alexa Development and Validation of a New Chromatographic Method for the Simultaneous Estimation of Serratiopeptidase, Aceclofenac and Paracetamol by RP-HPLC | Open Access Journals
ISSN: 2471-2698
Pharmaceutical Analytical Chemistry: Open Access
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Development and Validation of a New Chromatographic Method for the Simultaneous Estimation of Serratiopeptidase, Aceclofenac and Paracetamol by RP-HPLC

Navneet Kumar U1, Afroze A1,2*, Pradeepti C1, Shailendra K4, Naik KK3, Sharma M1, Swati P1, Sunil T1 and Sameer S1

1School of Pharmaceutical Sciences, Shoolini University, Bajhol, Solan, Himachal Pradesh, India

2Narayan Institute of Pharmacy, Jamuhar, Sasaram, India

3Nandha College of Pharmacy, Erode, Tamil Nadu, India

4Government Pharmacy College, Agam Kuan, Patna, Bihar, India

*Corresponding Author:
Afroze Alam
School of Pharmaceutical Sciences
Shoolini University, Bajhol, Solan-173 229
Himachal Pradesh, India
Tel: +919805185732/+918091974886
E-mail: [email protected]

Received Date: February 18, 2017 Accepted Date: March 07, 2017 Published Date: March 13, 2017

Citation: Navneet Kumar U, Afroze A, Pradeepti C, Shailendra K, Naik KK, et al. (2017) Development and Validation of a New Chromatographic Method for the Simultaneous Estimation of Serratiopeptidase, Aceclofenac and Paracetamol by RP-HPLC. Pharm Anal Chem 3: 122. doi: 10.4172/2471-2698.1000122

Copyright: © 2017 Navneet Kumar U, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Pharmaceutical Analytical Chemistry: Open Access

Abstract

Background: Method development, validation is an important parameter for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM) by RP-HPLC, is supposed to be a costly and tedious process. The present study revealed using cheap and cost effective solvent system for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM).

Objective: Development and validation of a new chromatographic method for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM) by RP-HPLC of marketed formulations.

Methods: Simultaneous determination of SERA, ACE and PCM were carried out by RP-HPLC at the wavelength 327 nm, flow rate 0.4 mL/min, and the mobile phase used was water: methanol in the ratio (50:50 v/v). Further validation parameters such as system suitability, linearity, accuracy, precision, specificity, LOD, LOQ and robustness were taken into account to carry out the validation of the method.

Results: Absorbance maxima for the simultaneous determination were selected by the UV spectrophotometer and that was found to be 327 nm in methanol and water. During the process of RP-HPLC, the linearity was obtained in the concentration range of 2-10 μg/mL for SERA, 100-500 μg/mL for ACE and 20-100 μg/mL for PCM. Correlation coefficient (r) for SERA, ACE and PCM in methanol and water was found to be 0.9817, 0.991 and 0.9949 respectively.

Conclusion: The RP-HPLC method was simple, accurate, precise, and rapid and can be used for the simultaneous determination of SERA, ACE and PCM in bulk and pharmaceutical dosage form. The method is also economical as RP-HPLC grade water methanol in a ratio of (50:50) was used to achieve all the validation parameters.

Keywords

Aceclofenac; Correlation coefficient; Paracetamol; Pharmaceutical dosage; RP-HPLC; Simultaneous estimation; Serratiopeptidase; Validation

Introduction

Paracetamol

Paracetamol (acetaminophen) is one of the most popular overthe- counter analgesic and antipyretic drugs. Paracetamol is available in different dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories. Paracetamol and its combined dosage form with the other drugs have been mentioned in many pharmacopeias [1,2]. Paracetamol (PCM) (4-hydroxyacetanilide) is a non-steroidal antiinflammatory drug and inhibits isoforms of cyclooxygenase, COX-1, COX-2 and COX-3 enzymes involved in prostaglandin (PG) synthesis. It is centrally and peripherally acting non-steroidal anti- inflammatory drug (Figure 1).

pharmaceutical-analytical-chemistry-Paracetamol

Figure 1: Structure of Paracetamol.

Serratiopeptidase

Serratiopeptidase (SERA) is Proteolytic enzyme, secreted from enterobacterium Serretia sp. E-15. Serratiopeptidase is found in the intestine of silkworm and allow the emerging moth to dissolve its cocoon. The culture of Serratia E- 15 bacteria produce Serratiopeptidase enzyme by the process of purification [3]. Serratiopeptidase hydrolyses histamine, bradykinin and serotonin, responsible for the oedematic status, thus producing anti-inflammatory effect [4,5]. Serratiopeptidase reduces swelling, improves microcirculation and expectoration of sputum, etc.

Aceclofenac

Aceclofenac (ACE), 2-[(2, 6-dichlorophenyl) amino] phenyl] acetyl] oxyacetic acid is used as anti-inflammatory drug. It is official in B.P. [6] and I.P. [7]. 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 [2]. A tablet dosage from containing all the three, (ACE 50 mg, SERA 5 mg and PCM 250 mg), is commercially available in the market and used as anti-inflammatory, analgesic and antipyretic agents. ACE, SERA and PCM are officially available in IP. Literature reviews revealed that UV Spectroscopy method have been reported for the determination of ACE, SERA and PCM individually in pharmaceutical dosage form [8]. Many UV [9,10] and HPLC [11-14] based methods have been reported for determination of these drugs alone as well as in combination with other drugs in pharmaceutical dosage form. Adidala et. al. has developed a simultaneous method for determination of these three drugs in relatively more expensive solvents such as methanol, acetonitrile and formic acids using RP- HPLC. Therefore, the present work was aimed to develop and validate an economical RP- HPLC method using simple methanol and water (50: 50) as mobile phase in isocratic flow for simultaneous determination of ACE, SERA and PCM in pharmaceutical dosage forms (Figure 2).

pharmaceutical-analytical-chemistry-Aceclofenac

Figure 2: Structure of Aceclofenac.

Materials and Methods

Chemical and reagents

ACE, SERA and PCM were procured from pharmaceutical industry Baddi, solan, HP, India. Commercial tablets of above combination used for analysis were procured from local pharmacy. HPLC grade methanol and water were procured from SD Fine-Chem Ltd, Mumbai.

Instruments

Agilent RP-HPLC 1200 series was used for the analysis. The system consists of binary pump, auto sampler and DAD detector manufactured by Germany manufacturer. The method was carried out on C18 Column (4.6 × 150 mm × 5 μm) as a stationary phase. Software EZChrome Elite was used throughout the experiment.

Preparation of mobile phase

The mobile phase comprised of water and methanol in the ratio (50:50). The container of solvent reservoir was rinsed first from the acid, base and from the solvent to be used. Vials were rinsed from the distilled water, HPLC grade water and methanol. The mobile phase was filtered through 0.42 μm nylon filter paper along with the ultrasonication for 20 minutes.

Preparation of standard stock solution

Standard Stock solutions of SERA, ACE and PCM were prepared separately by dissolving 20 mg of drug in 20 mL of methanol and volume was made up to 40 mL to get the concentration of 500 μg/ mL of solution of each drug. Preparation of standard stock solution of SERA, ACE and PCM were made by accurately mixing 1 mL of serratiopeptidase, 10 mL of aceclofenac and 50 mL of paracetamol from the stock solutions to get the concentration as described in the marketed formulations. Hence, dilutions were made accordingly to prepare a calibration graph.

Preparation of sample solution

The above prepared standard solution was used to prepare the further aliquots of SERA (2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL), ACE (20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL) and PCM (100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL). Filter out the samples from 0.22 μm nylon syringe filter for the further process.

Selection of analytical wavelength

Selection of wavelength was done on the basis of scanning a fixed concentration in the range of 200-800 nm for SERA, ACE and PCM. Wavelength of 327 nm was recorded using UV visible spectrophotometer. At the 327 nm SERA showed maximum absorbance while, ACE and PCM showed optimum absorbance, thus the elution exhibited accurate response at 327 nm using UV detector [15].

Optimized RP-HPLC chromatographic parameters

For optimization of the chromatographic conditions, parameters of HPLC such as flow rate, mobile phase ratio, injection volume, temperature, and wavelength were nearly fixed in a set number of experiments [16]. By making changes in all the above parameters, the best one was selected at which elution response was significant, accurate and recorded a sharp symmetric peak as shown in Table 2.

Formulation analysis

Accurately 230 mg of the powdered formulation was weighed dissolved in 10 mL of the solvent and subsequently ultrsonicated. From this a, mid conc. of 20 μg/mL was prepared and filtered out by 0.22 μm siring filter. The injection volume (10 μL) was taken and three peaks were distinctly observed at the wavelength 327 nm with flow rate 0.4 mL/minute [17,18].

Validation

Validation was performed as per International Council for Harmonization (ICH) guidelines. The following parameters should be considered as major and significant criteria for the method development and validation.

Linearity: Linearity was determined for paracetamol, aceclofenac and serratiopeptidase separately by plotting a calibration curve (Between peak area and their respective concentration). Evaluation of drug was performed with UV detector at 327 nm. The peak areas were recorded for all the peaks. Hence, standard calibration curves were plotted between peak area and concentrations [19,20].

System suitability: System suitability should be based on the criteria and parameters collected as a group that will be able to define the performance of the system.

Precision: Performed system precision by multiple injections of a homogeneous standard solution to indicate the performance of the HPLC instrument under the chromatographic condition and day tested [21]. The relative standard deviation shall be not more than 2.0 %. (ICH. Q2B [22]).

LOD and LOQ: Using average standard deviation of precision and slope of a straight-line coefficient, the values of LOD and LOQ were determined.

Robustness: The robustness study was done by making small changes in the optimized method & parameters.

Accuracy (Recovery study): % Recovery studies were carried out at three different levels of 50%, 100%, and 150% of standard solution in triplicate. Serratiopeptidase, Aceclofenac and Paracetamol were taken as samples [23].

Results

Optimized RP-HPLC chromatographic parameters

The chromatographic parameters had optimized for RP-HPLC and were shown in Table 1 and Figure 3.

pharmaceutical-analytical-chemistry-spectra

Figure 3: Overlay UV spectra of SERA, PCM and ACE.

Parameters Optimized conditions
Pump Binary Pump system
Detector Photo Diode array(DAD)
Sampler Auto Sampler
Column C18 column (4.6mm × 250mm, 5µm)
Mobile phase Water : Methanol (50:50)
Stationary Phase C18 column
Flow rate 0.4 mL/min
Run time 14 min
Vol. of injection 10µL
Detection Wavelength 327 nm

Table 1: Optimized HPLC parameter for method.

Linearity

To determine the linearity or calibration curve of PCM, ACE and SERA the samples solution of various concentration ranges were prepared as shown in Table 2. 10 μL of each concentration was injected into the RP-HPLC system separately (Figure 4).

pharmaceutical-analytical-chemistry-Chromatograms

Figure 4: Chromatograms for dilutions and formulation.

SERA ACE PCM
Rt(min) Conc. (µg/ml) Area Rt(min) Conc. (µg/ml) Area (mAU) Rt(min) Conc. (µg/ml) Area (mAU)
6.000 2 938 9.507 100 24544 7.807 20 52205
5.997 4 1200 9.547 200 33977 7.833 40 83955
5.947 6 1563 9.553 300 40046 7.847 60 110216
5.947 8 1812 9.573 400 46406 7.853 80 134120
6.347 10 1960 9.58 500 53436 7.867 100 156397
Slope
Intercept
r2
132.8   341.07   258.55
697.8 19018 19019
0.9817 0.991 0.9949

Table 2: Calibration curve reading.

Regression equation and correlation coefficient

It (y and R2) were determined and illustrated in Figure 5A, 5B and 5C by RP-HPLC of SERA, ACE and PCM.

pharmaceutical-analytical-chemistry-Calibration

Figure 5: Calibration curve of SERA (5A), ACE (5B) and PCM (5C) Determined by RP-HPLC.

System suitability

The System suitability should be based on the criteria and parameters collected as a group that was able to define the performance of the system Table 3.

Limit of detection (LOD) and Limit of quantification (LOQ)

A system precision was conducted by disposing multiple injections of a homogeneous standard solution which indicates the performance of the HPLC instrument under the chromatographic condition Tables 4 and 5.

Precision

Precision of the performance of the RP-HPLC was done under similar chromatographic condition for day 1 and day 2 the results were shown in Table 6.

Robustness

The robustness study was done by making small changes in the optimized method & parameters and no significant affect was recorded, even though, 5 mL change was brought about in mobile phase ratio. There was no significant impact observed on the retention time.

Accuracy (% Recovery study)

% Recovery studies were carried out at three different levels of 50%, 100%, and 150% of standard solution in triplicate in each level. The results are reported in terms of % recovery, RSD as shown in Table 7.

Discussion

Optimized RP-HPLC chromatographic parameters

To develop a cheap, precise, accurate and suitable RP-HPLC method for the simultaneous determination and estimation of PCM, ACE and SERA in marketed formulation tablet dosage form. The different mobile phases e.g. methanol and water in different proportions were used and finally methanol: water (50:50 v/v) selected as an appropriate proportion, which give significant retention time, acceptable peak parameters and suitable absorbance maxima (Figure 5) for PCM, ACE and SERA.

Calibration curve

Linearity or calibration curve of PCM, ACE and SERA: the sample solutions of various concentration ranges were prepared and the correlation coefficient (R2) of PCM, ACE and SERA were determined as 0.9949, 0.991 and 0.9817 respectively, shown in Table 2 which were much closed to required parameter for validation.

System suitability

The system suitability should be based on criteria and parameters collected as a group that will be able to define the performance of the system. The average peak areas of six injections were determined for SERA (1553), ACE (39982.2) and PCM (110240). The standard deviations were found for SERA (22.9957), ACE (235.553) and PCM (115.569). The % Regression standard deviations (%RSD) were determined for SERA (1.41661), ACE (0.58914) and PCM (0.10484). All the parameters significantly describe the excellent system suitability (Table 3).

Limit of detection (LOD) and Limit of quantification (LOQ)

A system precision was conducted by disposing multiple injections of a homogeneous standard solution which indicates the performance of the HPLC instrument under the chromatographic condition Table 4. The relative standard deviation shall not be more than 2.0 %. (ICH. Q2B). Limit of detection (LOD) for PCM, ACE and SERA were found to be 1.475 μg/mL, 2.279 μg/mL and 0.5714 μg/mL respectively and Limit of Quantification (LOQ) for PCM, ACE and SERA also were found to be 4.4698 μg/ml, 6.906 μg/mL and 1.7316 μg/mL respectively as shown in Table 5. It has been concluded from the data, the lowest value of LOD and LOQ could be used for the determination of same drugs.

Precision

A system precision was performed by subjecting injections of a homologous standard solution to indicate the performance of the HPLC instrument under the chromatographic condition and day tested for day 1 and day 2 as shown in Table 6 and %RSD were found less than 2.

Robustness

The robustness study was done by making small changes in the optimized method and parameters e.g. 5 mL change was brought about in mobile phase ratio. There was no significant impact observed on the retention time and tailing factor so we can use this method for further study as a robust method.

Accuracy (% Recovery study)

% Recovery studies were carried out at three different levels of 50%, 100%, and 150% of standard solution in triplicate. Serratiopeptidase, Aceclofenac and Paracetamol present in the sample solution were determined by adjusting the responses into the regression equation for Serratiopeptidase, Aceclofenac and Paracetamol at specific concentration levels. All The results were reported in terms of % recovery, RSD which found within the critical parameter.

Conclusion

The present study describes proposed RP-HPLC method for the simultaneous estimation of PCM, ACE and SERA in tablet dosage form are cheap, accurate, precise, linear, robust, simple and rapid. Acceptable regression values, RSD % and standard deviations are achieved which make it versatile and valuable for simultaneous determination of three drugs in tablet formulation. According to ICH guidelines of validation precision and accuracy have been within acceptable range. So, a large number of samples can be analyzed in short period of time. The results of this validated RP-HPLC method could be conveniently adopted for quality control analysis of PCM, ACE and SERA, simultaneously from tablet dosage forms.

References

Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Article Usage

  • Total views: 312
  • [From(publication date):
    July-2017 - Nov 25, 2017]
  • Breakdown by view type
  • HTML page views : 262
  • PDF downloads : 50
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

Agri & Aquaculture Journals

Dr. Krish

[email protected]

1-702-714-7001Extn: 9040

Biochemistry Journals

Datta A

[email protected]

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

[email protected]

1-702-714-7001Extn: 9042

Chemistry Journals

Gabriel Shaw

[email protected]

1-702-714-7001Extn: 9040

Clinical Journals

Datta A

[email protected]

1-702-714-7001Extn: 9037

Engineering Journals

James Franklin

[email protected]

1-702-714-7001Extn: 9042

Food & Nutrition Journals

Katie Wilson

[email protected]

1-702-714-7001Extn: 9042

General Science

Andrea Jason

[email protected]

1-702-714-7001Extn: 9043

Genetics & Molecular Biology Journals

Anna Melissa

[email protected]

1-702-714-7001Extn: 9006

Immunology & Microbiology Journals

David Gorantl

[email protected]

1-702-714-7001Extn: 9014

Materials Science Journals

Rachle Green

[email protected]

1-702-714-7001Extn: 9039

Nursing & Health Care Journals

Stephanie Skinner

[email protected]

1-702-714-7001Extn: 9039

Medical Journals

Nimmi Anna

[email protected]m

1-702-714-7001Extn: 9038

Neuroscience & Psychology Journals

Nathan T

[email protected]

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

Ann Jose

[email protected]

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

[email protected]

1-702-714-7001Extn: 9042

 
© 2008- 2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords