Microsoft Word - 33-42   33  Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020       Spectrophotometric Method for the Estimation of Ceftriaxone in Pure Form and Pharmaceuticals Abstract 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 2,5- dimethylphenol (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. Keywords: Ceftriaxone; Azo dye; Coupling; Spectrophotometric; 2,5-dimethylphenol; 4-tertbutylphenol. 1. 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]. O N S N N H O N OSHH N S H2N N N O O O OH Scheme 1. Structure of Ceftriaxone Sodium. Ibn Al Haitham Journal for Pure and Applied Science Journal homepage: http://jih.uobaghdad.edu.iq/index.php/j/index Doi: 10.30526/33.2.2441 Aya W. Ahmed  Salam A.H. Al-Ameri Department of Chemistry, College of Science, University of Mustansiriyah Ayawaleed47@yahoo.com Article history: Received 17 June 2019, Accepted 6 August 2019, Published in April 2020.   34 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020 Several analytical methods have been reported for the estimation of CFT in pure and pharmaceuticals such as spectrophotometric [5-8]. Spectrofluorimetry [9]. Infrared [10]. HPLC – UV [11-13]. Chromatography [14]. And HPLC- MS [15]. As well as in biological samples HPLC-UV [16- 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. Materials and Methods 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 ᵒC; 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 ᵒC; 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.   3. 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.     35 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020   Figure 1. Initial UV-Vis spectra for CFT azo dye with (A) 2,5-DMP and (B) 4-TBP reagents. 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].     Figure 2. Effect of different HCl concentration (volume) on the CFT azo dye formation via 2,5-DMP and 4-TBP reagents. 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]. 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 2.5 3 Volume of acid mL 4‐ TBP 2 ,5‐DMP   36 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020   Figure 3. Effect of different NaNO2 concentration (volume) on the CFT azo dye formation via 2,5-DMP and 4-TBP reagents. 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.     Figure 4. Effect of different reaction time on the CFT azo dye formation via 2,5-DMP and 4-TBP reagents. 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.     0 0.05 0.1 0.15 0.2 0.25 0.3 0 0.5 1 1.5 2 2.5 Volume of NaNO 2 ( mL) 2 ,5‐DMP 4‐ TBP 0 0.1 0.2 0.3 0.4 0 5 10 15 20 25 30 35 Time (min.) 2 ,5‐DMP 4‐ TBP   37 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020   Figure 5. Effect of different alkaline types (A) and concentrations (B) on the CFT azo dye formation via 2,5-DMP and 4- TBP reagents.   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.     Figure 6. Effect of different 2,5-DMP and 4-TBP reagents on the CFT azo dye formation. 3-2. Calibration Curve and Analytical Merits Calibration curves was constructed using 2.0 x 10 -3 M 2,5-DMP and 2.5 x 10-3 M 4-TBP solutions with a concentration ranges of 3.0 - 50 µg. mL-1 and 10- 30 µg. mL-1 of CFT solution via 2,5-DMP and 4-TBP respectively versus the absorbance resulted, as in Figure 7. The absorbance spectra were measured at λmax of 520 nm and 500 nm for CFT with 2,5-DMP and 4-TBP respectively, as in Figure 8. All the analytical data such as limit of detection (LOD), limit of quantification (LOQ) and relative standard deviation (%RSD) were summarized in Table 1. [21].     38 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020      Figure 7. Calibration graph of 10 – 30 and 3.0 – 50 µg. mL-1 of CFT with 2.0 x 10 -3 M 2,5-DMP and 2.5 x 10-3 M 4-TBP respectively.     Figure 8. UV-Vis spectra of Calibration graph of 10 – 30 and 3.0 – 50 µg. mL-1 of CFT with 2.0 x 10 -3 M 2,5-DMP and 2.5 x 10-3 M 4-TBP respectively.                                   y =  0.0134x ‐ 0.0152 R² = 0.998 y =  0.0079x +  0.0057 R² = 0.9989 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 10 20 30 40 50 60 Conc. ( µg. mL) Linear (4‐TBP) Linear (2,5‐DMP)   39 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020 Table 1. Analytical data of the proposed diazotization-coupling methods. Parameters 2,5-DMP 4-TBP Color red Orange Λmax, nm 520 500 Linearity range, µg. mL-1 (3-50) (10-30) Molar absorptivity, ε, L. mol-1.cm-1 4.377×103 7.446×103 Slope, b 0.0079 0.0134 Intercept, a 0.0057 -0.0152 Sandal sensitivity, S 0.1267 0.0745 coefficient of determination, % R² 99.89 99.8 correlation coefficient, r) 0.9994 0.9990 limit of detection, LOD, µg. mL-1 0.4820 0.2840 Limit of quantification, LOQ, µg. mL-1 1.6080 0.9454 At a linear concentration range between 3.0 -50 and 10 -30 µg. mL-1, the results showed a limit of detection and a limit of quantification of 0.482, 0.284 and 1.608, 0.945 µg. mL-1 with 2,5-DMP and 4- TBP respectively with recovery of 100.89% and 103.37%, with molar absorptivity of 4.377×103 and 7.446×103 L. mol-1.cm-1 for 2,5-DMP and 4-TBP respectively. 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. Table 2. The accuracy and precision of the developed method with 2,5-DMP and 4-TBP reagents. * Average for 5 determinations. ** Average for 15 determinations. Type of reagent  Conc. Taken, µg. mL-1  Conc. Found µg. mL-1  Relative Error%  %RSD *Recovery, %  **Average Recovery  2,5-DMP  1.0  0.98 -2.0 0.46 98.0  100.89 3.0 3.08 2.7 102.7 5.0  5.1 2.0 102.0  4-TBP  1.0  1.09 9.0 0.37 109.0  103.378 3.0 3.1 3.3 103.3 5.0  4.89 -2.2 97.8    40 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020 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. Table 3. Assay of CFT in pharmaceutical preparations using 2,5-DMP reagent. pharmaceuticals of CFT Conc.Taken µg. mL-1 Conc.Found µg. mL-1 %RE *Recovery % **Average Recovery Malaysia  6.0  6.002 0.03 100.03   100.57 10 10.11 1.1 101.1 Jordan 6.0 6.1 1.67 101.67 101.99 10 10.23 2.3 102.3 United State Emirate  6.0  5.897 -1.72 98.28  99.14 10 9.999 -0.01 99.99 * Average for 5 determinations, ** Average for 10 determinations for two concentration.   Table 4. Assay of CFT in pharmaceutical preparations using 4-TBP reagent. pharmaceuticals of CFT  Conc.Taken µg. mL-1  Conc. Found µg. mL-1  %RE *Recovery  %  **Average Recovery  Malaysia 6.0 6.011 0.18 100.18  101.39 10 10.26 2.6 102.6 Jordan 6.0 6.32 5.3 105.3 104.7 10 10.41 4.1 104.1 United State Emirate  6.0 5.994 -0.1 99.9  100.35 10 10.08 0.8 100.8 * Average for 5 determinations. ** Average for 10 determinations for two concentration.   41 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 33 (2) 2020 4. 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