03639045.2020.1821054 Metodo HPLC Paracetamol Imp J
03639045.2020.1821054 Metodo HPLC Paracetamol Imp J
03639045.2020.1821054 Metodo HPLC Paracetamol Imp J
To cite this article: Amira F. El-Yazbi, Karin M. Guirguis, Mona M. Bedair & Tarek S. Belal
(2020): Validated Specific HPLC-DAD Method for Simultaneous Estimation of Paracetamol and
Chlorzoxazone in Presence of Five of Their Degradation Products and Toxic Impurities, Drug
Development and Industrial Pharmacy, DOI: 10.1080/03639045.2020.1821054
Article views: 1
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Amira F. El-Yazbi 1, Karin M. Guirguis 2, Mona M. Bedair 1, Tarek S. Belal 1
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1 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of
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Alexandria, Elmessalah, 21521, Alexandria, Egypt
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Corresponding Author: Tarek S. Belal
E-mail: [email protected]
Abstract
This work demonstrates a specific and reliable HPLC with diode array detection (DAD)
method for the simultaneous estimation of paracetamol (PAR) and chlorzoxazone (CZ) in
presence of five of their degradation products and toxic impurities; namely; 4-aminophenol (AP),
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column (3.9 × 150 mm, 5 μm) with gradient elution of the mobile phase consisting of 0.05 M
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phosphate buffer pH 7.5 and methanol. The gradient elution started with 5% (by volume)
methanol ramped up linearly to 50% in 10 min, and then maintained at this percentage afterwards
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till the end of the run. The mobile phase was pumped at a flow rate of 1.0 mL/min. The multiple
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wavelength detector was adjusted at 244 and 285 nm to quantify PAR and CZ, respectively.
Additionally, the wavelength 270 nm was found suitable for monitoring separation of the entire
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mixture of PAR, CZ and their impurities. Seven peaks eluted with excellent resolution at
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retention times 3.4, 5.7, 8.0, 10.1, 10.8, 13.5 and 14.4 min for AP, PAR, NP, AT, ACP, CZ and
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CA respectively. Performance of the proposed method was validated with respect to linearity,
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range, precision, accuracy, specificity, robustness, detection and quantitation limits. Calibration
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curves were linear in the ranges of 10-75 and 10-100 g/mL for PAR and CZ, respectively with
correlation coefficients not less than 0.9998. The proposed method proved to be specific and
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stability-indicating by resolution of both drugs from their degradation products and toxic
impurities. Validated HPLC method was successfully applied to the analysis of PAR and CZ in
their combined capsules dosage form, and assay results were favorably compared with a
published reference HPLC method. DAD served as an efficient tool for peak identity and purity
verification.
Keywords: Paracetamol ; Chlorzoxazone ; HPLC-DAD ; Impurities ; Degradation products.
Introduction
Paracetamol (PAR) (Figure 1) is an analgesic antipyretic drug which was introduced into
medical practice many years ago and it is still accepted as a very effective treatment for the relief
of fever and several types of pain in adults and children (1). PAR has several related substances
and potential impurities that should be considered during assay of the bulk powder and final
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dosage forms. Four pharmacopeial related substances for PAR are included in this study: 4-
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aminophenol (AP), 4-nitrophenol (NP), acetanilide (AT) and 4-chloroacetanilide (CA) (2,3).
Figure 1 shows the chemical structures of these related substances. AP is the main hydrolytic
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degradation product of PAR, in addition, AP is known to be nephrotoxic and hepatotoxic (4).
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Similarly, CA is reported to be hepatotoxic and nephrotoxic as well (5). In regard to NP, it is one
of the most toxic and serious contaminant organic pollutants, which can easily irritate eyes, skin,
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and respiratory tract (6). On the other hand, AT was formerly used as an antipyretic drug. The use
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of AT was shortly discontinued because some patients developed a bluish color of the skin
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(cyanosis). This serious side effect was later identified to result from methemoglobinemia where
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AT interferes with the oxygen transport by hemoglobin (7). The toxicant properties of these
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compounds suggest that their separation and monitoring during analysis of the main drug is of
More focus can be found in the scientific literature for the simultaneous determination of
PAR and its main impurity AP. Methods used for this task included electrochemical
determination employing different sensors and electrodes (8-11), spectrophotometric (12, 13),
near infrared (NIR) spectroscopic (14), HPLC-DAD (15), UPLC/Q-TOF-MS (16), HPTLC (17)
and capillary electrophoresis (CE) methods (18, 19). Moreover, different chromatographic
techniques were applied for separation and determination of complex mixtures containing PAR
together with two or more of the its impurities. HPLC (5, 20, 21), UPLC (22), TLC–densitometry
(5) and GC/MS (23) were found useful for analysis of these multi-component mixtures.
Chlorzoxazone (CZ) (Figure 1) is a centrally acting skeletal muscle relaxant with sedative
properties (24). The main degradation product and a synthetic precursor of CZ is 2-amino-4-
chlorophenol (ACP) (25) (Figure 1). This compound is reported as nephrotoxic and a possible
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carcinogenic (26, 27). Selective determination of CZ and other drugs in different pharmaceutical
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mixtures in presence of their degradation products was carried out using HPLC-DAD (26, 28),
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HPLC with UV detection (29, 30) and TLC methods (26, 28, 31).
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Combination therapy is commonly used for management of different musculoskeletal
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painful conditions. PAR, non-steroidal anti-inflammatory drugs, opioid analgesics and skeletal
muscle relaxants are the main ingredients used in these combination therapies. The binary
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combination of PAR and CZ gained lots of attention and focus. Analysis of this mixture was
reported using various techniques such as: chemometric spectrophotometry (32-34), RP-HPLC
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(35-39), LC-MS/MS (40), packed-column supercritical fluid chromatography (SFC) (41) and
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HPTLC (42). In addition, few more specific methods were described dealing with the active
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ingredients together with the impurities. Simultaneous determination of PAR, CZ and the related
impurities AP, CA and p-chlorophenol was carried out using HPLC (43). A stability-indicating
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TLC-densitometric method for simultaneous determination of PAR, CZ and their toxic impurities
AP and ACP was published (44). Finally, traditional partial least squares (PLS) and advanced
artificial neural network (ANN) models were applied for the spectrophotometric determination of
both drugs together with their process-related impurities AP, NP, CA, ACP and 4-chlorophenol
(45).
Reviewing the literature exposed that there are no reports on the simultaneous
determination of PAR and CZ in presence of the five cited toxic impurities AP, NP, AT, CA and
ACP. This work introduces the development, validation and application of a simple, specific and
multipurpose HPLC-DAD method for the simultaneous determination of PAR and CZ in bulk
form and in their combined dosage form. The method can be considered stability indicating based
on the fact that AP and ACP are typical degradation products for PAR and CZ respectively.
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Moreover, separation of 5 potential impurities in a single run demonstrates the impurity profiling
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merit of the method.
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Experimental
Instrumentation
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The HPLC-DAD system consisted of Agilent 1200 series (Agilent Technologies, Santa
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Clara, CA, USA) (auto-injector, quaternary pump, vacuum degasser and diode array and multiple
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wavelength detector G1315 C/D and G1365 C/D) connected to a computer loaded with Agilent
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ChemStation Software. The column used was Waters Symmetry C8 (3.9 × 150 mm, 5 μm
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particle size).
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PAR was kindly supplied by European Egyptian Pharmaceutical Ind. Co., Alexandria,
Egypt and CZ was kindly donated by Pharco Pharmaceuticals Co., Alexandria, Egypt. The
related substances AP, NP, AT, CA and ACP were purchased from Sigma Aldrich, St. Louis,
MO, USA. HPLC-grade methanol (Fisher Scientific, UK), analytical grade of disodium hydrogen
phosphate anhydrous, ortho-phosphoric acid and high purity distilled water were used. The
General Procedure
A gradient mobile phase system consisting of 0.05 M phosphate buffer pH 7.5 (A) and
methanol (B) was used. The separation was achieved with a linear gradient program as follows: 5
% v/v B at zero time; ramped up to 50% B from 0 to 10 min; then kept constant at 50% B from
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10 to 20 min. After 20 min, the program was returned to the initial conditions and the analytical
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column was reconditioned for 3 min. The flow rate was 1.0 mL/min throughout the run. The
injection volume was 20 L. The eluant was monitored by DAD from 190 to 400 nm, and
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chromatograms were extracted at the wavelengths 244, 270 and 285 nm. All determinations were
performed at 25ºC.
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PAR (1000 g/mL) and CZ (1000 g/mL) stock solutions were separately prepared in
HPLC-grade methanol. The working standard solutions were prepared by dilution of aliquots of
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the stock solutions with distilled water to reach the concentration ranges 10-75 and 10-100
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g/mL for PAR and CZ, respectively. Triplicate injections were made for each concentration and
analyzed under the previously described HPLC conditions. Peak areas were recorded at 244 and
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285 nm for PAR and CZ, respectively. The peak areas were plotted against the corresponding
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concentrations to construct the calibration graphs and calculate the regression equations.
Stock solutions of the 5 impurities (1000 g/mL) were separately prepared in HPLC-
grade methanol. Aliquots of the related substances stock solutions were added to aliquots of the
two parent compounds stock solutions, and this mixed solution was diluted to volume with
distilled water. The mixture was chromatographed using the previously described HPLC
The content of 10 capsules were weighed and mixed. An accurately weighed portion of
shaking for 10 min then filtered into a 25 mL volumetric flask. The residue was washed with 2 ×
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5 mL portions of methanol and washings were added to the filtrate, then the solution was diluted
to volume with methanol to reach final concentrations of 1200 g/mL for PAR and 1000 g/mL
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for CZ. Aliquots of the stock sample solution were diluted with distilled water to obtain final
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concentrations within the specified ranges and treated as under “General Procedure” and
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recovered concentrations were calculated from similarly treated external standard solutions.
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Results and Discussion
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A simple liquid chromatographic method with diode array detection was mainly
developed for routine quality control analysis of PAR/CZ mixture. Another main consideration
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during optimization experiments was to make sure that the method is specific and effective
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enough to separate the studied drugs from their toxic impurities. Ideally, complete separation
among all peaks should be within reasonable analysis time to avoid excessive consumption of
solvents and long instrument operation hours. Different experimental conditions were studied and
optimized to achieve the best chromatographic separation among all peaks. For optimization of
the analytical column, a couple of reversed-phase columns were tested: Waters Symmetry C18
(3.9 × 150 mm) and Waters Symmetry C8 (3.9 × 150 mm). The most successful separation
among all the eluting peaks including the five potential impurities was accomplished by the
Waters Symmetry C8 column; therefore, it is the column of choice for this separation.
Various mobile phases were investigated taking into consideration system suitability
parameters. The mobile phase combination of choice was 0.05 M phosphate buffer pH 7.5 and
methanol. Phosphate buffer was substituted by other aqueous phases such as phosphoric acid
solution or water. Additionally, acetonitrile organic modifier was examined either alone or with
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methanol. In these experiments, insufficient separation or overlapping between some peaks were
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observed especially between CA and CZ peaks as well as between ACP and NP peaks. Not only
the composition of the mobile phase was important for separation of this multi-component
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mixture, but also pH of the phosphate buffer was found crucial for optimum chromatographic
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separation. Several pH values of the phosphate buffer were tried (pH 4, 5 and 6) where CZ and
CA showed overlapping peaks and almost all the peaks were tailed. Obvious improvement was
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noticed upon using phosphate buffer pH 7. Baseline separation with resolution value 1.65 was
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obtained between CZ and CA peaks with better peak shapes. The best separation was achieved at
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pH 7.5 with resolution values not less than 3.3 among all eluting peaks. Excellent separation,
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tolerable peak asymmetry and reasonable retention times were accomplished with gradient
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system starting with 5% (by volume) methanol increased linearly to 50% in 10 min, and then
fixed at this % untill the end of the run. Flow rate was kept constant at 1.0 mL/min with the
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Quantitation was achieved using the diode array detection based on peak area
measurement. PAR exhibits an absorption band all over the range 200–300 nm with maximum
absorption at 244 nm. On the other hand, CZ is considered a weak UV absorbing compound at
the previously mentioned wavelength and shows maximum absorption at about 285 nm.
Consequently, 244 and 285 nm were found suitable for recording peak areas of PAR and CZ,
respectively. In addition, the wavelength 270 nm was found suitable for monitoring PAR, CZ and
excellent separation of the two analytes PAR and CZ together with their studied impurities.
Figure (2) exhibits a typical chromatogram for separation of this complex mixture. The elution
order starts with those of higher polarity AP and PAR, followed by NP, AT, ACP, CZ and finally
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CA. Their retention times were 3.36, 5.68, 7.99, 10.05, 10.84, 13.46 and 14.40 min respectively.
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System suitability parameters were calculated and they were found acceptable (Table 1).
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Method validation
The validation of the proposed method was accomplished using the validation of analytical
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procedures of International Conference on Harmonization (ICH) guidelines (46).
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Concentration ranges, linearity and detection and quantification limits
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Linearity was accomplished by plotting the measured peak areas at 244 nm and at 285 nm
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for PAR and CZ, respectively, versus the corresponding concentrations. Table 2 demonstrates the
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analytical parameters and data of the proposed method such as concentration ranges, linear
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deviations of the intercept (Sa) and slope (Sb). Results of the correlation coefficient values
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(0.9998) indicated good linearity with deviation around the slope (Sb%) found to be less than
1%.
Low limit of detection (LOD) and limit of quantification (LOQ) values of PAR and CZ
estimated by the signal-to-noise ratio method (Table 2) confirm the sensitivity of the proposed
Three replicate determinations for each concentration of three different concentration levels
for each drug within one day were performed for assessing intra-day precision and accuracy of
the used method. While, for the inter-day precision and accuracy, three replicate determinations
of the same three concentration levels for each drug were determined on three different days. The
obtained recoveries were satisfactory with RSD % and Er % < 2.0 % indicating good precision
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and accuracy of the proposed method for the determination of PAR and CZ (Table 3).
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Specificity
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The successful separation of PAR and CZ with excellent resolution from their related
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substances and potential impurities indicated the high specificity of the proposed method (Figure
2). Additionally, the diode-array detection confirmed method’s specificity as it allowed peak
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purity verification where co-elution of any of the inactive ingredients during assay of the dosage
Robustness
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The effect of small changes in different steps of the procedure was performed in order to
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study the robustness of the proposed method. Changes in column temperature (± 5ºC), methanol
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% in the mobile phase (± 2%), flow rate (± 0.05 mL/min) and detection wavelengths (± 2 nm)
were tested. Results obtained showed that such small changes did not cause any significant effect
on peak areas or retention times of all peaks (Table 4). Additionally, these minor experimental
changes did not affect the resolution among eluting peaks. Resolution between any two
successive peaks was never less than 1.99 which implies excellent baseline separation (Table 4).
Stability of solutions
PAR and CZ standard and sample solutions were found to be stable within 6 h at room
the studied analytes were stable for at least one week when stored refrigerated at 4ºC except for
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Assay of capsules dosage forms
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The proposed HPLC-DAD method was applied for the determination of PAR and CZ
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combination in Relax® capsules pharmaceutical formulation. Figure 3 demonstrates the obtained
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chromatogram for such dosage form with PAR and CZ eluted at their specific retention times
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without the appearance of any interfering peaks from the inactive ingredients of the assayed
capsules. Peak purity was assessed by scanning the absorption spectra of each peak at different
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points in order to assure the absence of co-eluted inactive components (Figure 4). Since they are
superimposable, then the peak is considered pure and homogenous with no signs of co-elution of
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any extra substance. Results obtained (Table 5) showed good accuracy and precision with
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accepted % recovery, SD and RSD% values. Moreover, determination of PAR and CZ with the
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reported HPLC method (43) was applied performed. Statistical comparison using the Student’s t-
and the variance ratio F-tests of the obtained data with those of the proposed method showed that
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the calculated values did not exceed the theoretical ones at the 95% confidence level. Therefore,
no significant differences between the obtained recoveries (Table 5) of the two methods. Thus,
the proposed method is applicable to the assay of the binary mixture of PAR and CZ in capsules
A simple, specific and rapid gradient elution HPLC procedure for the assay of PAR and
CZ in their pharmaceutical formulation is described in this work. Water was used as a solvent
with the minimum requirement of organic solvents during preparation of most of the solutions
used. This promotes the developed method to be environment-friendly, simple and cost-effective.
Moreover, the single step of changing the mobile phase composition linearly with keeping the
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flow rate constant adds to the method simplicity. Upon comparison with other reported methods
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(32-45) for the analysis of PAR-CZ mixture, the developed method showed better specificity for
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the determination of such combination, in addition to the separation of the two drugs from five of
their toxic impurities and reported degradation products. Therefore, the proposed method can be
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applied for stability indicating assay and impurity profiling purposes. Also, using DAD allows
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peak identity and purity confirmation which imparts the proposed method an additional
advantage over methods that use the universal UV detector. Reliability of the proposed method
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was assured by examining different validation parameters and the successful application to
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capsules formulation.
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ACP AT CA NP
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(ACP).
DAD1E,Sig=270,4Ref=off,TT(KARIN1-11-2017\MIX-3-PH7500003.D)
mAU
2
140 3
120 6
100
80
7
4
5
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1
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40
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20
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0 2 4 6 8 10 12 14 16 18 min
Figure 2: HPLC chromatogram at 270 nm for a mixture containing 50 g/mL of each of (1) 4-
aminophenol (AP), (2) paracetamol (PAR), (3) 4-nitrophenol (NP), (4) acetanilide (AT), (5) 2-
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amino-4-chlorophenol (ACP), (6) chlorzoxazone (CZ) and (7) 4-chloroacetanilide (CA)
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DAD1A, Sig=244,4Ref=off, TT(KARIN6-11-2017\C2-60PAR-50CZ30.D)
mAU
PAR A
400
350
300
250
200
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150
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100
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50
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0
0 2 4 6 8 10 12 14 16 18 min
DAD1B, Sig=285,4Ref=off, TT(KARIN6-11-2017\C2-60PAR-50CZ30.D)
mAU
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120
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100
PAR
80
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60
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40
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20
0
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0 2 4 6 8 10 12 14 16 18 min
Figure 3: HPLC chromatograms at 244 nm (A) and 285 nm (B) of a solution containing 60
µg/mL PAR and 50 g/mL CZ obtained from Relax® capsules.
*DAD1,5.651(110Fl,-)Ref=5.477&6.624of C2-60PAR-50CZ30.D
*DAD1,5.691(390Fl,-)Ref=5.477&6.624of C2-60PAR-50CZ30.D A
*DAD1,5.724(548Fl,-)Ref=5.477&6.624of C2-60PAR-50CZ30.D
*DAD1,5.784(411Fl,-)Ref=5.477&6.624of C2-60PAR-50CZ30.D
*DAD1,5.851(175Fl,-)Ref=5.477&6.624of C2-60PAR-50CZ30.D
Norm.
500
400
300
t
200
ip
100
cr
0
220 240 260 280 300 320 340 360 380 nm
us
*DAD1,13.457(198Fl,-)Ref=13.244&14.471of C2-60PAR-50CZ30.D
*DAD1,13.504(523Fl,-)Ref=13.244&14.471of C2-60PAR-50CZ30.D B
*DAD1,13.551(711Fl,-)Ref=13.244&14.471of C2-60PAR-50CZ30.D
*DAD1,13.644(497Fl,-)Ref=13.244&14.471of C2-60PAR-50CZ30.D
an
*DAD1,13.751(257Fl,-)Ref=13.244&14.471of C2-60PAR-50CZ30.D
Norm.
M
600
500
d
400
e
300
pt
200
100
ce
0
220 240 260 280 300 320 340 360 380 nm
Ac
Figure 4: Purity plots for 60 μg/mL PAR (A) and 50 μg/mL CZ (B) obtained from analysis of
Relax® capsules.
Table 1: System suitability parameters for the separated compounds in this study
%RSD
Capacity Theoretical
Tailing Selectivity Resolution of
Compound tR ± SD (min) factor Plates
Factor () (Rs) Peak
(k) (N)
Area
AP 03.36 ± 0.004 1.24 03295 1.08 0.11
PAR 05.68 ± 0.003 2.79 15746 1.08 1.69 11.18 0.10
NP 07.99 ± 0.018 4.31 20469 1.04 1.40 11.30 1.29
t
ip
AT 10.05 ± 0.007 5.70 33705 1.17 1.26 09.42 0.21
ACP 10.84 ± 0.006 6.23 37916 1.21 1.08 03.58 0.67
cr
CZ 13.46 ± 0.006 7.97 43633 1.14 1.24 10.86 0.54
CA 14.40 ± 0.012 8.61 33030 1.15 1.07 03.31 1.15
us
an
M
e d
pt
ce
Ac
Table 2: Analytical parameters for the determination of PAR-CZ mixture using the proposed
HPLC-DAD method
Parameter PAR CZ
t
ip
Sbb 0.413 0.240
RSD% of the slope (Sb%) 0.647 0.653
cr
Correlation coefficient (r) 0.99991 0.99989
us
Sy/xc 21.424 18.630
LODe (μg/mL) 1.191 1.705
f an
LOQ (μg/mL) 3.968 5.685
M
e d
pt
ce
Ac
Table 3: Precision and accuracy for the analysis of PAR and CZ in bulk form using the proposed
HPLC-DAD method.
Nominal value Found ± SDa
RSD(%)b Er(%)c
(μg/mL) (μg/mL)
t
ip
Between-day 50 50.06 ± 0.349 0.700 -0.12
75 75.02 ± 0.671 0.890 -0.03
cr
20 19.99 ± 0.114 0.570 –0.05
us
Within-day 50 50.01 ± 0.002 0.004 -0.02
75 74.77 ± 0.048 0.060 –0.31
CZ an
20 20.30 ± 0.112 0.550 -1.50
Between-day 50 50.61 ± 0.869 1.720 -1.22
M
75 74.85 ± 1.399 1.870 –0.20
a
Mean ± standard deviation for three determinations.
d
b
% Relative standard deviation.
c
e
% Relative error.
pt
ce
Ac
Table 4: Evaluation of robustness of the proposed HPLC-DAD method.
Peak area ± SD
Retention Resolution RSD
Parameter Compound PAR at 244 nm and
time ± SD (Rs ± SD) %
CZ at 285 nm
AP 03.35 ± 0.336
Methanol PAR 05.68 ± 0.418 09.26 ± 0.495 3322.13 ± 8.21 0.25
percentage NP 08.52 ± 0.427 11.59 ± 0.123
in the mobile AT 10.06 ± 0.436 05.90 ± 0.046
phase ACP 10.83 ± 0.426 02.94 ± 0.051
± 2% CZ 13.71 ± 0.655 10.07 ± 0.218 1919.40 ± 10.62 0.55
CA 14.42 ± 0.755 02.06 ± 0.050
t
AP 03.34 ± 0.147
ip
PAR 05.69 ± 0.201 09.28 ± 0.075 3291.43 ± 86.23 2.62
NP 08.53 ± 0.248 11.59 ± 0.064
cr
Flow rate
AT 10.07 ± 0.254 05.85 ± 0.120
± 0.05
ACP 10.84 ± 0.257 0 2.92 ± 0.056
mL/min
us
CZ 13.68 ± 0.297 09.95 ± 0.235 1893.31 ± 19.72 1.04
CA 14.39 ± 0.342 02.05 ± 0.035
AP 03.34 ± 0.143
PAR
NP
05.69 ± 0.248
08.53 ± 0.485
an
09.21 ± 0.533
11.36 ± 0.741
3329.17 ± 23.31 0.70
Column
AT 10.06 ± 0.301 05.73 ± 0.822
temperature
M
ACP 10.83 ± 0.331 02.86 ± 0.087
± 5 °C
CZ 13.69 ± 0.496 09.77 ± 0.187 1919.34 ± 36.95 1.93
CA 14.40 ± 0.529 01.99 ± 0.124
d
Working
PAR 3382.57 ± 112.24 3.32
e
wavelength
CZ 1892.06 ± 23.06 1.22
± 2 nm
pt
ce
Ac
Table 5: Application of the proposed HPLC-DAD method to the analysis of PAR-CZ mixture in
capsules dosage form (Relax capsules).
Proposed method Reference method
Parameters (HPLC Method)
PAR CZ PAR CZ
%Recovery ± SDa 100.12 ± 0.68 99.72 ± 0.20 99.84 ± 1.07 99.31 ± 0.44
RSD%b 0.68 0.20 1.07 0.44
t 0.48 1.93
F 2.48 4.84
t
a
ip
Mean ± standard deviation for five determinations.
b
% Relative standard deviation.
Theoretical values for t and F at p = 0.05 are 2.31 and 6.39, respectively.
cr
us
an
M
e d
pt
ce
Ac