Kinetic Spectrophotometric Determination of Certain Cephalosporins in Pharmaceutical Formulations

A simple, reliable, and sensitive kinetic spectrophotometric method was developed for determination of eight cephalosporin antibiotics, namely, Cefotaxime sodium, Cephapirin sodium, Cephradine dihydrate, Cephalexin monohydrate, Ceftazidime pentahydrate, Cefazoline sodium, Ceftriaxone sodium, and Cefuroxime sodium. The method depends on oxidation of each of studied drugs with alkaline potassium permanganate. The reaction is followed spectrophotometrically by measuring the rate of change of absorbance at 610 nm. The initial rate and fixed time (at 3 minutes) methods are utilized for construction of calibration graphs to determine the concentration of the studied drugs. The calibration graphs are linear in the concentration ranges 5–15 μg mL−1 and 5–25 μg mL−1 using the initial rate and fixed time methods, respectively. The results are validated statistically and checked through recovery studies. The method has been successfully applied for the determination of the studied cephalosporins in commercial dosage forms. Statistical comparisons of the results with the reference methods show the excellent agreement and indicate no significant difference in accuracy and precision.


Experimental
2.1. Apparatus. Spectronic Genesys 2PC. Ultraviolet/Visible spectrophotometer (Milton Roy Co, USA) with matched 1 cm quartz cell was used for all measurements connected to IBM computer loaded with winspec application software.

Materials and Reagents.
All the materials were of analytical reagent grade, and the solutions were prepared with double-distilled water. Samples of cephalosporin were generously supplied by their respective manufacturers and were used without further purification.
International Journal of Analytical Chemistry Potassium permanganate solution should be freshly prepared and its molarity was checked titrimetrically.

Preparation of Standard Solution.
Stock solution containing 1 mg mL −1 of each cephalosporins was prepared in double-distilled water, working standard solutions containing 100 μg mL −1 were prepared by suitable dilution of the stock solutions with double-distilled water. Dissolved in about 20 mL methanol, swirled, and sonicated for 10 minutes, the resultant mixture was filtered into round bottom flask for removal of any pharmaceutically "inert" ingredients (cellulose, disaccharides) that could be subject to oxidation by permanganate. The residue was washed thoroughly with about 5 mL methanol and the combined filtrate as well as washing solutions was subjected to evaporation under vacuum till dryness. The residue lifted was dissolved in about 20 mL distilled water and filtered into 100 mL volumetric flask. The filter paper was washed thoroughly with double distilled water, and then the combined filtrate as well as washing solutions was mixed well and completed to volume with the same solvent to obtain solution of 1.0 mg mL −1 . The final solution was diluted quantitatively with the same solvent to obtain working standard solution of 100.0 μg mL −1 , then the general procedure was followed.

Procedure for Capsules and Suspension.
The contents of 20 capsules were evacuated and well mixed. Then an accurately weighed amount equivalent to 100.0 mg evacuated capsules or dry powder suspension of each drug was transferred into a 100 mL beaker, and then the procedure was continued as described under tablets.

Procedure for Vials.
A 100.0 mg quantity of each vial was transferred into a 100 mL volumetric flask, dissolved, and completed to the mark with double-distilled water to obtain solution of 1.0 mg mL −1 . Further dilutions with double-distilled water were made to obtain sample solutions (100.0 μg mL −1 ), then the general procedure was followed.

Results and Discussion
Potassium permanganate as strong oxidizing agent has been used in oxidimetric analytical method for determination of many compounds [36][37][38][39]. During the course of the reaction, the valence of manganese changes. The heptavalent manganese ion changes to the green color (Mn VI), while in neutral and acidic medium, the permanganate is further reduced to colorless (Mn II). The behavior of permanganate was the basis for its uses in development of spectrophotometric method. The absorption spectrum of aqueous potassium permanganate solution in alkaline medium exhibited an absorption band at 530 nm. The additions of any of the studied drugs to this solution produce a new characteristic band at 610 nm ( Figure 1). This band is due to formation of manganate ion, which resulted from the oxidation of cephalosporin by potassium permanganate in alkaline medium. The intensity of the color increases with time; therefore a kinetically based method was developed for determination of cephalosporins in their pharmaceutical dosage formulations. The different variables that affect the formation of manganate ion were studied and optimized.

Effect of Potassium Permanganate Concentration.
The absorbance increases substantially with increasing the concentration of potassium permanganate ( Figure 2). Maximum absorbance was obtained when 2.5 mL of 6 × 10 −3 M of potassium permanganate was used. Thus, the adoption of 3 mL of potassium permanganate in the final solution proved to be adequate for the maximum concentration of cephalosporin used in determination process (the concentration of the final assay was 1.8 × 10 −3 M).

Effect of Sodium Hydroxide Concentration.
Maximum absorption was obtained when 1 mL of 0.75 M NaOH was used. Over this volume no change in absorbance could be detected. So 1.2 mL of 0.75 M of NaOH was used as an optimum value (Figure 3).

Effect of Temperature.
At room temperature the reaction rate increases substantially with time, although heating the solution was found to increase the rate of the reaction however but MnO 2 was precipitated, therefore room temperature was selected as the optimum temperature.

Stoichiometry and Reaction
Mechanism. The stoichiometric ratio between potassium permanganate and each of investigated cephalosporins was determined by Job's method [ 40,41] and was found to be 1 : 1 ( Figure 4). Cephalosporins were found to be susceptible for oxidation with alkaline potassium permanganate producing a green color peaking at 610 nm. Therefore, the reaction mechanism is proposed on the basis of the literature background (39)     of permanganate with fixed concentration of investigated cephalosporins. The plot of initial rate (ΔA/Δt) against initial absorbance was linear passing through origin indicating that the initial order of the reaction with respect to permanganate was 1. The order with respect to investigated cephalosporins was evaluated by measuring the rate of the reaction at several concentrations of cephalosporins at a fixed concentration of permanganate reagent. This was done by plotting the logarithm of initial rate of the reaction versus logarithm of molar concentration of investigated cephalosporins and was found to be 1. However under the optimized experimental conditions, the concentrations of cephalosporins were determined using relative excess amount of potassium permanganate and sodium hydroxide solutions. Therefore pseudo-zero-order conditions were obtained with respect to their concentrations.

Initial Rate Method.
The initial rate of the reaction would follow pseudo-first-order and were found to obey the following equation: where ν is the reaction rate, A is the absorbance, t is the measuring time, K is the pseudo-first-order rate constant, C is the molar concentration of cephalosporins, and n is the 6 International Journal of Analytical Chemistry    order of the reaction. The logarithmic form of the above equation is written as follows: Regression analysis using the method of least square was performed to evaluate the slopes, intercepts, and correlation coefficient. The analytical parameters and results of regression analysis are given in Table 2. The value of n (≈1) in the regression equation confirmed that the reaction of cephalosporins with the potassium permanganate was pseudo-first-order with respect to cephalosporins concentration. The limits of detection (LOD) were calculated and results obtained confirmed good sensitivity of the proposed method and consequently their capabilities to determine low amount of cephalosporins.

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International Journal of Analytical Chemistry

Fixed Time Method.
In this method, the absorbance of the reaction solution containing varying amount of cephalosporins was measured at preselected fixed time. Calibration plots of absorbance versus the concentration of cephalosporins at fixed time were established for each investigated cephalosporins. The regression equation, correlation coefficients, and detection limits are given in Table 3. The lowest detection limit was obtained at fixed time of 15 minutes. However the fixed time of 3 minutes showed a wider concentration range for quantification.
According to international conference of harmonization (ICH) guideline for validation of analytical procedures [42], the detection limit is not required to be part of validation procedure for assay. Therefore on the basis of wider concentration range and less time of analysis, the fixed time of 3 minutes was recommended for determination. [42] is established by confirming that the analytical procedure provides a suitable degree of precision, accuracy, and linearity when applied to the sample containing amount of analyte within or at the extreme of the specified range of the analytical procedure [43,44]. In this work, concentrations ranging from 7.8 × 10 −6 M to 4.31 × 10 −5 M were studied for the investigated drugs in the initial rate method and concentration ranging from 5 to 25 μg mL −1 were studied for the investigated drugs in the fixed time method (at preselected fixed time of 3 minutes). The whole set of experiments were carried out through this range to ensure the validation of the proposed procedure. Linear calibration graphs were obtained for all the studied drugs by plotting the logarithm of initial rate of the reaction versus logarithm of molar concentration of analyte in the sample (in initial rate method) within the specified range ( Figure 7):

Validation of the Proposed Method. Concentration range
or by plotting the absorbance of the studied drugs versus the drug concentration (in fixed time method) within the specified range ( Figure 8). Linearity was studied for both initial rate and fixed time method indicated by the values of correlation coefficient (r) and determination coefficient (r 2 ) for both method (Tables 2  and 3).
Accuracy [44] was checked at three concentration levels within the specified range, six replicate measurements were recorded at each concentration levels. The results were recorded as percentage recovery ± standard deviation (Tables  4 and 5).
Precision [44] was checked at three concentration levels, eight replicate measurements were recorded at each concentration level. The results are summarized in (Table 6). The calculated relative standard deviation were all below 2.2% indicating excellent precision of the proposed procedure at both level of repeatability and intermediate precision.
Specificity and interference the proposed procedure was performed in visible region away from the UV-absorption region of investigated drugs (228-300 nm), and the interference by reducing sugars in tablets was eliminated by extraction with methanol prior to analysis. However interference was observed from L-arginine formulated with cephradine in velosef vial. Therefore, the proposed procedure could not be used for analysis of cephradine in presence of L-arginine, except after separation by a suitable separation technique. Limit of detection (LOD) [42] was calculated based on standard deviation of response and the slope of calibration curve [43]. The limit of detection was expressed as [44] where σ is the standard deviation of intercept. S is the slope of calibration curve.
The results were summarized in (Tables 2 and 3) indicating good sensitivity of the proposed method. According to USP XXV validation guidelines [44], the calculated LOD values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated drug concentration by LOD equations were actually detected in these experiments. Limit of quantitation (LOQ) was calculated based on standard deviation of intercept and slope of calibration curve. In this method, the limit o quantitation is expressed as [44] The results were summarized in (Tables 2 and 3) indicating good sensitivity of the proposed method. According to USP XXV validation guidelines [45], the calculated LOQ values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated drug concentration by LOQ equations were actually quantitated in these experiments.

Application to Pharmaceutical Dosage
Forms. The initial rate and fixed time methods of the proposed kinetic spectrophotometric method for determination of investigated cephalosporins have been tested on commercial pharmaceutical dosage forms. The concentration of investigated cephalosporins was computed from its responding regression equations. The results of proposed method (initial rate and fixed time) were statistically compared with those of reported methods [3][4][5], in respect to accuracy and precision. The obtained mean recovery values were 99.2-100.67 ± 0.6226-1.69% (Table 7), which ensures that there is no interference of other additives present in the studied formulations.
In the t-and F-tests, no significant differences were found between the calculated and theoretical values of both the proposed and the reported methods at 95% confidence level. This indicates good precision and accuracy in the analysis of investigated cephalosporins in pharmaceutical dosage forms.

Conclusion
The initial rate and fixed time methods can be easily applied for determination of investigated cephalosporins in pure and dosage forms that do not require elaborate treatment and tedious extraction of chromophore produced. The proposed method (initial rate or fixed time) is sensitive enough to enable determination of lower amounts of drug, these advantages encourage the application of proposed method in routine quality control of investigated cephalosporins in industrial laboratories. Finally our method provides advantages of improving selectivity, avoiding interference of colored and/or turbidity background of samples because it measures the increase in absorbencies with time against blank treated similarly.