Validated Spectrofluorimetric Method For The Determination of Cefoxitin Sodium in Its Pure Form and Powder for Injection via Derivatization with 4-Chloro-7-nitrobenzo-2-oxa-1 , 3-diazole ( NBD-Cl )

Objectives: An accurate and precise spectrofluorimetric methodwas developed and validated for the determination of βlactam antibiotic named; cefoxitin sodiumin its pure form and powder for injection. Methods: Based on nucleophilic substitution reaction of target drug with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to form a highly fluorescent fluorophore measured at 540 nm after excitation at 460 nm. Results: Under optimum condition, the proposed method obeys Beer’s law in range (0.5-7 μg mL) and the reaction mechanisms were presented. Conclusion: The method was validated according to ICH guideline for accuracy, precision and was successfully applied for the determination of the drug in its pure form and powder for injection. The obtained results were statistically compared with those of the reported method and found to be in good agreement.


INTRODUCTION
Cefoxitin sodium is a semisynthetic cephamycin antibiotics classified as a second generation Cephalosporin, chemically named Sodium 3carbamoyloxymethyl-7-methoxy-7-[2-(2-thienyl) acetamido]-3-cephem-4-carboxylate 1 .The most novel of chemical feature of cefoxitin sodium is the possession of an alpha-oriented methoxyl group in place of the normal H atom at C-7. Figure (1), this increased steric bulk conveys very significant stability against β-lactamases 2 .It is produced by Streptomyces lactamdurans and used for the treatment of infections caused by anaerobic and mixed aerobic anaerobic infections, such as pelvic inflammatory disease and lung abscess 3,4 .Literature survey reveals that HPLC methods were developed for the determination of cefoxitin sodium in pharmaceutical formulations 5 and in biological fluids [6][7][8][9] , TLC method 10 , LC-MS/MS 11 and a flow injection chemiluminescent method was also reported 12 .Colorimetric methods were used for the determination of cefoxitin sodium in pharmaceutical formulations and in biological fluids [13][14][15] , first and second derivative UV spectroscopy 16,17 and a stability indicating method by spectrofluorimetric analysis 18 was also described for its analysis.Khalid et al, recently developed different spectrophotometric method for determination of cefoxitin sodium in the presence of its alkali-induced degradation product 19,20 .

Chemicals and reagents
Cefoxitin Sodium 98.8% was kindly supplied by Pharco B International Co., Cairo, Egypt.Lot no.12052036 Primafoxin ® 1gm vial labeled to contain 1gm of cefoxitin sodium per vial, Batch No. (109), the product of PharcoB international Co., Egypt, were purchased from local pharmacies.
Water used throughout the procedures was freshly double distilled.

Standard solutions
Stock solution (1mg mL -1 ) was prepared by dissolving 100 mg of cefoxitin sodium in 80 mL water, and the volume was then completed to 100 mL with water.The solution was found to be stable for at least two weeks when stored at 5°C in the dark 16 .
Working solution (0.1 mg mL -1 ) was obtained by dilution of the stock solution with water.

Linearity and construction of calibration curves
Aliquots from stock standard solution of cefoxitin sodium were accurately measured and transferred into a test tube set to prepare different concentration covering the linearity range (0.5-7 µg/mL), then 1mL (0.1% NBD-Cl) was added followed by 1.5 mL of (0.2M) NaHCO3.The reaction mixtures were allowed to proceed in thermostatically controlled water bath at 60 °C for 30 minutes, and then cooled to room temperature.After cooling, the reaction mixture was acidified by adding 1mL of 1M HCl, and completed to volume with water.The relative fluorescence intensity was measured at λem.= 540 nm after excitation at λex.= 460 nm.

Application to pharmaceutical preparation
An accurately weighed quantity of well mixed powder from three vials of Primafoxin ® 1gm equivalent to 100 mg of cefoxitin sodium was transferred into a 100-mL volumetric flask.The powder was dissolved by shaking with 50 mL water.Then volume was adjusted with water to obtained stock solution labeled to contain (1mg mL -1 ) cefoxitin sodium, which was further diluted to contain (0.1 mg mL -1 ).Cefoxitin sodium then analyzed by the corresponding regression equation for the proposed method.

RESULTS AND DISCUSSION
Cefoxitin sodium doesn't has a native fluorescence, so its derivatization with fluorigenic reagent was necessary for spectrofluorimetric determination.

Effect of reagent volume
The influence of NBD-Cl concentration was studied using different volumes of 0.1% (w/v) NBD-Cl solution ranging from (0.25-2 mL), it was found that 1mL of 0.1% (w/v) NBD-Cl produce the highest FI and beyond which the FI decreased.

Effect of NaHCO3 concentration
The reaction of cefoxitin sodium with NBD-Cl should be carried out in alkaline medium (pH ~8.3) in order to generate the nucleophile from cefoxitin sodium.The influence of NaHCO3was studied using different volumes of 0.2 M NaHCO3 solution ranging from (0.25-2.5 mL), it was found that 1.5 mL (0.2 M) NaHCO3produces the highest FI and above and beyond which the FI decreased,

Effect of temperature
The influence of temperature the reaction was carried out at different temperatures (2570 C), it was found that the reaction was dependent on the temperature and the FI increased as the temperature increased and the maximum FI was obtained at 60 C (Figure 6).This result was coincident with the result reported previously by H. W. Darwish et al 25 .

Effect of reaction time
In order to determine the time required for completion of the reaction, the reaction was carried out at different reaction time interval (5-40 min.).The results indicated that the optimum time was 30 min (Figure 7).

Effect of HCl concentration
Addition of HCl 21 to the reaction mixture before measurement of the FI was necessary for remarkably decreasing the background fluorescence (duo to the hydrolysis product of NBD-Cl to the corresponding hydroxyl derivative namely, 7-hydroxy-4 nitrobenzoxadiazole (NBD-OH) 32 .The fluorescence of NBD-OH was found to be quenched in strong acidic medium (pH ≤ 1), where the reaction product was not affected, the reaction was carried out using different volumes of 1M HCl ranging from (0.25-2mL).The optimum concentration of HCl required for acidification was found to be 1 mL of 1M HCl (Figure 8).

Effect of diluting solvent
To select the most appropriate solvent for diluting the reaction solution, different solvents involve: water, methanol, ethanol, propanol, acetone, acetonitrile were studied.The highest FI was obtained upon using water or methanol but water was used as a diluting solvent because it is environmental friendly (Figure 9).

Stability of fluorescent fluorophore
The effect of time on the stability of the Fluorescent cefoxitin-NBD fluorophore was studied by measuring the FI at different time intervals.It was found that the FI values remain constant for at least 24 hour at room temperature.
The optimum variables affecting the reaction of cefoxitin sodium with NBD-Cl were summarized in Table 1.24 hr.at room temp.

Stoichiometry and mechanism of the reaction 36-3
The stoichiometry of the reaction between cefoxitin sodium and NBD-Cl was investigated by the limiting logarithmic method 34 , where, two sets were prepared, one of which containing variable concentration of (NBD-Cl) ranging from (6×10 -3 -3×10 -2 M) while, constant drug concentration containing (3×10 -3 M), the second set contained variable concentration of the drug ranging from(1×10 -4 -1×10 -2 M), while constant concentration of (NBD-Cl) containing (1×10 -3 M), Figure 10, A plot of log FI against log concentration of NBD-Cl and cefoxitin sodium, two straight lines were obtained.The slopes were 0.8245 and 0.9368 indicating the 1:1 ratio for the reaction (owing to the molar reactivity of the reaction is 0.8245/0.9368).This ratio means one molecule of the drug reacts with one molecule of NBD-Cl.Methods validation 39 The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines in terms of linearity, range, LOD, LOQ, accuracy and precision.

Linearity and range
The method obeys the Beer's law in the studied range of 0.5-7 µg mL -1 , Table 2, illustrated the regression parameters of the calibration curve and correlation coefficient of the drug analyzed.

Limits of detection and quantitation
LOD was found to be 0.048μg mL -1 , while LOQ was found to be 0.160μg mL -1 , as shown in Table 2.

Accuracy and precision
Accuracy of the proposed procedure (%R) was found to be 99.84.Intra-day precision (repeatability day precision) as % RSD was found to be 1.551, while interday precision (intermediate precision) was found to be 1.036, table (2).Good %R confirms excellent accuracy.Recovery study by standard addition technique: Validity of the proposed method was performed by adopting standard addition technique with mean recovery of added ± SD of 100.78 ± 0.610 %. Results are presented in Table 3

CONCLUSION
Because cefoxitin sodium has no native fluorescence, this work introduced an accurate spectrofluorimetric method for the determination of cefoxitin sodium in its pure form and powder for injection based on nucleophilic substitution reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to form a highly fluorescent yellow fluorophore.The proposed method is suitable for the routine analysis of cefoxitin sodium in quality control and clinical laboratories.

Figure 3 .
Figure 3. Excitation and emission spectra of the reaction product of cefoxitin sodium (7 µg mL -1 ) with 0.1% NBD-Cl.Optimization of experimental conditionsDifferent experimental parameters affecting the fluorescence intensity were studied and optimized.

Figure 4 .
Figure 4. Effect of volume of 0.1% NBD-Cl on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 5 . 5 .
Effect of volume of 0.2 M NaHCO3 on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 6 .
Figure 6.Effect of heating temperature (°C) on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 7 .
Figure 7. Effect of heating time at 60 o C on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 8 .
Figure 8.Effect of volume of 1M HCl on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 9 .
Figure 9.Effect of diluting solvent on the on the fluorescence intensity of cefoxitin-NBD fluorophore at λem 540 nm.

Figure 10 .
Figure 10.Stoichiometry of the derivatization reaction between cefoxitin sodium and NBD-Cl using limiting logarithmic method.

Figure 11 .
Figure 11.The Proposed reaction pathway between cefoxitin-sodium and NBD-Cl.

Table 2 . Linearity studies and regression equation of the proposed spectrofluorimetric method.
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