INTRODUCTION:

Cefaclor (Fig.1) is chemically, (6R,7R)-7-[[(2R)-(2-Amino-2-phenyl acetyl) amino]-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid [1]. It is a second generation, semi synthetic cephalosporin antibiotic. It is a broad spectrum, beta lactamase resistance antibiotic, effective against a wide range of gram positive and gramnegative respiratory tract pathogens.

Its mechanism of action based on binding to specific penicillin-binding proteins (PBPs) located inside the bacterial cell wall; it inhibits the third and last stage of bacterial cell wall synthesis.

Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins. It is possible that cefaclor interferes with an autolysin inhibitor. It is mainly used for the treatment of certain infections caused by bacteria such as pneumonia and ear, lung, skin, throat, and urinary tract infections^1,2.

Figure 1 Chemical Structure of Cefaclor

A thorough literature survey has revealed that HPLC method for Cefaclor with combination of other drugs and individually in dosage forms and in biological samples^3-15, spectrofluorimetric method¹⁶, spectrophotometric methods for cefaclor indivially in dosage forms, combined with other cephalosporins and UV spectroscopic method for other cephalosporin derivatives^17-25have been reported in various reputed journals. Costlier and volatile solvents were used as mobile phase solvent system and the time of analysis was more in some reported methods.

Some spectrophotometric methods are recently described in the literature for analysis of cephalosporins in raw material and finished product involve the use of organic solvents and reagents to produce colour chromogens, which do not contribute to the generation of this kind of waste by the chemical or industries. Therefore, it is necessary to reduce the use of organic and toxic solvents in order to reduce the impacts of their activities on the environment. So, the main objective of this study was, therefore, to develop a simple, selective, precise, less time consuming, environmental safe and economical method with a wide linear range and good sensitivity for assay of Cefaclor in raw drug as well as in finished formulation.

MATERIAL AND METHODS:

Instruments and chemicals:

The present spectrophotometric study was carried out on a Systronics 2201, UV-Visible double-beam spectrophotometer with matched 1 cm path-length quartz cells. Absorption spectra were recorded on a medium scan speed, setting slit width to be 1 nm. Cefaclor was received as a gift sample from Orchid Chemicals and Pharmaceutical Ltd, Chennai, India. The other chemicals like sodium hydroxide, hydrochloric acid, and hydrogen peroxide used were of Merck – AR grade and double distilled water was used throughout the experiment.

Preparation of standard stock solution:

A stock solution was prepared by dissolving 100 mg of Cefaclor pure drug in 100 mL of distilled water to get standard stock solution (1 mg/mL).

Construction of the calibration curve:

Aliquot volumes containing the drug were quantitatively transferred from standard stock solution, serially diluted to a set of 10 mL volumetric flasks, and made up to the mark with distilled water to obtain final concentrations of 5-50 μg/mL of Cefaclor. The absorbance was measured at 262.4 nm and standard calibration curve was constructed by plotting the absorbance versus the concentration of the drug. Ultimately, the corresponding regression equation was also derived.

Validation of the proposed method:

The validation of the method was tested with respect to linearity, specificity, accuracy and precision according to ICH Q2B recommendations.

Accuracy (Percentage Recovery):

Accuracy and precision of the method were evaluated with the help of percent recovery, standard deviation (SD) and percent relative standard deviation (RSD) by using standard addition method. The accuracy was evaluated by applying the proposed method to analyse the formulation with known amounts of drug²⁶. Three levels of standard drug (10%, 20% and 30%) were spiked individually with the formulation and analysed in six replicates during same day.

Precision:

Precision of the method was studied as repeatability, intra-day and inter-day precision in six replicates during same day (Intra-day precision or repeatability) and six consecutive days (Inter day precision or inter mediate) using 20 mg/mL concentration of standard drug solutions²⁷. The %RSD values were found to be less than 2% for both intra-day and inter-day analysis which indicating high precision of the method. The results were shown on Table 3.

Linearity:

The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range²⁸.A linear correlation was found between absorbance at l max and various concentration of Cefaclor. The linearity curve was obeyed Beer’s law in the range from 5-50 mg/mL and it was described by regression equation (y=mx+c) and correlation coefficient (r²) which were displayed on the graph. Molar absorptivity, Sandell sensitivity, standard error on slope, confidence limit of slope (95%), standard error on intercept, confidence limit of intercept (95%), LOD and LOQ were calculated.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

For the determination of LOD and LOQ, the method is based on residual standard deviation of regression line and slope. To determine LOD & LOQ the specific calibration curve was studied using the sample containing analyte in the range of detection limit and quantitation limit using the formula 3.3 σ/S and 10σ /S²⁹.

Forced degradation study:

Forced degradation studies were performed to demonstrate the optimized method is stability indicating^30,31. About 2 mL aliquot of standard stock solution of Cefaclor (1 mg/mL) was taken in four replicate in a volumetric flask (100 mL) and mixed with 10 mL of 0.1N HCl (acid hydrolysis) or 0.1N NaOH (alkaline hydrolysis) or 5% H₂O₂ (oxidative degradation) and set aside for 1 h at room temperature. Solution was diluted up to mark with distilled water. For photolytic degradation, a solution of drug (20 µg/mL) was prepared as per the procedure under construction of the calibration graph and was exposed to UV radiation of wavelength 254 nm and of 1.4 flux intensity for 24 h in a UV chamber. For thermal degradation solid drug of Cefaclor was kept in an oven at 100°C for 24 h. After cooling to room temperature, 20 µg/mL concentrated drug solution was prepared as per above mentioned method. Finally, absorbance of all the above prepared solutions which were kept under various stress conditions such as acid and alkaline hydrolysis, oxidative degradation, photolytic degradation and thermal degradation were measured at 262.4 nm against respective solvent as blank in each case.

Application of developed method to analyse drug formulation:

For the estimation of Cefaclor from various brands of tablet formulation (procuring from market), a portion of tablet powder equivalent to 100 mg of the drug from each brand were accurately weighed and transferred into 100 mL volumetric flask. The drug was dissolved by adding 70 mL of distilled water and sonicated for 15 min. The final volume was made with distilled water and filtered. Aliquot containing suitable concentration (20 µg/mL) of Cefaclor was analyzed as described under construction of the calibration graph. The nominal content of the drug in tablet formulation for each brand was determined using the corresponding regression equation and results of %RSD of drug content were statistically compared with reported method^32,33.

RESULTS AND DISCUSSION:

As shown in Fig 2(a) and (b), the absorption spectrum of Cefaclor in distilled water shows a maximum absorption (λ max) at 262.4 nm and at this wavelength, distilled water didn’t show any significant absorbance. Therefore further analysis was carried out at 262.4 nm.

Least square regression equation of Cefaclor in aqueous medium has shown the R²value very closer to 1 indicated high degree of correlation between two variables, such as absorbance and concentration (Fig 3). Beer’s law obeyed over the range of 5-50 mg/mL, high values of molar absorptivity and low values of sandell sensitivity and LOD revealed that proposed method is highly sensitive. LOD and LOQ values for the drug were found and all the parameters of standard curve were displayed in Table 1.

Figure 2(a): Spectrum for 20mcg/mL solution of Cefaclor

Figure 2(b): Overlay spectrum for Cefaclor (λ max at 262.4 nm)

Table 1: Calibration Curve Data

Parameters	Values
l max(nm)	262.4
Linearity Range (µg/mL)	5-50
LOD (µg/mL)	1.0917
LOQ (µg/mL)	3.3075
Molar absorptivity (L mol^-1cm^-1)	0.817 × 10³
Sandell’s sensitivity (mg/cm²/0.001absorbance unit)	0.02792
Slope	0.02175
Standard Error on Slope	0.0002450
Confidence Limit of Slope (95 %)	0.02120 – 0.02231
Intercept	0.008545
Standard Error on Intercept	0.007247
Confidence Limit of Intercept (95 %)	0.007847 – 0.02494
Correlation Co-efficient	0.9989
Standard deviation of Residuals	0.01285

Fig 3: Calibration curve for Cefaclor

Table 2: Accuracy (% Recovery) Study Data

S. No	Level of Standard drug added	% Recovery*	SD*	% RSD	S. E	95 % CI
1	10%	100.83	0.6251	0.61	0.3609	99.27-102.38
2	20%	99.90	0.6133	0.61	0.3541	98.38-101.43
3	30%	101.45	0.5205	0.51	0.3005	100.16-102.75

*(n=3), % RSD = Percentage Relative Standard Deviation, S.E = Standard Error, 95 % CI = 95 Percent Confidence Interval

Table 3: Inter-day and Intra-day Precision Study Data

S. No	Repeatability				Intermediate Precision
S. No	% content*	% RSD*	S. E	95 % CI	% content*	%RSD*	S. E	95 % CI
1	100.58	0.35	0.2088	99.68-101.48	99.88	0.57	0.3292	98.46-101.30
2	98.59	0.80	0.4601	96.61-100.57	99.14	0.43	0.2485	98.07-100.21
3	99.07	0.69	0.3969	97.36-100.78	99.50	0.95	0.5490	97.18-101.91
4	99.56	0.23	0.1330	98.99-100.14	99.62	0.36	0.2107	98.72-100.53
5	98.25	0.41	0.2359	97.23-99.26	99.64	0.55	0.3172	98.27-101.01
6	88.10	0.77	0.3921	86.41-89.78	98.70	0.73	0.4216	96.88-100.51

*(n=3), % RSD = Percentage Relative Standard Deviation, S.E = Standard Error, 95 % CI = 95 Percent Confidence Interval

Accuracy of the method was ascertained by investigating the mean % recovery between measured actual concentration and taken concentration for Cefaclor at three concentration levels covering the specified range. The values of % recovery very close to 100% demonstrated high accuracy of the proposed method (Table 2). To check precision of the proposed method, drug was analysed six times within a day (intra-day precision) and in six consecutive days (inter-day precision) and %RSD values were ≤ 0.80 (intra-day) and ≤ 0.95 (inter-day) indicating high precision (Table 3) of developed method.

Commercially available solid dosage forms were analysed by the above proposed method for its percentage drug content. The performance of the said method was judged by comparison with reference method statistically. No significant difference was found by applying t- and F-tests at 95% confidence level indicating good accuracy and precision (Table 4). Calculated t-values and F-values did not exceed tabulated values of 2.776 (t) and 6.39 (F) at 95 % confidence level and for four degrees of freedom (Table 4) which indicating close similarity between proposed and reported method.

Table 4: Statistical comparison of proposed method with reference method

Brand name	% RSD for % content*		Statistical Data
Brand name	Proposed method	Reference method	Variance-ratio F-test	Student’s t-test
Distaclor (125 mg)	0.4856	0.5447	1.249	0.7850
Vercef (250 mg)	0.5815	0.5915	1.039	0.4753
Halocef (250 mg)	0.4852	0.4784	1.008	2.598

(*n=3)

The stability study of the analyte was performed by forced degradation studies. Cefaclor was subjected to various stress conditions like acid, alkaline, hydrogen peroxide induced degradation, thermal and photolytic condition. Analysis was performed by measuring absorbance of Cefaclor after subjecting to stressed conditions at λ max of pure drug. Percentage degradation was calculated by the formula, % degradation = (Expected concentration-Actual concentration)/Expected concentration × 100 and percentage recovery also were calculated for each case (Table 5). Results revealed no specific change in absorbance of drug solution kept under UV stressed, refrigeration and room temperature, so its percentage recovery is closer to 100 % which indicates the drug stability. The analyte showed slight degradation with acid hydrolysis and oxidative degradation condition. But, there was significant change in absorbance after alkaline treatment, confirming Cefaclor is susceptible to alkaline hydrolysis (% degradation of 26%).

Table 5: Results for stability of drug under forced degradation study

Parameters Studied	Conc. Taken (mg/mL)	Conc. Found (mg/mL)	% Degradation	% Recovery
Acid hydrolysis	20	18.5	7.5%	92.5%
Alkaline hydrolysis	20	14.8	26.0%	74.5%
Oxidative degradation	20	18.1	9.5%	90.5%
UV degradation	20	19.1	4.5%	95.5%
At refrigeration	20	19.6	2.0%	98.0%
At room temperature (after 6 hr)	20	19.0	5.0%	95.5%

CONCLUSION:

The proposed method has distinct advantages over other exiting method regarding less cost, time saving and also the method requires only the wavelength scan. Furthermore, the results of the present work revealed that it is simple, sensitive with enough precision and accuracy. So method provides rapid analysis for determination of percentage purity of drug available from various sources without any tedious procedure and also used in analysis of stability study samples.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 07.02.2019 Accepted on 15.04.2019

Asian J. Pharm. Res. 2019; 9(2): 75-79.

DOI: 10.5958/2231-5691.2019.00012.1