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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 3  |  Page : 458-465

Development and validation of chemometric-assisted spectrophotometric method for the simultaneous estimation of aceclofenac, paracetamol, and chlorzoxazone with impurities


1 Department of Pharmaceutical Chemistry, MAEERS Maharashtra Institute of Pharmacy, MIT Campus, Pune, Maharashtra, India
2 Department of Pharmaceutical Chemistry, School of Pharmacy, MIT World Peace University, Pune, Maharashtra, India
3 Department of Pharmaceutical Quality Assurance, PES Modern College of Pharmacy (for Ladies), Pimpri-Chinchwad, Maharashtra, India
4 Department of Pharmaceutical Chemistry, MIT World Peace University, Pune, Maharashtra, India
5 Department of Petroleum Engineering, School of Petroleum Engineering, MIT World Peace University, Pune, Maharashtra, India
6 Department of Quality Assurance, School of Pharmacy, MIT World Peace University, Pune, India

Date of Submission13-Apr-2022
Date of Decision18-May-2022
Date of Acceptance28-Jun-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Vishnu Choudhari
Department of Quality Assurance, School of Pharmacy, MIT World Peace University, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_92_22

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  Abstract 


Background: Analysis of tertiary mixtures of analytes along with their impurities with simple and cost effective manner is always of interest. Utility of chemometric techniques are growing in pharmaceuticals, it improve speediness in the analysis and also provide analytical solutions with reduce the number of steps in the analytical method. In this study UV-Visible spectrophotometry coupled with principle component regression (PCR) and partial least square (PLS) multivariate methods was applied for estimation of three drugs in their formulation. Method: The calibration and validation sets were prepared in linear concentration range of three drugs and major impurities of paracetamol and aceclofenac. The series of sets were prepared using multilevel multifactorial design. Leave- One-Out (LOO) cross validation technique was employed to get essential number of Latent variables (LVs) that provides the greatest predictive ability. The developed method was studied for qualitative and quantitative analysis of titled drugs and validated as per regulatory guidelines. Results: The results showed the values of coefficient of determination (R2) for all drugs and impurities was higher than 0.99 indicating high acceptability. The obtained RMSE values were relatively low. Coefficient of determination and RMSE values indicate good accuracy and precision, respectively. Conclusion: Proposed method was successfully used for analysis of aceclofenac, paracetamol and chlorzoxazone in tablet dosage form and major impurities of aceclofenac, paracetamol in bulk.

Keywords: Aceclofenac, analytical method validation, chemometric, chlorzoxazone, impurities, paracetamol, partial least square, principal component regression, spectrophotometric


How to cite this article:
Gunjal R, Gajbhar A, Vichare V, Sutar A, Deshmukh M, Choudhari V. Development and validation of chemometric-assisted spectrophotometric method for the simultaneous estimation of aceclofenac, paracetamol, and chlorzoxazone with impurities. Biomed Biotechnol Res J 2022;6:458-65

How to cite this URL:
Gunjal R, Gajbhar A, Vichare V, Sutar A, Deshmukh M, Choudhari V. Development and validation of chemometric-assisted spectrophotometric method for the simultaneous estimation of aceclofenac, paracetamol, and chlorzoxazone with impurities. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Oct 5];6:458-65. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/458/356160




  Introduction Top


Aceclofenac (ACF) is chemically, ([2-{2, 6-dichlorophenyl] amino} phenylacetooxy acetic acid) [Figure 1]a, is a nonsteroidal anti-inflammatory agent with prominent anti-inflammatory and analgesic activities. ACF inhibits action of cyclooxygenase enzyme. Paracetamol (PAR) is chemically, N-acetyl-p-aminophenol [Figure 1]b, it acts by blocking COX-2 mostly in the central nervous system.[1] Chlorzoxazone (CHX) is chemically 5-chloro-2-hydroxy benzoxazole [Figure 1]c, it inhibits muscle spasm. This combination of three drugs is widely prescribed for the treatment of pain associated with the muscle spasm. Diclofenac-free acid (DFA) [Figure 1]d and p-aminophenol (PAP) [Figure 1]e are major impurities of ACF and PAR, respectively.
Figure 1: (a) Structure of aceclofenac. (b) Structure of paracetamol. (c) Structure of chlorzoxazone. (d) Structure of diclofenac-free acid. (e) Structure of para aminophenol

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Various liquid chromatography (LC) and LC–mass spectrometry (LC-MS) methods are reported for the determination of ACF from tablet,[2] its metabolite,[3],[4] from binary and ternary combination with other drugs.[5],[6],[7],[8],[9] Various methods for individual determination of PAR by LC-MS in plasma,[10],[11] in plasma with metabolites,[12] in binary combination with ultraviolet (UV) spectrophotometry are available.[13],[14] Various LC and LC-MS methods for determination of Paracetamol in binary and ternary combination with other drugs from plasma and various formulations are reported.[15],[16],[17],[18],[19],[20],[21] Methods are also reported for the estimation of CHX from plasma and formulations individually and in combination with other drugs.[22],[23] Literature survey also indicates that there are methods reported for estimation of ternary combinations of the titled analytes from bulk, formulations, and plasma by LC, ultra–high-performance LC, and LC-MS methods.[24],[25],[26],[27],[28],[29],[30],[31],[32],[33] Literature survey also revealed that there is no UV spectrophotometric method available for the estimation of the titled analytes and impurities. The UV absorption spectra of ACF, PAR, and CHX in methanol at their corresponding ratio in the combined dosage form show strong overlap [Figure 2]a. Thus, direct simultaneous spectrophotometric determination of the three drugs in the mixture is not possible. Furthermore, there is no UV-spectrophotometric supported chemometric-based method available for quantitative analysis of titled analytes and their impurities. Thus, the aim of the work was to develop UV spectrophotometric method and validate as per regulatory guidelines. Efforts were directed in this work for simultaneous determination of these three drugs in ternary mixture using chemometric techniques in presence of major impurities as well as degradation products DFA and para-aminophenol of ACF and PAR, respectively.[34],[35],[36],[37],[38],[39]
Figure 2: (a) Overlay spectra of ACF, PAR, and CHX in methanol. (b) Overlay spectra of ACF, PAR, and CHX with their selected impurities DFA and PAP in methanol. ACF: Aceclofenac, PAR: Paracetamol. CHF: Chlorzoxazone, DFA: Diclofenac-free acid

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Currently, multivariate methods are extensively used in quantitative spectral analysis of compounds having highly overlapping spectra. In multivariate calibrations, measurement of absorbance at many points in the wavelength range is characterized by the higher speed of data processing and minimizing of calibration models error. Chemometrics, introduced in 1972 by Svan Wold is the science of relating measurements acquired on a chemical systems or processes to another state of the system involving the application of mathematical or statistical methodologies.

Complex spectra of mixtures of analytes are processed effectively for simultaneous analyses of components of mixture easily by chemometric methods and these methods currently finding wide applications in the pharmaceutical industry.[34],[35],[36] These chemometric methods include principal component regression (PCR), and partial least squares (PLS).


  Methods Top


All the chemicals and reagents used were of analytical grade. Acemiz-MR tablets (Lot No 162J7AGI) manufactured by Lupin Ltd., Mumbai, India, containing ACF IP 100 mg, PAR IP 325 mg, and CHX USP 250 mg in each tablet were procured from local pharmacy.

Instrumentation and software

Cary 100, Double beam UV–Visible spectrophotometer were used to record spectra. All measurements were carried out over the wavelength range of 200–400 nm, spectral bandwidth adjusted to 1 nm, scanning speed was 100 nm/min. PLS, PCR modeling, and data analysis were performed using a trial version, Unscrambler X (10.5.1); numerical calculations were performed using Microsoft Excel 2010.

One component calibration

Linearity range of each of the analyte was determined by performing one component calibration. Concentration range determined (μg/ml) was for ACF (1–5), PAR (3–15), and CHX (2.5–12.5). Similarly, impurities DFA (0.5–2.5) and PAP (1.5–7.5) were also studied for linear dynamic ranges. The linear dynamic range for all the analytes was determined by linearity plot of drug amount and the corresponding absorbance values. Overlain spectra of analytes ACF, PAR, and CHX at concentration of 2, 6, and 5 μg/ml, respectively, is presented in [Figure 2]a. [Figure 2]b represents overlain spectra of ACF, PAR, and CHX along with their selected impurities DFA and PAP in methanol.

Preparation of standard stock solution and working stock solution

Standard stock solution (SSS) containing 1000 μg/ml of each of ACF, DFA, PAR, PAP, and CHX were prepared separately by dissolving accurately weighed quantities of standard analytes in methanol. The 5 mL of SSS of each of ACF, DFA, PAR, PAP, and CHX diluted separately to 50 ml with methanol to get second SSSs having 100 μg/ml of analytes. Second SSS suitably diluted to obtain working standard solutions with concentration in linearity range as defined before for all the analyte drugs and impurity standards or degradation products mentioned before in methanol.

Construction of calibration and validation set

The multilevel multifactor design used for the construction of calibration as well as validation set.[35] The sets were having analyte amount selected in concentration range of 1–5 μg/ml (ACF), 3–15μg/ml (PAR), and 2.5–12.5μg/ml (CHX) and impurities 0.5–2.5 (DFA), 1.5–7.5 (PAP) for calibration set. Of 25 sets, 8 mixtures were selected for validation set. Composition of these mixture solutions in the form of analytes and selected impurities is presented in [Table 1]. All the absorbance spectra recorded in wavelength range of 220–320 nm, in this range data captured at interval of 1 nm. The spectra were saved in MS-Excel format and further processed by Unscrambler software for model generation with latent variables (LVs) while processing; which is necessary to obtain a good prediction. Leave-one-out cross-validation method was used to obtain necessary number of LVs as given by the following equation:
Table 1: Concentration aceclofenac, paracetamol, chlorzoxazone and their impurities diclofenac-free acid and para-amino phenol in the training and calibration sets for the multivariate calibrations

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Where,



RMSECV = Root mean square error of cross validation

Cpre = predicted concentration of validation set

Cact = actual concentration of calibration set

Ic = Total number of samples in calibration set

After the PCR and PLS models have been constructed, it was found that the optimum number of LVs were three factors for both PCR and PLS as depicted in [Figure 3]. For the validation of generated models, concentration in validation set predicted by using proposed models as depicted in [Table 2].
Figure 3: (a) Explained variance in PCR. (b) Explained variance in PLS. PCR: Principle component regression, PLS: Partial least square

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Table 2: Predicted results for validation set by principle component regression and partial least squares method

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Assay of marketed formulation

For formulation assay, 20 tablets accurately weighed and finely powdered. Powder equivalent to 100 mg of ACF suitably processed to extract the analytes by sonication for 10 min and filtered through Whatman filter paper number 41. Filtrate transferred to flask, volume adjusted to the mark in 100 mL volumetric flask by passing solvent through the filter assembly. Resulting solution was diluted suitably to get target analyte concentration (TAC) of 3 μg/ml of ACF, 9.75 and 7.5 μg/ml of PAR and CHX in methanol. The diluted solution was analyzed by proposed methods.

Accuracy study

The recovery study carried out at three levels. Levels selected for the study were 50%, 100%, and 150% of TAC. For the study, calculated amount of analytes from standard solutions spiked into sample and processed as per the proposed method procedure. Amounts in solutions predicted using developed PCR and PLS models.

Precision

Precision was determined at three concentration levels, i.e., at 2, 3, 4 μg/ml for ACF and corresponding concentrations of PAR and CHX in three replicates at each level.


  Results Top


Assay results of the three titled drug analytes by PCR and PLS are presented in [Table 3]. The data indicate that the assay values for all the three analytes were in the range of 100.46%–101.29% w/w and % relative standard deviation (RSD) was always less than 1.45 by PCR method. When the same solutions were estimated by PLS, assay values for all the three analytes were in the range of 100.84–101.41% w/w and % RSD values were always <1.42.
Table 3: Assay result for aceclofenac, paracetamol, and chlorzoxazone in tablet by proposed method

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Accuracy data of the analyte by proposed methods at 50%, 100%, and 150% and are presented in [Table 4]. The data indicate that the % recovery of ACF was in the range of 98.68%–101.29% w/w and % RSD was always <1.04 by PCR and PLS methods at all the selected three recovery levels. The recovery of PAR and CHX was in the range of 99.29%–101.58% w/w and % RSD was always less than 0.75 by PCR and PLS methods at all the selected three recovery levels.
Table 4: Accuracy data of analytes by proposed models

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Precision data of all the three analytes determined in the linearity range from standard mixture at three concentration levels as described in the method section; results of the precision obtained for ACF, PAR, and CHX by PCR and PLS models are presented in [Table 5]. The amount of analyte determined during the precision study from the solutions was well in agreement as per requirements of precision with % RSD determined was <2. Concentration range of the analytes used for the study, wavelength range used for spectra scanning is given in [Table 6]. The table also gives a summary of the method sensitivity, method precision, accuracy, and linearity parameters
Table 5: Precision results obtained for aceclofenac, PARA, and chlorzoxazone by principle component regression and partial least squares models

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Table 6: Summary of calibration and method validation results

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  Discussion Top


As shown in [Figure 2]a and [Figure 2]b, overlay of absorption spectra of ACF, PAR, and CHX in methanol show complete and clear overlapping of the spectra of analytes. Therefore, simultaneous determination of ACF, PAR, and CHX using standard UV–Visible spectrophotometry was difficult because of observed considerable overlapping of the spectral curve. To solve the overlapping spectra among ACF, PAR, and CHX, the quantitative analysis was performed with the help of PCA and PLS calibration. This technique is carried out in three steps, namely calibration, validation, and prediction of unknown samples.

Then, few wavelengths were optimized during PCR and PLS modeling to obtain the wavelengths, which were able to provide the best correlation between actual value of ACF, PAR, and CHX and its predicted value. Finally, the wavelength of 210–320 nm was preferred for quantification ACF, PAR, and CHX simultaneously.

Multivariate calibration is finding more and more useful for spectral analysis; this is because the simultaneous treatment of many wavelengths greatly improves the precision as well as predictive ability of the experiments. Further considering full-spectrum imparts the ability to achieve improved precision. Improved precision is possible because there is a signal averaging effect when much spectral intensity is included. In signal, averaging process noise elements in the acquired spectra are nullified. Out of total solutions, 17 sets of solutions were used for calibration and 8 were used for validation [Table 1]. The best results were obtained with the wavelength intervals of λ= 1 nm in selected solvent. The developed methods found to be accurate and precise as results are close to 100% and precise with % RSD <2.

Limitations of the study

Limitations may be associated with changes in the instrument performances. As instrument performance erodes results may vary and this can be controlled by having stringent limits for UV instruments and can be monitored and corrected by increased frequency of instrument calibration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 2], [Figure 1], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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