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Comparison of HPLC and UV Spectrophotometric Methods for the Determination of Cefaclor Monohydrate in Pharmaceutical Dosages | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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Comparison of HPLC and UV Spectrophotometric Methods for the Determination of Cefaclor Monohydrate in Pharmaceutical Dosages

Alfeen Mohammad A*

Analytical Chemistry Department, Faculty of Science, Al-Baath University, Homs, Syria

*Corresponding Author:
Mohammad Anas Alfeen
Lecturer and Chemist
Department of Analytical Chemistry
Faculty of Science, Al-Hamra Street
Homs, Syrian Arab Republic
Tel: 963968628582
E-mail: [email protected]

Received date: December 21, 2015; Accepted date: January 15, 2016; Published date: January 18, 2016

Citation: Mohammad A A (2016) Comparison of HPLC and UV Spectrophotometric Methods for the Determination of Cefaclor Monohydrate in Pharmaceutical Dosages. Med chem 6:019-022. doi:10.4172/2161-0444.1000317

Copyright: © 2016 Mohammad AA. 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.

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This paper describes the development and evaluation of a HPLC and UV spectrophotometric methods to quantify Cefaclor Monohydrate in Oral suspensions and Capsules. HPLC analysis were carried out using a C18 Knauer column and a mobile phase composed of Triethylamine:methanol:Acetonitrile:water (2:10:20:68) v/v%, with a flow rate of 1.0 mL/min and UV detection at 265 nm. For the spectrophotometric analysis, water was used as solvent and the wavelength of 264 nm was selected for the detection. Both methods were found to quantify Cefaclor monohydrate in Oral suspensions and Capsules accurately. Therefore HPLC and UV methods presented the most reliable results for the analyses of Oral suspension and Capsules.


HPLC; Cefaclor monohydrate; Spectrophotometric methods; Antibacterial activity


Cefaclor monhydrate (CAS 56238-63-2) (Figure 1) is a second generation cephalosporin with high antibacterial activity; it has enhanced in vitro activity against clinically important Gram-positive and Gramnegative microorganisms [1]. The chemistry of cephalosporins has been widely explored because of their extensive medical applications [2]. Several analytical procedures are available in literature for the analysis of antimicrobial. These methods are spectrophotometry [3-13], high performance liquid chromatography [14-19], capillary electrophoresis [20], fluorimetry [21-24], polarography [25-29], titrimetry [30], and bioassay [31,32]. Spectrophotometric assay for determination of other cephalosporins as ceftazidime has been described [33] but no method for Cefaclor monohydrate had been previously described.


Figure 1: Structure of Cefaclor monohydrate.

The purpose of this study was to develop and validate analytical methods to quantify Cefaclor monohydrate in Capsules and Oral suspensions, using HPLC and UV spectrometry. The results obtained by these methods were statistically compared, by using analysis of variance (ANOVA). In addition, the reliability and feasibility of them were evaluated focusing on routine quality control analysis.


Reagents and materials

Cefaclor monohydrate reference standard was kindly donated by Parabolic Indian Ltd. The Capsules and Oral Suspensions were purchased from Medico Labs-Homs-Syria and Oubari Company- Aleppo-Syria. Ultra Pure Water was purified by using a Millipore system (Bedford, MA). Methanol, Acetonitrile, and Triethylamine (HPLC grade) was obtained from Merck (Fairfield, OH).

Instruments and analytical conditions

All HPLC measurements were made on a Waters 1525 Binary HPLC Pump, consisting of a 7725i manual injector with a 20 μL loop (Rheodyne, Torrance, CA), integrated UV detector UV-vis (Milford, MA). The system employed a 250 mm × 4.6 mm C18 column Wat 054275 (Milford, MA) and particle size of 5 μm guard column. The detector was utilized at 265 nm and UV spectra from 200 to 400 nm were recorded on line for peak identification. The mobile phase consisted of Triethylamine:methanol:Acetonitrile:Ultra Pure water (2:10:20:68) v/v%, at a flow rate of 1.0 mL/min. The injection volume was 20 μL. Ultraviolet spectrophotometric analyses were carried out on a UV-Vis Shimadzu UV mini 1240 (Shimadzu, Kyoto, Japan) spectrophotometer, in a 1 cm quartz cubette. The wavelength of 264 nm was selected for the quantitation of Cefaclor monohydrate and the measurements were obtained against water as a blank.

Preparation of standard and sample solutions

The standard stock solutions were prepared by dissolving 10 mg of Cefaclor monohydrate reference standard in 10 mL of water to get a concentration of 1 mg/mL. An aliquot of 100 μL of the obtained solution was transferred to a 10 mL volumetric flask. The volume was adjusted with Ultra Pure water for spectrophotometric and chromatographic analysis, resulting in solutions of 10 μg/mL.

The sample solutions were prepared by dissolving 10 mg of Cefaclor monohydrate powder for Capsules or Oral suspensions in 10 mL of water to get a concentration of 1 mg/mL. An aliquot of 100 μL of this solution was transferred to a 10 mL volumetric flask. The volume was adjusted with water for spectrophotometric analysis or mobile phase for chromatographic analysis, to obtain a solution at 10 μg/mL of Cefaclor.


The optimized spectrophotometric and chromatographic methods were completely validated according to the procedures described in ICH guidelines Q2 (R1) for the validation of analytical methods [34].


Standard solutions containing 1000 μg/mL of Cefaclor monohydrate in water were prepared, in triplicate. Aliquots of these solutions were diluted in water. Eight different concentrations, corresponding to 1.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 and 60 μg/mL of Cefaclor (for UV analysis) and Twelve different concentrations, corresponding to 0.1, 0.5, 1.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0 and 80.0 μg/mL of Cefaclor (for HPLC analysis). Calibration curves with concentration versus peak area or absorbance were plotted for each method and the obtained data were subjected to regression analysis using the least squares method.


The intra-day precision was evaluated by analyzing six samples (n=6), at the test concentration of 10 μg/mL, using the UV and the HPLC methods. Cefaclor monohydrate contents and the relative standard deviations (RSD) were calculated.


Cefaclor monohydrate reference standard was accurately weighed and added, at three different concentrations. At each concentration, samples were prepared in triplicate and the recovery percentage was determined by UV and HPLC methods.


The robustness of the method was determined by the variation of the analyst and mobile phase flow rate. The flow rate was checked in 0.8 mL to 1.0 mL.

Analysis of cefaclor monohydrate powder for capsules and oral suspension

Samples of Medaclor, Oraclor were analyzed by the validated HPLC and UV methods. The sample solutions for the HPLC and UV analyses were prepared as described previously. The Cefaclor monohydrate contents were determined by using the two methods and the obtained results were statistically compared by using ANOVA test and Tukey’s multiple comparison test, applied at 0.05 significance level.

Results and Discussion

During the chromatographic method development, Ultra Pure Water showed to be a more adequate organic solvent than Methanol, regarding the Cefaclor monohydrate retention. A typical chromatogram obtained is as shown by Figure 2.


Figure 2: A typical chromatogram showing the separation of Cefaclor monohydrate (50.0 μg/mL) standard solution (A) and sample solution (B).

After the evaluation of the Cefaclor monohydrate UV spectrum in various solvents (Ultra Pure water, methanol, (Ultra Pure Water: Methanol) (50:50) v/v%, hydrochloric acid 0.1M, and sodium hydroxide 0.1 M). In the range of 200-400 nm (Figure 3), the wavelength of 264 nm was chosen due to the adequate molar absorptivity of Cefaclor monohydrate in this region and to minimize possible interference from other compounds and solvents in the samples.


Figure 3: Ultraviolet region spectrum in the Cefaclor monohydrate reference substance at 10 mg/ml in: Ultra Pure Water (A), Methanol (B), Ultra Pure Water: Methanol (50:50) v/v% (C), Hydrochloric acid 0.1 M (D) and sodium hydroxide 0.1 M (E).


A linear relationship was found between the Cefaclor monohydrate concentrations and the response of both HPLC and UV methods. The regression analysis data are presented in Table 1. High regression coefficient (r2) values were obtained (0.9995 and 0.9996, respectively). A random pattern of the regression residues was found and no significant deviation of linearity was detected in the assayed range.

Regression parameters HPLC UV
Regression coefficient (r2) 0.9995 0.9996
Slope ± standard error 0.199 ± 0.20 0.025 ± 0.0017
Intercept ± standard error 0.205 ± 0.11 0.006 ± 0.010
Relative standard error (%) 1.13 1.78
Concentration range (µg/mL) 0.1-80.0 1.0–60.0
Number of points 12 8

Table 1: Overview of the Linearity Data Obtained for Cefaclor monohydrate by the Chromatographic and Spectrophotometric Methods.

The precision data obtained for the evaluated methods are demonstrated in Table 2. Both methods presented RSD values lower than 2.0%, assuring a good precision.

Validation  parameters HPLC UV
Intra-day precision, n=6 (RSD%) 1.13 1.78
Accuracy, n=9 (mean recovery, %) (10 µg/mL) 100.1 100.82

Table 2: Validation Paramaters of the Evaluated Methods for Cefaclor monohydrate Determination.

Accuracy (Table 2) was investigated by means of a standard addition experiment. Both chromatographic and spectrophotometric methods exhibited mean recoveries (n=9) close to 100% demonstrating an adequate accuracy.

The difference in the retention time, the peak area and the analyst (for a given Cefaclor monohydrate concentration) caused by the aforementioned minor alterations were insignificant (Table 2).

Analysis of capsules and oral suspensions cefaclor monohydrate

The validated chromatographic and spectrophotometric methods were applied to the analysis of Cefaclor monohydrate in Medaclor, Oraclor (Table 3). ANOVA test revealed a statistically significant difference between the results obtained for injectable samples, from the distinct methods, at a confidence level of 0.05. Chromatographic analysis showed to be the most sensitive and selective method, and might be applied successfully for Cefaclor monohydrate trace analysis and quantitation in biological matrices. We cannot discharge, however, the analyses time and cost. The spectrophotometric method is clearly less expensive and requires shorter analysis time, besides the ease of handling and lower residues generation.

Analyst Area Mean ± SEM RSD (%)
1 691545
688479 ± 0.27 0.72
2 691563
634243 ± 1.25 3.31

Table 3: Robustness of the HPLC Method for Cefaclor monohydrate by Varying the Analyst.

Since the use of Cefaclor monohydrate as a potent antimicrobial drug is widespread, the development and validation of simple and reliable methods are essential to assure the quality of the raw materials and pharmaceutical formulations marketed nowadays. A simple method to identify and precisely quantify these drugs may be an important tool to avoid treatment inefficacy and development of resistance due to the exposition to sub therapeutic doses [35].


HPLC and UV spectrophotometry were found to be adequate methods to quantify Cefaclor monohydrate in Capsules and Oral suspensions solutions; the chromatographic and spectrophotometric methods presented the most reliable results. Since these methods are fast and simple, they may be successfully applied to quality control analyses, with the aim of quantifying and identifying Cefaclor monohydrate in pharmaceutical products.


Author thank Medico Labs, Homs, Syria for providing Cefaclor reference substance. This work was supported by Faculty of Science, Al-Baath University, Homs, Syria.


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