Article Wjpps 1425131684
Article Wjpps 1425131684
Article Wjpps 1425131684
1*
Sri Shivani College of Pharmacy, Warangal, Telangana.
2
Synapse Life Sciences, Hanamkonda, Telangana.
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.
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.
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).
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.
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
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.
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.
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.
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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