Spectrophotometric Determination of Ceftiofur Hydrochloride Using N-Bromosuccinimide and p-Dimethylaminobenzaldehyde

Simple, accurate and reproducible UV-Visible spectrophotometric methods were established for the assay of ceftiofur based on the formation of oxidation and condensation products. Method A involves the oxidation of the ceftiofur (CEFT) with N-bromosuccinimide (NBS) and determination of the unconsumed NBS with p-N-methylaminophenol (PMAP)-sulfanilamide (SA) (PMAP-SA) reagent. Method B includes the determination of unreacted NBS using a known excess of celestine blue (CB) and measuring the remaining dye. Condensation of p-dimethylaminobenzaldehyde (PDAB) with the drug was the basis of method C. Determination of CEFT in bulk form and in pharmaceutical formulations was also incorporated.

Ceftiofur(CEFT) has worldwide approvals for respiratory disease in swine, ruminants and horses and has also been approved for foot rot and metritis infections in cattle.A very few physicochemical methods have been reported in the literature for the assay of CEFT in biological fluids and pharmaceutical formulations.Most of them are based on spectrophotometric methods 2,3 , HPLC [4][5][6][7][8] , GC 9,10 , fluorimetry [11][12][13] , LC-MS 14 , GC-MS [15][16][17] , TLC 18 and Mass 19 .The analytically useful functional groups in CEFT includes 2-amino-4-thiazoyl, β-lactam, carboxyl and double bond in dihydrothiazine have not been fully exploited for designing suitable spectrophotometric methods and so still offer a scope to develop more visible spectrophotometric methods with better sensitivity, selectivity, precision and accuracy.A reported UV-Visible spectrophotometric method has been adopted for the determination of CEFT in pharmaceutical formulations (Tablets) and used as reference method to compare the results obtained by the proposed methods.

Experimental
An Elico UV-Visible digital spectrophotometer with 1cm matched quartz cells were used for the spectral and absorbance measurements.An Elico LI-120 digital pH meter was used for pH measurements.

Recommended procedures Method A
Aliquots of standard CEFT solution (1.0-5.0 mL, 200 µg mL -1 ) were transferred into a series of 25 mL calibrated tubes.Then 0.5 mL (8.75x10 -1 M) of acetic acid and 2 mL (4.94x10 -3 M) of NBS solutions were added and kept aside for 15 min at room temperature.Then 1.5 mL (8.71x10 -3 M) of PMAP solution was added.After 2 min 2.0 mL (1.16x10 -2 M) of SA solution was added.The volume was made up to the mark with distilled water as blank.The absorbance was measured after 10 min at 520 nm against distilled water.A blank experiment was also carried out without the drug.The decrease in the absorbance and in turn the drug concentration was obtained by subtracting the absorbance of the test solution from the blank.The amount of CEFT was computed from its calibration graph Figure 1.

Method B
Aliquots of standard CEFT solution (0.5-3.0 mL, 20 µg mL -1 ) were transferred into a series of 25 mL calibrated tubes.Then 1.25 mL (5.0 M) of HCl and 2.5 mL (5.618x10 -4 M) of NBS were added.The volume was brought to15 mL with distilled water.After 10 min, 10 mL (5.50x10 -4 M) of CB solution was added and mixed thoroughly.The absorbance was measured after 5 min at Test vs Reagent blank Reagent Blank vs. Distilled water Spectrophotometric Determination of Ceftiofur Hydrochloride 765 540 nm.The blank (omitting drug) and dye (omitting drug and oxidant) solutions were prepared in a similar manner and their absorbance was measured against distilled water.The decrease in absorbance corresponding to consumed NBS and in turn the drug concentration was obtained by subtracting the decrease in absorbance of the test solution (dye-test) from that of the blank solution (dye-blank).The amount of CEFT was computed from its calibration graph Figure 2.

Method C
To each of 10 mL calibrated tubes, aliquots (0.5-2.5 mL, 400 µg/mL) of methanolic standard drug solution, 2.0 mL of PDAB and 3.0 mL of conc H 2 SO 4 were added successively and the total volume in each flask was brought to 9 mL by adding methanol and placed in hot water bath for 25 min.Then the flasks were cooled and made up to the mark with methanol and the absorbance was measured after 5 min at 540 nm against a reagent blank prepared in a similar way.The concentration of the drug sample was computed from Beer-Lambert's plot Figure 3.

Chemical reactions of methods -Method A
The oxidant (NBS) reacts with CEFT and forms an oxidation product (Scheme 2).The unreacted NBS then reacts with metol (PMAP as sulphate) giving rise to p-N-methylbenzoquinone monoamine (PMBQMI) which in turn forms charge transfer complex with sulfanilamide (SA).

Method B
As in method A, in this method NBS reacts with CEFT and forms an oxidation product (Scheme 3).The unreacted NBS then quantitatively decolorizes celestine blue, by disrupting the chromophores and auxochromes present in celestine blue.

Method C
The NH 2 group in thiazolyl moiety of CEFT reacts with PDAB to form a colored species, azomethine.

Validation of methods
The optimum conditions for the color development of methods A, B and C were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species.
The optical characteristics such as Beers law limits, molar absoptivity and Sandells sensitivity for the methods (A-C) are given Table 1.The precision of the method to the drug was found by measuring the absorbance of 6 separate samples containing known amounts of drug and the results obtained are incorporated in Table 1.Regression analysis using the method of least squares was made to evaluate the slope (b), intercept (a) and correlation coefficient (r) and standard error of estimation (Se) for each system.The accuracy of the methods was ascertained by comparing the results by proposed and reference methods (UV) statistically by the t-and F-tests Table 2.The comparison shows that there is no significant difference between the results of studied methods and those of the reference ones.The similarity of the results is obvious evidence that during the application of these methods the excipients present in pharmaceutical formulations do not interfere in the assay of proposed methods.As an additional check of accuracy of the proposed methods, recovery experiments were carried out.The recovery of the added amounts of standard drug was studied at 3 different levels.Each level was repeated 6 times.From the amount of drug found, the percentage recovery was calculated from the amount of drug found.
The high λ max values of all the proposed methods have a decisive advantage since the interference from the associated ingredients should be generally less at higher wavelengths than at lower wavelengths.Thus the proposed visible spectrophotometric methods are simple and sensitive with reasonable precision, accuracy and constitute better alternatives to the existing ones to the routine determination of CEFT in bulk forms and pharmaceutical formulations.

Conclusions
The proposed methods exploit the various functional groups in CEFT molecule.The decreasing order of sensitivity (∈ max ) among the proposed methods is Method B > Method A> Method C, respectively.The concomitants which do not contain the functional groups chosen in the present investigation do not interfere in the color development by the proposed methods.Thus the proposed methods are simple, sensitive and selective with reasonable precision and accuracy and constitute better alternatives to the reported ones in the assay of CEFT in bulk drugs and pharmaceutical formulations.

Scheme 1 .
Scheme 1.Structure of ceftiofur.A reported UV-Visible spectrophotometric method has been adopted for the determination of CEFT in pharmaceutical formulations (Tablets) and used as reference method to compare the results obtained by the proposed methods.

Table 1 .
Optical and regression characteristics, precision and accuracy of the proposed methods for CEFT.

Table 2 .
Assay of CEFT in pharmaceutical formulations.