

Table 22: % degradation data for 100/500/500 mg tablets
Stress Condition % % %

Acid degradation
degradation degradation degradation

16.2 0.1 3.2
0.1N HCl, 1hr at 60°C
Alkali degradation
95.1 9.9 7.0
1N NaOH, 1hr at 80°C
Peroxide degradation
17.5 0.5 1.9
6% H2O2, 2hrs at 80°C
Thermal degradation
11.3 1.4 1.4
2 hrs at 80°C
Photolytic degradation
12.0 1.4 1.6
1.2 m.lux hrs

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Table 23: % degradation data for 100/500/15 mg tablets
Aceclofenac Paracetamol
Stress Condition % %

Acid degradation 0.1N HCl, 1hr at 60°C 10.7 3.6
degradation degradation

Alkali degradation 1N NaOH, 1hr at 80°C 98.6 19.2
Peroxide degradation 6% H2O2, 2hrs at 80°C 17.4 2.5
Thermal degradation 2 hrs at 80°C 8.2 3.0
Photolytic degradation 1.2 m.lux hrs 8.2 3.0
Solution Stability Studies:

The mobile phase was prepared as per the proposed test method and kept at room temperature for a period of two days in
Mobile phase stability at room temperature:

well closed condition. The system suitability solutions were prepared and injected into the HPLC system at initially and
periodically after 1day. The system suitability parameters were evaluated. The mobile phase was also observed for Physical
changes like haziness or precipitation. System suitability results are tabulated in Table 24.
Table 24: Mobile phase stability at room temperature
Observations

The % RSD of peak area Aceclofenac 0.2 0.1
Sr. No. System Suitability parameter Limits
Initial Day-1

response for five replicate Paracetamol 0.2 0.2 NMT 2.0
injections Chlorzoxazone 0.1 0.1
1

Aceclofenac 6737 6704
Theoretical plates Paracetamol 3013 3227 NLT 1000
Chlorzoxazone 10870 10725
2

Aceclofenac 1.9 1.8
Tailing factor Paracetamol 1.3 1.2 NMT 2.0
Chlorzoxazone 1.0 1.0
3

Standard and test preparations were prepared as per the proposed test method and the stock solutions were kept at room
Analytical Solution Stability (Standard and Test preparation) at room temperature:

temperature. The solutions were diluted freshly at each time and injected into the HPLC system at initially and at different
time intervals. The % difference in area was calculated against the fresh standard solution injected at each time interval.
The % difference in area response was evaluated against the initial assay value. The results are tabulated in Table 25, 26

Table 25: Stability of Aceclofenac Standard and test solution at room temperature
and 27.

Aceclofenac Standard Aceclofenac Test

3549534 NA 3598615 NA
Time (Hours) Area Response % Difference Area Response % Difference

3539237 0.3 3578216 0.6
Initial

3540881 0.2 3609559 0.3
4 Hrs 35 min

3559542 0.3 3597509 0.0
8 Hrs

3546836 0.1 3575222 0.7
12 Hrs 15 min

3540089 0.3 3575272 0.6
16 Hrs 20 min

3557104 0.2 3571046 0.8
20 Hrs 25 min

3528644 0.6 3562606 1.0
23 Hrs

3532159 0.5 3581784 0.5
24 Hrs 30 min

3581358 0.9 3581157 0.5
29 Hrs

3630421 2.3 3581119 0.5
33 Hrs
40 Hrs 50 min

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Table 26: Stability of Paracetamol Standard and test solution at room temperature
Paracetamol Standard Paracetamol Test

9533772 NA 9674499 NA

9485553 0.5 9681641 0.1
Initial

9529119 0.0 9720005 0.5
4 Hrs 35 min

9562369 0.3 9713140 0.4
8 Hrs

9558274 0.3 9703185 0.3
12 Hrs 15 min

9551274 0.2 9705705 0.3
16 Hrs 20 min

9554327 0.2 9673281 0.0
20 Hrs 25 min

9551111 0.2 9659821 0.2
23 Hrs

9541020 0.1 9661378 0.1
24 Hrs 30 min

9619965 0.9 9674547 0.0
29 Hrs

9760962 2.4 9661793 0.1
33 Hrs

Table 27: Stability of Chlorzoxazone Standard and test solution at room temperature
40 Hrs 50 min

Chlorzoxazone Standard Chlorzoxazone Test

14101688 NA 14905697 NA

14074515 0.2 14896823 0.1
Initial

14105493 0.0 15018775 0.8
4 Hrs 35 min

14182706 0.6 14980781 0.5
8 Hrs

14175597 0.5 14922091 0.1
12 Hrs 15 min

14185354 0.6 14925356 0.1
16 Hrs 20 min

14165758 0.5 14885819 0.1
20 Hrs 25 min

14150058 0.3 14871542 0.2
23 Hrs

14064098 0.3 14908388 0.0
24 Hrs 30 min

14290784 1.3 14911371 0.0
29 Hrs

14499323 2.8 14930540 0.2
33 Hrs
40 Hrs 50 min
BLANK CHROMATOGRAM

483


STANDARD CHROMATOGRAM

TEST CHROMATOGRAM

LINEARITY OF DETECTOR RESPONSE OF ACECLOFENAC

484


LINEARITY OF DETECTOR RESPONSE OF PARACETAMOL

LINEARITY OF DETECTOR RESPONSE OF CHLORZOXAZONE

LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF ACECLOFENAC

485


LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF PARACETAMOL

LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF CHLORZOXAZONE

AS SUCH SAMPLE CHROMATOGRAM HIGHER STRENGTH

486


AS SUCH SAMPLE PURITY PLOT HIGHER STRENGTH

AS SUCH SAMPLE CHROMATOGRAM LOWER STRENGTH

487


AS SUCH SAMPLE PURITY PLOT LOWER STRENGTH

ACID DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

488


ALKALI DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

PEROXIDE DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

THERMAL DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

489


PHOTOLYTIC DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

The complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust and
CONCLUSION

stability indicating. This method has wide applicability and useful for regular quality control analysis of formulation samples.

1. Granberg RA, Rasmuson AC, Solubility of paracetamol in pure solvents, Journal of Chemical & Engineering Data, 1999, 44
REFERENCES

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490

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