Spectrophotometric Method for the Estimation of Ceftriaxone in Pure Form and Pharmaceuticals

Ceftriaxone sodium were one of the widely antibacterial drugs used. Azo dye derivatization of diazonium salt that formed via the reaction between ceftriaxone with hydrochloric acid and sodium nitrite was developed for the on-research drug analysis then coupling with each one 2,5dimethylphenol (2,5-DMP) and 4-tertbutylphenol (4-TBP) respectively in the alkaline media. The developed diazonium coupling methods include an optimization study. The results show a limit of detection and limit of quantification 0.482, 0.284 μg/mL, and 1.607, 0.945 μg/mL using 2,5-DMP and 4-TBP reagents respectively. Moreover, the recovery % obtained was 100.89%, and 103.37% at linear concentration range 3.0 – 50, and 10 – 30 μg/mL, with molar absorptivity of 4.377×103, 7.446×103 L. mol-1. cm-1 using 2,5-DMP and 4-TBP reagents respectively. An acceptable accuracy represented by the relative standard deviation (RSD%) was achieved of a 0.46 and 0.37 for 2,5-DMP and 4-TBP respectively. The proposed method was successfully used for the determination of ceftriaxone sodium in pure and in pharmaceuticals.


Introduction
Cephalosporins consist of two -incorporated rings system of beta-lactam and dihydrothiazine [1]. Ceftriaxone sodium (CFT) is one of the third generation cephalosporins and one of the most widely used antibiotics to treat both bacterial infections Gram positive and Gram negative [2,3]. The molecular formula is C18H18N8O7S3, Scheme I. [4]. Several analytical methods have been reported for the estimation of CFT in pure and pharmaceuticals such as spectrophotometric [5][6][7][8]. Spectrofluorimetry [9]. Infrared [10]. HPLC -UV [11][12][13]. Chromatography [14]. And HPLC-MS [15]. As well as in biological samples HPLC-UV [16][17][18][19][20]. The idea of this work is to develop a simple, sensitive and effective method for the validation of the Ceftriaxone in pure and commercial preparation as a quality control purpose.

2-1. Chemicals and Equipment:
All chemicals used were of analytical reagent grade with high purity. Sodium nitrite, hydrochloric acid, potassium hydroxide, analytical standard of ceftriaxone sodium, analytical standard of 2,5-DMP and 4-TBP were purchased from Sigma-Aldrich, Al Qiffaf Scientific Company -Baghdad, Iraq. Double distilled water was used throughout the experiments for preparation of the reagents and samples.
For all absorbance detection, a double beam dual chopper, UV-Vis spectrophotometer, Varian, Cary 100, Australia was used pH S-3E Precision acidity Meter, Ray Magnetic Instrument Factory, Shanghai, China. Mettler AE 200 Electronic Balance, Germany and shaker.

2-2. Preparation of a Ceftriaxone Sodium Stock Solution
A 1000 µg. mL -1 of standard CFT was prepared by dissolving 0.1g in 100 mL of distilled water then prepared a diluted solution.

2-3. General Procedures: 2-3-1. Coupling the Ceftriaxone with the 2,5-DMP reagent:
In an ice bath at zero to 5 ᵒ C; in a 20 mL volumetric flasks take a 0.5 mL of 1000 µg. mL -1 of standard CFT solution then add 2.0 mL of 0.57M HCl and 0.5 mL of 0.14M NaNO2 which lead to forming a diazonium salt after leaving the solution for 10 minutes which coupling with 1.5 mL of 2.0 x 10 -3 M 2,5-DMP reagent in alkali media by using 1.5 mL of 0.45 M KOH. The Azo dye formed was diluted with distilled water to the mark. The colored product solution was measured at 520 nm.

2-3-2. Coupling the Ceftriaxone with the 4-TBP Reagent:
In an ice bath at zero to 5 ᵒ C; in a 20 mL, volumetric flasks a 0.5 mL of 1000 µg. mL -1 of standard CFT solution was taken then a1.5 mL of 0.57M HCl and 1.0 mL of 0.14 M NaNO2 was added that led to form a diazonium salt after leaving the solution for 10 minutes which coupling with 1.5 mL of 2.5 x 10 -3 M 4-TBP reagent in alkali media by using 2.0 mL of 0.45 M KOH. The Azo dye formed was diluted with distilled water to the mark. The product colored solution was measured at 500 nm.

Result and Discussion
Initial tests were performed via the spectrophotometric method for the estimating of CFT by using 2,5-DMP and 4-TBP reagents and a red color of the azo dye formed was illustrate, Figure 1 A, B.

3-1. Method Optimization
This spectrophotometric method for CFT estimation including diazonium coupling was optimized. The optimization parameters are; HCl and NaNO2 concentrations also the alkaline type and concentration.

3-1-1. Hydrochloric acid volume
The volume of hydrochloric acid that is important to the nitrous acid resulting with sodium nitrite which reacts with the amine group of ceftriaxone to form the diazonium salt.
The increase of acid volume was effect on the resulting azo dye absorbance. The optimum volume was 1.0 and 1.5 mL of 0.57 M HCl for 2,5-DMP and 4-TBP respectively, Figure 2. The intensity was reduced at higher or additional acid concentration which due to the incompetently converted of the primary amine [21].

3-1-2. Sodium Nitrate Volume
Sodium nitrate with HCl produced nitrous acid, which was responsible for the diazonium salt forming that was administered for the formation of azo dye. Increasing amounts of 0.14 M sodium nitrate were studied in the range of 0.25 -2.0 mL. The best of the volume of sodium nitrate were 0.5 and 1.0 mL and the product dyes measured at 500 nm and 520 nm for 4-TBP and 2,5-DMP reagents respectively as in Figure 3. The excess of the NaNO2 volume caused interferences contamination that led to reduce the absorbance [22].

3-1-3. Reaction Time
The reaction time was studied over a period of zero to 30 minutes to discover the ideal time for the diazonium salts and the developed azo dyes affect the absorbance intensity. The results illustrated that the 10 minutes was a sufficient time as in Figure 4.

3-1-4. Base type and Concentration
The alkaline media was so important to remove hydrogen atom of the phenolic compounds, which changed to very active species called phenoxide ion that coupling with diazonium salts to form azo dyes. A range of bases such as NaOH, KOH, NH4OH and Na2CO3 at the concentrations range of 0.23, 0.35, 0.45 and 0.62 M. Potassium hydroxide was chosen as a best with 2,5-DMP and 4-TBP respectively as in Figure 5-A.
Additionally, optimum volume of KOH was studied in a range of 0.5 -2.5mL which showed that the 1.5 and 2.0 mL with 2,5-DMP and 4-TBP respectively were the appropriate volumes which gave a good dye with higher absorbance as in Figure 5-B.

3-1-5. Volume of the Reagents 2,5-DMP and 4-TBP
Various volumes of 2.0 x 10 -3 M 2,5-DMP and 2.5 x 10 -3 M 4-TBP reagents were used to select the appropriate volume, which was combined with CFT diazonium salt to form a dark azo dye that gave a high intensity and higher absorbance. The results showed that the 1.5 mL of 2,5-DMP and 4-TBP is the best volume, as in Figure 6.

3-3. Accuracy and Precision
The precision and accuracy were calculated by the analysis a five replicates for three different CFT concentrations [21]. The precision was estimation by the determine of percent relative error %R.E whereas the accuracy was determination by calculating the percent relative standard deviation %RSD. An reasonable precision with acceptable accuracy was obtained which illustrated a 0.46 and 0.37 for 2,5-DMP and 4-TBP as in Table 2.

3-4. CFT: Reagent Stoichiometry
A continuous variation (Job) method [22]. Was applied for the estimation of CFT: reagent stoichiometry. The solutions were prepared at an equal concentration of 2.0 × 10 -3 M for CFT with 2.5-DMP and 2.5 × 10 -3 M for CFT and 4-TBP. This method was applied by increasing the volumes from 0.1 to 0.9 mL of CFT and reducing the volumes from 0.9 to 0.1 mL of reagents to a final volume of 1.0 ml. The results showed a conjugated ratio is of 1:1 between CFT with two reagents, Figure 9, 10. Figure 9. Continuous variation method for CFT: Reagent stoichometry with 2,5-DMP at 520nm and with 4-TBP at 500nm.

3-5. Application of the developed method for the CFT estimation in pharmaceuticals
This developed method was successfully used for the CFT determine in commercial pharmaceuticals for diverse products from Malaysia, Jordan and United Arab of Emirate at a concentration of 6.0 and 10 µg. mL -1 . The results were summarized in Tables 3, 4. * Average for 5 determinations, ** Average for 10 determinations for two concentration.

Conclusion
This study is successfully employed for the CFT determination in pharmaceuticals, which gave an increase in the analytical sensitivity, selectivity and precision. The procedure in spectral mode was simple, inexpensive and accurate.