WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Adidala et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 4, Issue 03, 1008-1023. Research Article ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

SIMULTANEOUS ESTIMATION OF PARACETAMOL,
ACECLOFENAC AND SERRATIOPEPTIDASE IN COMBINED
TABLET DOSAGE FORM

Adidala Raghuram Reddy1*, K. Rajamani1, Arrabelli Mounika2,

Gouru Santhosh Reddy2

1*
Sri Shivani College of Pharmacy, Warangal, Telangana.
2
Synapse Life Sciences, Hanamkonda, Telangana.

Article Received on ABSTRACT

22 Dec 2014,
A Simultaneous estimation of RP-HPLC method was developed and
Revised on 17 Jan 2015, validated for the estimation of paracetamol, aceclofenac and
Accepted on 10 Feb 2015
serratiopeptidase in tablet dosage form using C18 column (250mm
4.6mm, 5µ) with mobile phase consisting of Acetonitrile: Water
*Correspondence for
(70:30v/v) and 0.1% Glacial acetic acid with a flowrate of 1.0ml/min
Author
Adidala Raghuram and UV detection at 227nm. Recovery was observed 99.1% to 99.4%
Reddy for Mankind, 98.64% to 99.1% for Nelnac, 98.9% to 99.5% Lupin
Sri Shivani College of Tablets. Accuracy of drugs was observed to be within the limits of
Pharmacy, Warangal,
98% to 102% by mean of 3 determinations. Precision of drugs was
Telangana.
observed to be less than 2.0 of %RSD by mean of 6 determinations.
Linearity was observed over the concentration range 1–50 µg/ml (r2=0.998) with
regression equation y = 36941x – 61362 for Paracetamol, Aceclofenac 1–50 µg/mL
(r2=0.997) with regression equation y = 42784x + 23799 and Serratiopeptidase 1-50 µg/mL
(r2= 0.998) with regression equation y = 1904x + 22854. LOD and LOQ of Paracetamol,
aceclofenac and serratiopeptidase were found to be 2.27µg/ml, 1.1µg/ml, 3.62µ/ml and
6.88µg/ml, 3.33µ/ml, 10.9µg/ml respectively. The method was validated as per ICH
guidelines.

KEYWORDS: Aceclofenac, Paracetamol, Serratiopeptidase, HPLC, Method development,

Method validation.

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INTRODUCTION
Paracetamol
Paracetamol is chemically N-(4-hydroxyphenyl) acetamide. It is a centrally and peripherally
acting non-opioid analgesic and antipyretic. Paracetamol (acetaminophen) is one of the most
popular over-the-counter analgesic and antipyretic drugs. Paracetamol is available in different
dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories. Dosage forms of
paracetamol and its combinations with other drugs have been listed in various
pharmacopoeias.[1,2]

The combination of paracetamol with dipyrone is used as an antipyretic, analgesic and anti-
inflammatory drug.

Numerous methods have been reported for the analysis of paracetamol and its combinations
in pharmaceuticals or in biological fluids. Paracetamol has been determined in combination
with other drugs using titrimetry,[3,4] voltammetry,[5] fluorimetry,[6] colorimetry,[6] UV-
spectrophotometry,[7−9] quantitative thin-layer chromatography (TLC),[10] high-performance
liquid chromatography (HPLC)[11−16] and gas chromatography (GC)[17] in pharmaceutical
preparations.

Aceclofenac
Aceclofenac (ACF), 2-[(2, 6-dichlorophenyl)amino]phenyl]acetyl] oxyacetic acid is used as
anti-inflammatory drug. It is official in B.P.[18] and I.P.[19] Aceclofenac is a non-steroidal anti-
inflammatory drug (NSAID). Aceclofenac has higher anti-inflammatory action than
conventional NSAIDs. It is a cytokine inhibitor. Aceclofenac works by blocking the action of
a substance in the body called cyclooxygenase. Cyclooxygenase is involved in the production
of prostaglandin (chemical in the body) which causes pain, swelling and inflammation.
Aceclofenac is the glycolic acid ester of Diclofenac.[20]

Serratiopeptidase
Serratiopeptidase (Serratia E-15 protease, also known as serralysin, Serratiopeptidase,
Serratia peptidase, serration peptidase or serrapeptidase) is a proteolytic enzyme (protease)
produced by enterobacterium Serretia sp. E-15. Serratiopeptidase is present in the silkworm
intestine and allow the emerging moth to dissolve its cocoon. Serratiopeptidase is produced
by purification from culture of Serratia E – 15 bacteria.[21-23] It can reduce the levels of dead
tissue in the circulatory system, promoting smoother healthier flowing blood.

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Serratiopeptidase fights fibrin build up in the cardiovascular system, organs and muscle
tissue. In addition, histological studies also show it to be a powerful anti-inflammatory. It
also is an anti-edemic, preventing swelling and fluid retention.[24]

Many UV[28, 29] and HPLC[25-27,31-36,39] based methods have been reported for estimation of
these drugs alone as well as in combination with other drugs in pharmaceutical dosage form.
But no method had yet been reported for simultaneous estimation of these two drugs using
HPLC in bulk drug and pharmaceutical dosage forms. Therefore, the present work was aimed
to develop and validate a new RP- HPLC method for simultaneous estimation of paracetamol,
aceclofenac and serratiopeptidase in pharmaceutical dosage forms.

EXPERIMENTAL
Materials and reagents
Paracetamol, Aceclofenac and Serratiopeptidase were procured from Green waves chemicals
Pvt. Ltd., Bubeneshwar, India. Commercial tablets of above combination (Nelnac, Mankind,
Lupin) used for analysis was procured from local pharmacy. HPLC grade acetonitrile and
water were procured from Finar chemicals limited, Ahmedabad.

Instrumentation
RP-HPLC was performed using Shimadzu HPLC system consisting of a pump LC-10AD,
rheodyne sample injection port with 20 microlitre loop, SPD-10A UV-Visible detector,
N2000 software, column used was Welchrome C18 (250 x 4.6mm, 5μ).

Chromatographic conditions
A reverse phase column [Welchrome C18 (250 x 4.6mm, 5μ particle size)], equilibrated with
mobile phase [Acetonitrile: Water (70:30 v/v) and 0.1% Glacial acetic acid was used. Mobile
phase flow rate was maintained at 1ml/min and effluents were monitored at 227nm. The
sample was injected using 20 microlitre fixed loop rheodyne injector and run time was 10
mins.

Standard Solution preparation

About 100 mg of pure samples of paracetamol, aceclofenac and serratiopeptidase were
accurately weighed and transferred to a 100 ml volumetric flask. Then they were dissolved in
mobile phase and the solution was made up to the required volume. Each ml of stock solution

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contained 1000 g/ml. 10 ml of this stock solution was diluted to 100 ml with mobile phase
to give 100 g/ml solution (Working Stock).

Preparation of sample solution from dosage form

Twenty tablets were weighed and pulverized. The tablet powder equivalent to 100 mg of
paracetamol, aceclofenac and serratiopeptidase was transferred to a 100 ml volumetric flask
and dissolved in mobile phase and the content was made up to mark with mobile phase. Then
the sample solution kept in sonicater for 15 min and the solution was filtered through 0.45µm
filter paper.

Assay
With the optimized chromatographic conditions mentioned early, a steady base line was
recorded. After the stabilization of baseline, inject the sample solution of a concentration 30
µg/ml of each paracetamol, aceclofenac and serratiopeptidase respectively. Each solution was
run at an interval of 10 minutes and the peak areas were found and amount of the drug and
percentage of assay was calculated by regression equations which were tabulated in Table-
4,5,6 and chromatograms were recorded and presented in Figure-8,9,10.

Validation of HPLC method

The proposed RP-HPLC method was validated as per ICH guidelines.

Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, degradants,
matrix, etc.

No peaks were obtained while running the solution containing placebo ingredients which
proves that the method is specific. Chromatogram is shown in Figure-4.

Linearity
Linearity was determined for paracetamol, aceclofenac and serratiopeptidase separately by
plotting a Calibration curve of peak area against their respective concentration. From the
calibration curve it was found that the curve was linear observed over the concentration range
1–50 µg/ml (r2=0.998) with regression equation y = 36941x - 61362 for Paracetamol,
Aceclofenac 1–50 µg/mL (r2=0.997) with regression equation y = 42784x + 23799 and

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Serratiopeptidase 1-50 µg/mL (r2= 0.998) with regression equation y = 1904.x + 22854.
Results were shown in the Table-7,8,9 and Figure-5,6,7.

Precision
Precision study was performed to find out intraday and interday variations. The intraday and
interday precision study of paracetamol, aceclofenac and serratiopeptidase was carried out by
estimating the correspondence response 3 times on the same day and on 2 different days for 3
different concentrations of paracetamol, aceclofenac and Serratiopeptidase and the results
were reported in terms of % relative standard deviation (%RSD). However, all results fall
within acceptance limits (RSD < 2), as shown in Table-11.

Accuracy
The accuracy of the method was determined by calculating the recovery studies at three
levels by taking mean of three determinations of three standard concentrations(low, medium
and high). Mean value of each concentration was determined from regression equation of
calibration curve. The recoveries results of paracetamol, aceclofenac and serratiopeptidase in
pharmaceutical preparation are shown in the Table-10.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ for paracetamol, aceclofenac and serratiopeptidase were separately
determined by based on calculating the signal-to-noise ratio (S/N is 3.3 for LOD and 10 for
LOQ) and from the calibration curves the standard deviation of the y-intercepts and slope of
the regression lines were used. σ is standard deviation of response (y – intercept) and S is
the slope of calibration plot. Results are shown in the Table-12.

Robustness
The robustness study was done by making small changes in the optimized method parameters
like ±1nm change in wavelength and ±1ml/min change in flow rate in flow rate. There was no
significant impact on the retention time and tailing factor. Results were tabulated in Table-
13,14.

RESULTS AND DISCUSSION

To develop a precise, accurate and suitable HPLC method for simultaneous estimation of
paracetamol, aceclofenac and serratiopeptidase in tablet dosage form different mobile phases
such as acetonitrile and water in different proportions were used and finally acetonitrile:

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water (70:30v/v) and 0.1% Glacial acetic acid was selected as an appropriate mobile phase,
which give good retention time and acceptable peak parameters for paracetamol, aceclofenac
and serratiopeptidase. The linear relationship was carried out between the peak area and
concentration from a range of 1-50µg/ml for paracetamol, aceclofenac and serratiopeptidase.
The linearity can be expressed as correlation coefficient 0.998, 0.997 and 0.999 for
paracetamol, aceclofenac and serratiopeptidase respectively. Chromatogram of standard
drugs was shown in Figure-11. Validation is performed as per ICH guidelines. It was
assessed at 3 concentration levels %RSD obtained was less than 2% for both drugs. The
results of precision are shown in Table-11. System suitability parameters for proposed
method are shown in Table-15. Assay of tablets paracetamol, aceclofenac and
serratiopeptidase was evaluated. Three replicate determinations were carried out on tablets.
Percentage purity was found to be 98.64%, 98.9% and 99.1% for Nelnac tablets and 99.32%,
99.1% and 99.4% for Mankind tablets and 99.5%, 98.9% and 99.2% for lupin tablets. Data of
standard graph were tabulated in Table No.1 and 2. Results of tablet analysis were shown in
Table-4,5,6. Robustness studies were carried out after deliberate alterations of flow rate and
wavelength. It was observed that there are no varied changes changes of retention times of
peak of interest.

Table No.1 Paracetamol Standard Graph data

Concentration (µg/ml) Peak Area

10 297790
20 665610
30 1061575
40 1414786
50 1864463

Figure No.1 Standard Graph of Paracetamol

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Table No.2 Aceclofenac Standard Graph data

Concentration (µg/ml) Peak Area
10 410951
20 827011
30 1297163
40 1756228
50 2196919

Figure No.2 Standard Graph of Aceclofenac

Table No.3 Serratiopeptidase Standard Graph data

Concentration(µg/ml) Peak Area
10 38125
20 61331
30 76221
40 99581
50 119916

Figure No.3 Standard Graph of Serratiopeptidase

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Table No.4 Data of Assay (Nelnac)

Amount % Amount Acceptance
Drug Label Claim
estimated estimated Criteria
Paracetamol 325mg 320.6 98.64
Aceclofenac 100mg 98.9 98.9 98 to 102
Serratiopeptidase 10mg 9.91 99.1

Table No.5 Data of Assay (Mankind)

Amount % Amount Acceptance
Drug Label Claim
estimated estimated criteria
Paracetamol 325mg 322.8 99.32
Aceclofenac 100mg 99.1 99.1 98 to 102
Serratiopeptidase 15mg 14.91 99.4

Table No.6 Data of Assay (Lupin)

Amount % Amount Acceptance
Drug Label Claim
estimated estimated criteria
Paracetamol 325mg 323.4 99.50
Aceclofenac 100mg 98.9 98.9 98 to 102
Serratiopeptidase 15mg 14.88 99.2

Figure No.4 Specificity Chromatogram of the Developed Method

Table No.7 Linearity Graph data of Paracetamol

Concentration (µg/ml) Peak Area
1 11632
2.5 25753
5 88990
10 290816
20 669234
25 883965
30 1061576
40 1428758
50 1765685

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Figure No.5 Linearity Graph of Paracetamol

Table No.8 Linearity Graph data of Aceclofenac

Concentration (µg/ml) Peak Area
1 115828
2.5 107715
5 287315
10 432971
20 827011
25 1043942
30 1297163
40 1756228
50 2196919

Figure No.6 Linearity Graph of Aceclofenac

Table No.9 Linearity Graph data of Serratiopeptidase

Concentration Peak Area
1 25066
2.5 28698
5 33673
10 39185
20 61658
25 69867
30 78541
40 99618
50 118833

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Figure No.7 Linearity Graph of Serratiopeptidase

Table No.10 Data of Accuracy

Theoretical Obtained
%
Drug Concentration concentration
Recovery
(µg/ml) (µg/ml)
10 9.824 98.24
Paracetamol 30 29.793 99.31
50 49.31 98.62
10 9.916 99.16
Aceclofenac 30 29.862 99.54
50 49.56 99.12
10 9.836 98.36
Serratiopeptidase 30 29.523 98.41
50 49.285 98.57
*Mean of 3 Determinations

Table No.11 Data of Precision

Theoretical Intra day Inter day
Drug
Concentration Mean % RSD Mean % RSD
Paracetamol 10 µg/ml 296025 1.42 295574 1.17
Aceclofenac 10 µg/ml 419358 1.12 418782 1.41
Serratiopeptidase 10 µg/ml 38721 0.97 38677 0.64
*Mean of Six determinations

Table No.12 Data of Limit of Detection and Quantification

Parameter Paracetamol Aceclofenac Serratiopeptidase
Limit of
2.27 1.10 3.62
Detection(µg/ml)
Limit of
6.88 3.33 10.99
Quantification(µg/ml)

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Table No.13 Data of Robustness (Flow rate)

Theoretical
Mean Rt
Flow rate Drug Concentration %RSD
(Peak area) value
µg/ml
10 557628 3.22 0.95
Paracetamol
30 1003243 3.22 1.02
10 120115 7.89 1.16
0.9ml/min Aceclofenac
30 426195 7.89 1.09
10 47919 4.37 1.3
Serratiopeptidase
30 36029 3.96 1.24
10 256287 2.62 1.04
Paracetamol
30 751605 2.63 1.5
10 112176 6.44 1.38
Aceclofenac
1.1ml/min 30 383992 6.43 1.18
10 42296 3.58 1.29
Serratiopeptidase
30 56221 3.57 1.32

Table No.14 Data of Robustness (Wavelength)

Theoretical Mean
Rt
Wavelength Drug Concentration (Peak %RSD
value
µg/ml area)
10 150894 2.87 1.25
Paracetamol
30 455989 2.89 0.97
10 143083 5.58 1.29
222 nm Aceclofenac
30 621568 7.09 1.41
10 115031 4.27 1.06
Serratiopeptidase
30 231146 4.28 0.52
10 477001 2.90 1.32
Paracetamol
30 1646236 2.90 1.26
10 105643 5.59 1.09
Aceclofenac
232 nm 30 449806 7.10 0.73
10 45492 4.33 0.94
Serratiopeptidase
30 240445 4.31 1.29

Table No.15 System suitability parameters for HPLC method

Parameters Paracetamol Aceclofenac Serratiopeptidase Acceptance Limits
Theoretical plates 3535.878 3420.016 3420.016 > 2000
Tailing factor 1.109 1.232 1.232 < 2.0
Assymetry factor 1.281 1.466 1.466 < 2.0

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Figure No.8 Chromatogram of Tablet Dosage form (Nelnac)

Figure No.9 Chromatogram of Tablet Dosage form (Mankind)

Figure No.10 Chromatogram of Tablet Dosage form (Lupin)

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Figure No.11 Chromatogram of Standard drugs

CONCLUSION
The present paper describes proposed RP-HPLC method for the simultaneous estimation of
paracetamol, aceclofenac and serratiopeptidase in tablet dosage form is accurate, precise,
linear, robust, simple and rapid. Acceptable regression values, RSD % and standard
deviations which make it versatile and valuable for simultaneous estimation of three drugs in
tablet formulation. Acceptable values of precision and accuracy have been obtained as per
ICH guidelines of validation. The run time is relatively short i.e. within 10 mins. So, A large
number of samples can be analysed in short period of time. The results of this developed RP-
HPLC method could be conveniently adopted for quality control analysis of paracetamol,
aceclofenac and serratiopeptidase simultaneously from tablet dosage forms.

ACKNOWLEDGEMENT
The authors are thankful to Green waves chemicals Pvt. Ltd., Bubeneshwar, India for
providing sample of paracetamol, aceclofenac and serratiopeptidase for project work. The
authors are grateful to Sri Shivani College of Pharmacy, Affiliated to Kakatiya University,
Warangal, Telangana and Synapse Life Sciences, Warangal, Telangana for providing all the
facilities to carry out the work.

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