Batch and flow injection spectrophotometric methods for determination of Ceftazidime in pharmaceutical formulations

: It is generally accepted that there are two spectrophotometric techniques for quantifying ceftazidime (CFT) in bulk medications and pharmaceutical formulations. The methods are described as simple, sensitive, selective, accurate and efficient techniques. The first method used an alkaline medium to convert ceftazidime to its diazonium salt, which is then combined with the 1-Naphthol (1-NPT) and 2-Naphthol (2-NPT) reagents. The azo dye that was produced brown and red in color with absorption intensities of ƛmax 585 and 545nm respectively. Beer's law was followed in terms of concentration ranging from (3-40) µg .ml -1 For (CFT-1-NPT) and (CFT-2-NPT), the detection limits were 1.0096 and 0.8017 µg.ml -1 , respectively, and the molar absorptivity was 0.7926×10 4 and 0.5466×10 4 L.mol -1 .cm -1 . The Flow Injection Analysis (FIA) method is used to estimate ceftazidime and in the second procedure record measurements using the UV-Visible approach. The Flow injection allows for exact drug estimation under ideal experimental conditions. The concentrations were in the range of (3-50) µg .ml -1 For (CFT-1-NPT) and (CFT-2-NPT), the detection limits were 0.8102, 1. 2809µg.ml -1 , and the molar absorptivity was 0.9565×10 4 ,0.7106×10 4 L.mol -1 .cm -1 , respectively. The proposed two methods for determination Ceftazidime in Pharmaceutical formulation were successfully applied, as these methods were characterized by simplicity, speed, accuracy, and low cost.


Introduction
Antibiotics are substances or compounds that kill or inhibit the growth of bacteria . 1 . And the antibiotics are antimicrobial substances used to treat infections caused by bacteria, fungus, and parasites. 2 . Ceftazidime is a third-generation semi-synthetic cephalosporin that inhibits Gram-negative and Gram-positive bacteria, including pseudomonas aeruginosa. 3 Chemically, Ceftazidime (CFT) is [7- (Fortaz,Tazicef) 4 is the trade name. • It is used to treat infections caused by Pseudomonas aeruginosa, which include infections of the bones and joints, cystic fibrosis (respiratory tract infections), and cystic fibrosis gastroenteritis (intestinal infections) 5,6 Many researches have been conducted to estimate the presence of this medication in pharmaceutical formulations such as : Two Oxidative Visible Spectrophotometric Methods 7 RP-UPLC Method 8 Poly Diphenylamine Partially Oxidized for Sensitive Electrochemical Applications 9 Using resorcinol and 2-methyl phenol, spectrophotometric detection of ceftazidime in pharmaceuticals 10 The utility of a fluorescamine-based technique for very sensitive spectrofluorimetric measurement of Ceftazidime and Vancomycin in pharmaceuticals and real human plasma was investigated using flow injection analysis (FIA). 11 Flow injection analysis is one of many analytical chemistry techniques that have been widely embraced and adopted in a variety of analytical fields. Diazotization and coupling processes have been widely used to determine a wide range of pharmaceuticals and pharmaceutical preparations, including the determination of the ceftazidime compound employing the reaction between the diazotized drug and the coupling reagent 4-tert-butylphenol 12 . Many papers have been written about diazotization reactions and their connection to the flow injection system [13][14][15][16][17] .The proliferation of these domains is owing to the ease with which they can be linked or coupled with a variety of techniques such as chromatic Flame atomic absorption 18,19 , electrochemical detection 20 , chemical fluorescence 21 . Through the (FIA) technology, which was characterized by high repetition and conformity, as well as its simplicity, speed, and low cost, the (FIA) technology has demonstrated its high efficiency. 22,23 . (FIA) is a quantitative method for assessing or estimating minerals and materials using injection flow such as Manganese in plants 24 , cholesterol 25 and Mebeverine Hydrochloride 26 In this paper, new methods for determining ceftazidime in its pure and pharmaceutical formulations have been developed using diazotization and coupling reactions with 1naphthol and 2-naphthol reagents, and then using these reactions with the flow injection technique in the presence of a visible and ultraviolet detector, and their applications in the scientific field to estimate these drugs in their pure and pharmaceutical formulations have been developed.

Apparatus;
All spectral and absorbance measurements were executed on an Advanced microprocessor UV-VIS spectrophotometer single beam LI-295 recording spectrophotometer using 1 cm quartz cells.
Flow injection configuration a three-channels manifold was employed with, Peristaltic pump (AlLITEA, C4, made in Sweden) with polyvinyl chloride tube (0.8) internal diameter as in fig.2.

Reagents;
Pharmaceutical preparations: Ceftazidime Pharmaceutical preparations were purchased from commercial sources, and Reagents were all analytical reagent grade chemicals.
Ceftazidime (CFT) (1000 µg/ml ) ; ( Laboratorios TORLAN Barcelona -Spain ) A stock solution (1000 µg/ml) 9.1478×10 -5 M of ceftazidime was prepared by dissolving 0.1 gm of CFT in distilled water. Then transfer it to a conical flask 100ml and complete the volume to the mark.
1-Naphthol (1-NPT);1-Naphthol (50µg/ml ) 3.471×10 -4 M was prepared by dissolving 0.1 g (BDH) distilled water with a small amount of 25% NaOH were added to ensue complete dissolution . Then transfer it to a conical flask 50ml and complete the volume to the mark.
2-Naphthol (2-NPT): 2-Naphthol (50µg/ml ) 3.471×10 -4 M was prepared by dissolving 0.1 g (AR) distilled water with a small amount of 25% NaOH were added to ensue complete dissolution . Then transfer it to a conical flask 50ml and complete the volume to the mark. Different concentrations of reagents from (0.0694×10 -3 to 0.832×10 -3 ) M (300 -1200) µg/ml used in the injection method were prepared by drawing different volume (1-12) ml from 2000µg/ml for reagents completing the volume of the marker in a 20 ml conical flask .
Sodium nitrite NaNO 2 1% (0.1449 M) 1g sodium nitrite (BDH) was dissolved in distilled water and diluted to the desired concentration in a 100 mL conical flask.
HCl Solution 1:1 (36.46 M ) In a 100 ml conical flask, 50 mL of 11.64 M concentrated hydrochloric acid (ADR) was diluted with distilled water NaOH 25% solution ( 6.2505 M) 25 g sodium hydroxide (BDH) was dissolved in distilled water and diluted to the desired concentration in a 100 mL conical flask.

Pharmaceutical Ceftazidime;
Commercially available pharmaceutical preparations were used Ceftazidime Roth ( Pharma Roth GmbH 65189 Wiesbaden Germany) , YENIZidime (yenipharma company Turkey -Ankara) and TOTTIZIM (Made in ITALY) ( 1000µg/ml ) 0.1 g dissolved in distilled water and fill to the mark in a 100 ml conical flask General procedure of diazotization reaction; An aliquot of a sample solution containing CFT (1ml ) from 1000µg/ml was transferred into 20.0 mL of conical flasks and then cooled in an ice bath maintained at -5.0 ºC to this solution, 1ml of HCl followed by 1.0 mL from NaNO 2 1%(w/v), were added to the mixture and left to stand for 10 min . Then, 1 mL of 1-naphthol and 2-naphthol was added to flask, then 1ml of NaOH 25% solution was added to the mixture, and diluted to the mark with double distilled water(D.W). The azo dye that formed was monitored at λmax 585and 545nm respectively General procedure of Flow Injection method; Using a three-channels peristaltic pump, the first channel contains a mixture of hydrochloric acid and sodium nitrite This mixture is passed through the injection valve, containing 100 μL of ceftazidime, to form the diazonium ion, which is passed into a 100 cm reaction coil. the second channel contains the reagent (1-NPT or 2-NPT ) that coupled with diazonium ion to form diazonium salt, the third channel contains 5% of NaOH which is added to the product, her the colored product (Brown and red respectively ) appears which will pass to the UV-Visible reagent for absorption and is measured the resulting brown and red colors were measured at λmax 585 and 545 nm for the two detectors, respectively.

Result and discussion
Absorption spectra Diazotized CFT was coupled with 1-Naphthol and 2-Naphthol in an alkaline solution to produce brown and red colored chromophore. The absorbance of the azo dyes were recorded at wavelengths of 585 and 545nm against a blank solution that which is prepared in the same way as samplethe same additions -except for drugs , and were obtained with a maximum absorbance under optimal conditions. Figs. 3, 4.

Part 1: Optimization of experimental conditions
To achieve the optimum detection limit and highest sensitivity, the many aspects impacting the spectrum characteristics of the dyes generated by coupling nitrogenous drug with organic reagent were examined. All of these tests were carried out in 20ml volumetric flasks with (1000µg/ml) 9.1478×10 -5 M of CFT standard solution.    The impact of different types of bases on absorption; The effect of different bases, 25 percent w/v [KOH, NaOH, Na 2 CO 3 ], on the absorption of azo dye has been tested, and it was found that the KOH solution showed the highest absorption for both the reagent and the CFT-2-NPT, but the solution was unstable and continuation of bubbles, therefore NaOH was used. as shown in Table  2.

Effect of the sodium hydroxide volume;
The effect of varied sodium hydroxide volumes on absorbance were tested for the volume range (0.5-2ml), with 1,0.5ml gave the maximum absorption for CFT-1-NPT and CFT-2-NPT, respectively, Fig.8.

Figure 8. Effect of the NaOH 25%
The Effect of reagent volume on diazonium salt: With 1 ml of 1000µg/ml ceftazidime solution, several quantities of (50µg/ml) 3.4710-4 M of reagents 1-NPT and 2-NPT were examined (0.5-1.00ml). The reagent with the highest absorbance was 0.5ml. The absorbance signal remained steady after that. Fig.9.

Figure 9. The mole-ratio plot for diazotized ceftazidime to 1-NPT and 2-NPT
The possible reaction mechanism can be written as in the Fig.10.

Data analysis
The absorption signals were measured against a series of ceftazidime concentrations to plot a calibration curve. Fig 11 and Table 3 exhibit the developed method's calibration curve and analytical figures.
.    The effect of NaNO 2 1%volume; The effect of the volume of the sodium nitrite on the absorbance of the colored products Fig .13. The effect of different volumes of sodium nitrite was studied that range from 25 to 5 ml, 20 and 15 ml that was gave the highest absorption for reagents respectively.

Figure 13. Effect volume of NaNO2
The effect of reagents concentration; At constant CFT concentrations, the effects of 1-NPT and 2-NPT concentrations on absorbance were investigated. CFT was injected into the HCl stream in fixed volumes (100 mL) at 1000 µg/mL.  The effect of different concentration of NaOH.
The effect of different concentration of NaOH was studied, the range from (5 to 25%), with a fixed concentration of acid and drug, and it 5% showed the highest absorption intensity to both of reagents. Fig. 15. After determining the optimal experimental conditions, the calibration curve was ploted as it is shown in Fig. 16 and Table 4 calibration curve and analytical information, respectively

Conclusion:
Because the 1-NPT and 2-NPT reagents are methods that are available and inexpensive, the research includes two simple, sensitive, fast, and inexpensive methods for estimating ceftazidime. The first (diazotization-coupling) entails converting ceftazidime into a colored dye that can be measured using a UV-method Vis spectrophotometer. The second step involves using flow injection to preconcentrate colored dye. This is the first method for extracting CFT from a flow injection. These methods have been used to estimate ceftazidime in pharmaceuticals with great success.