Development of Simple Green Spectrophotometric and Conductometric Methods for Determination of Cephalosporins in Pure, Pharmaceutical Dosage forms and Human Urine

Five Simple, accurate and rapid spectrophotometric and conductometric methods were developed for the determination of four third generation cephalosporins, namely, cefotaxime sodium (I) , cefoperazone sodium (II), ceftazidime pentahydrate (III) and cefdinir (IV) in pure active ingredient, pharmaceutical dosage forms and human urine. Method A: is based on the reaction of the sulphide ions produced from the alkaline hydrolysis of the cited four drugs with Paminophenol (PAP). This reaction results in a thionine dye (phenothiazine derivative) formation which exhibits maximum absorbance at 545 nm. Method B: is based on oxidation of drug (I and III) with a known excess of n-bromosuccinimide (NBS) in acidic medium followed by the determination of unreacted amount of n-bromosuccinimide with metol and sulphanilic acid. The purple-red reaction product exhibits maximum absorbance at 520 nm. Method C: is based on the formation of yellow chelate between drug (IV) and palladium (II) chloride in buffered medium (pH 3.5) with an absorption maximum at 314 nm. Method D: is based on the reaction of drug (IV) with aqueous ninhydrin to give yellow colored product in the presence of bicarbonate with an absorption maximum at 433 nm . Method E: A conductometric method is based on the reaction of the four cited drugs with phosphotungstic acid (PTA) forming an ion associate in aqueous medium. Validation of the proposed methods was carried out. All proposed methods were successfully applied for the commercial dosage forms of the cited drugs. Method C was successfully applied for the determination of cefdinir in human urine.


Instruments
Shimadzu recording spectrophotometer UV 1201 equipped with 10 mm matched quartz cells and conductometer model 470 portable conductivity / TDS meter, 25 DEG.C-C10 dip-type cell with a cell constant, K cell of 1.09 were used. Digital analyzer pH meter (USA) was employed for pH measurment.

Reagents and materials
All chemicals and materials were of high analytical grade, and double distilled water was used through the work. 9-Metol (Sigma Aldrich ,Germany) (0.2 g%, w/v) aqueous solution.
11-Palladium(II) chloride (Sigma, Milwukee, WI,USA) was prepared as 2×10 -3 M by dissolving 35.5 mg of palladium(II) chloride in 1 mL of concentrated hydrochloric acid and diluting to 50 mL with distilled water, with the aid of heat and then the solution was cooled and diluted to 100 mL with distilled water. 12-Ninhydrin (Sigma, Milwukee, WI,USA) was prepared as 0.5% aqueous solution. 13-Phosphotungstic acid (Winlab , UK) was prepared as 10 -2 M aqueous solution. 14-Sulphuric acid , hydrochloric acid and sodium hydroxide were obtained from El-Nasr Chemical Company,   (I, II, III and IV) respectively were transferred and the solutions were completed with 1M NaOH to the mark . The flasks were heated in a boiling water bath for 50 minute for drug (I and II) and 60 minute for drug (III and IV) and then cooled to room temperature. One mL of each of these solutions was transferred into another three sets of 10 mL volumetric flasks. Two milliliters of zinc acetate solution then the specified volume of paminophenol and Ammonium iron (III) sulphate solutions were added to each flask. The flasks were shaken for 30 seconds and allowed to stand for the specified time. The volumes were completed to 10 mL with distilled water. Absorbance was measured at 545 nm against blank solution ( Figure 1).

Method B
Into two sets of 10 mL volumetric flasks, accurate volumes of the standard solution of each drug containing (0.02-0.32) and (0.04-0.32) mg of drug (I and III) respectively were transferred . To each flask 0.8 and 2 mL of NBS were added for drug (I and III) respectively. The content was mixed well . The flasks were kept for 15 and 10 min for drug (I and III) respectively with intermittent shaking. Then, the specified volume of metol was added . After 1 minute, the specified volume of sulphanilic acid was added to each flask, The flasks were kept aside for 3 minutes. Then, the volume was diluted to the mark with bidistilled water and mixed well. The absorbances were measured at 520 nm ( Figure 2).

Method C
To a set of 10 mL volumetric flasks, accurately measured aliquots of standard drug (IV) solution in the range of (0.03 -0.26) mg were transferred. 2 mL buffer solution of pH 3.5 were then added, then 0.5 mL of 2 M potassium chloride and 1 mL Pd(II) chloride solution. The solutions were allowed to stand at room temperature (25 ᵒC) for 10 minutes, then diluted to volume with bidistilled water, absorbance was measured at 314 nm against blank solution ( Figure 3).

Method D
To a set of 10 mL volumetric flasks, accurately measured aliquots of standard drug (IV) solution in the range of (0.04 -0.3) mg were transferred and completed to 5 mL with bidistilled water. 1 mL of ninhydrin solution followed by 1 mL of saturated solution of sodium bicarbonate were added to each flask. The flasks were heated in a boiling water bath for 15 minutes, cooled and then diluted to volume with bidistilled water. Absorbance was measured at 433 nm versus blank solution ( Figure 3).

Method E
Aliquots of drug solution containing (3 -30 mg) were transferred to a 50 mL calibrated flasks. Volumes were made up to the mark using bidistilled water and transferred to a beaker. Titration with 10 -2 M phosphotungstic acid was performed. The conductance was measured subsequent to each addition of phosphotungstic acid solution and after stirring for 2 minutes, the conductance was corrected for dilution [7] using the following equation.
Where Ω -1 obs is the observed electrolytic conductivity, v1 is the initial volume and v2 is the volume of phosphotungstic acid solution added. A graph of corrected conductivity versus the volume of added phosphotungstic acid solution was constructed and end-point was estimated (Figure 4).

.Method A and E :
The conent of one vial was transferred into 100 mL volumetric flask and the volume was completed with distilled water .Accurate volume of vial equivalent to 300 and 150 mg for method A and C respectively was transferred to 50 mL volumetric flask .The volume was completed with double distilled water and the procedure was completed as under general procedure .

For Method B:
The conent of one vial was transferred into 100 mL volumetric flask and the volume was completed with 0.2 M and 0.05 M HCl for drug (I and III) respectively. Accurate volume of vial equivalent to 10 mg for drug (I and III) was transferred to 50 mL volumetric flask .The volume was completed with 0.2 M and 0.05M HCl for drug (I and III) respectively and the procedure was completed as under general procedure .

For cefdinir capsules:
The contents of 10 capsules were removed and their weight was determined accurately. The combined contents were mixed and a quantity equivalent to 10 mg for method C and D and equivalent to 150 mg for method A and E was transferred into 50 mL volumetric flasks, extracted with 1 mL 1 M NaOH and shaken with 10 mL bidistilled water, then filtered and diluted to 50 mL with bidistilled water. The assay was completed as under general procedure.

For cefdinir suspension:
An accurately measured volume of the freshly reconstituted oral suspension equivalent to 10 mg for method C and D and equivalent to 150 mg for method A and E was transferred into 50 mL volumetric flasks, extracted with 1 mL 1 M NaOH and shaken with 10 mL distilled water, then filtered and diluted to 50 mL with distilled water. The assay was completed as under general procedure. O c t o b e r 1 8 , 2 0 1 3

Prepartion and analysis of human urine samples ( for method C)
Human urine samples were collected freshly from healthy volunteers. Blank urine pool was diluted 1:1 with double distilled water, then spiked with the appropriate amounts of stock solution to prepare samples. The assay was completed as described above.

RESULTS AND DISCUSSION
Many of the reported methods suffered from poor sensitivity, use of expensive organic solvents and extraction step. The use of organic solvent as the reaction medium is undesirable. Green or environmentally-friendly analytical methods are promising in recent years. Modern analytical methods need to be green without losing accuracy and sensitivity.The aim of the present work was to develop five new sensitive, cost effective methods for the determination of cephalosporins in pure drug , in pharmaceutical preparations and human urine. Method (A) is based on the use of PAP in sulphuric acid and aqueous medium. Method (B) is based on redox reaction in acidic and aqueous medium . Method (C) is based on use of the palladium (II) chloride in walpole acetate buffer pH 3.5, unlike other methods which are based on the use of palladium (II) chloride in DMF [6]. No spectrophotometric method was reported for cefdinir determination in human urine, so method (C) is advantageous in its determining in human urine. Method (D) is based on use of aqueous ninhydrin in bicarbonate medium, unlike other methods which are based on the use of methanolic or ethanolic ninhydrin [8] . Method (E) is based on direct titration of the cited drugs with PTA in aqueous medium. So, all proposed methods are free from usage of hazardous and expensive chemicals. Since inexpensive and easily available chemicals are used, the developed methods are green low cost analytical methods for cefdinir.

Method development (Method A)
Para amino phenol (PAP) was widely used in many analytical methods for pharmaceutical compounds determination. It was used for determination of cimetidine, famotidine , nizatidine , ranitidine hydrochloride [9] and cephalexin [10].
Method A is based on the alkaline hydrolysis of the cited drugs producing the sulphide ions which react with PAP and ferric ions by ring closure redox reaction giving red thionine dye (phenothiazine derivative) .

Optimization of the reaction conditions
The effect of hydrolysis time, effect of PAP volume, ammonium iron (III) sulphate volume and effect of reaction time were studied. It was found that 50 minutes hydrolysis time for (I and II) and 60 minutes for (III and IV), 4.5 mL of PAP for (I and II) , 3 mL for (III) and 4 mL for (IV) (Figure 5), 1.5 mL of ammonium iron (III) sulphate for (I,II and III) and 2 mL for (IV) and 1 minute for (I and II) and 3 minutes for (III and IV) were sufficient to give maximum absorbance. The color produced was stable for 1 hour.

Method developemt (Method B)
NBS-metol-primary arylamine combination was used for the determination of the oxidant, thereby permitting the indirect assay of many oxidisable substances including drugs in which the drug is oxidized with a known excess of NBS and, after the reaction, the unreacted NBS reacts with metol and primary arylamine and the purple color formed is measured and correlated to drug concentration.
Many pharmaceuticals have been estimated by this approach using NBS as oxidant and sulphanilic acid as primary arylamine .e.g. aspartame [11] and pioglitazone hydrochloride [12].
In this method, the application of NBS-metol-primary amine combination to the determination of (I and III) is described.
The method is based on the oxidation of the drug by a known excess of NBS in acidic medium and subsequent determination of the unreacted NBS by interacting with metol and the primary aromatic amine, sulphanilic acid. The studied drugs when added in increasing amounts to a fixed amount of NBS, consume NBS and consequently, there will be a concomitant fall in the NBS concentration. This is observed as a proportional decrease in the absorbance of the reaction mixture on increasing the concentration of drugs .The following scheme illustrates the proposed reaction mechanism.
Scheme (3): Proposed reaction mechanism of the studied drugs and NBS-metol-primary amine combination.

Optimization of the reaction conditions
The effect of acid type, molarity of hydrochloric acid, NBS volume, metol volume, sulphanilic acid volume, reaction time between drug and NBS, waiting time after addition of sulphanilic acid and time after dilution were studied. It was found that 0.2 M and 0.05 M hydrochloric acid, 0.8 and 2 mL of NBS solution, 0.5 and 1 mL of metol solution (Figure 6), 0.5 and 1 mL of sulphanilic acid solution, 15 and 10 minutes reaction time with NBS and 3 minutes waiting time after addition of sulphanilic acid were sufficient to give maximum absorbance. Solutions of ceftazidime can be measured immediately after dilution with water giving stable absorbances. For cefotaxime sodium, the solution should be stand for 5 minutes after dilution then measured due to increasing the absorbances in the first five minutes after dilution. All absorbances for both drugs are stable for 30 minutes. O c t o b e r 1 8 , 2 0 1 3

Method development (Method C)
The use of palladium (II) chloride as a complexing agent for drugs quantitation is very wide. Palladium(II) chloride was found to form complexes of square or 5 -co-ordinate shape [13]. The chelate complex of palladium (II) ions is watersoluble and does not need extraction procedure. Several drugs were determined spectrophotometrically by measuring the color intensity of their complexes with palladium (II) ions, e.g. metoclopramide, promazine [14] and cefotaxime , cefuroxime and cefazolin [6].
Reaction of Pd(II) chloride with drug (IV) produced yellow complex which was soluble in walpole acetate buffer pH 3.5.
The absorption spectra showed a maximum absorbance at 314 nm .

3.3.1.Optimization of the reaction conditions
Effect pH, effect of KCl , effect of reagent volume, effect of reaction time , effect of temperature and effect of order of addition were studied. It was found that 2 mL of Walpole acetate buffer pH 3.5 , 0.5 mL of 2 M KCl , 1 mL 2 × 10 -3 M Pd(II) chloride (Figure 7), 10 minutes were sufficient to give maximum absorbance with cefdinir. Temperatures higher than room temperature caused absorbance decrease. The most suitable order of addition was found to be drug , buffer, KCl and Pd(II) chloride. The color produced was stable for 30 minutes.

3.3.2.Composition of the complex
The stoichiometry of the complex between cefdinir and Pd(II) chloride was studied by applying Job's method of continuous variation [15] using an equimolar ( 5 × 10 -4 M) solutions of cefdinir and Pd(II) chloride . The total volume of drug and Pd(II) chloride was kept at 2 mL then the procedure was completed as under the above mentioned procedure. The results obtained showed that the stoichiometric ratio of the complex was (1 : 1) (reagent:drug) (Figure 8). O c t o b e r 1 8 , 2 0 1 3 Vd is the volume taken from drug molar solution. Vr is the volume taken from Pd (II) chloride molar solution.

Formation Constant of the Reaction Product
The formation constant (Kf) of the complex was calculated using the following formula [16]: The Gibbs free energy change of the reaction (ΔG) was found to be -2.6 × 10 4 K.J./mole. It has a negative value which indicates the spontaneous nature of the reaction [16].

Method developemt (Method D)
Ninhydrin (triketohydrindane hydrate) is a carbonyl reagent which can form a purple condensation product which can be measured spectrophotometrically .So, it was applied in the pharmaceutical assay of different nitrogenous compounds such as some penicillins [17] and tranexamic acid [8]. A modified approach for ninhydrin green use was developed for lisinopril determination based on the formation of a yellow colour product with aqueous ninhydrin in the presence of bicarbonate with an absorption maximum at 420 nm [18]. So the aim of this work is to develop simple green method for cefdinir determination by the reaction with aqueous ninhydrin in bicarbonate medium giving yellow color measured at 433 nm. The green use of ninhyrin leads to short heating time (15 minutes) and avoidance of organic solvent use, so reduces cost.

Optimization of the reaction conditions
The effect of pH, volume of NaHCO3, concentration of ninhydrin and heating time were studied. Different molarities of NaOH were used to study the effect of pH on the reaction. No color product was formed in NaOH medium, so the reaction is specific in bicarbonate medium .1 mL of NaHCO3, 1 mL of ninhydrin ( Figure 9) and heating for 15 minutes were sufficient to give maximum absorbance and stable yellow color product. The developed yellow color was stable for 1 hour. O c t o b e r 1 8 , 2 0 1 3

Method E ( conductometric method)
Conductometric titration is one of the simplest analytical techniques used in drug standardization laboratories. Precipitimetric conductometric titrations using phosphotungstic acid as a titrant are commonly used for the quantitative determination of different compounds eg. reproterol HCl , pipazethate HCl , salbutamol sulphate [19].
The present method aims to introduce new conductometric method for the determination of the cited drugs which is very simple in application and of low expenses but as the same time having a high degree of accuracy and precision when compared to the reported methods .
The conductance measured before any addition of the titrant (volume of phosphotungstic acid equals zero) is due to the formation of RNHx + and OH − by hydrolysis. During titration, replacement of the RNHx + ions by mobile H + occurs resulting in ion associate formation and the conductivity increases. The conductivity continues to increase rapidly after the endpoint. Curve break is observed at molar ratio of 3 : 2 (drug-reagent) for all drugs except for ceftazidime pentahydrate which has a curve break at drug-reagent molar ratio of 1:1.
Representative titration curve is shown in (Figure 4) indicating two straight lines intersecting at the end point. After the end point, sudden change in the slope occurs.

Optimization of the reaction conditions
The optimum conditions for performing the titration in a quantitative manner were elucidated as described below.

3.5.1.1.Titration medium.
Preliminary experiments in aqueous solutions of both drug and reagent, drug and reagent solutions in ethanol-water (50%, v/v) mixture, methanolic solutions of both drug and reagent, drug and reagent solutions in methanol-water (50% v/v) mixture and drug and reagent solution in acetone-water (50% v/v) mixture. Aqueous medium led to higher conductance and most sharp end point for all drugs.

Reagent's concentration.
The optimum concentration of phosphotungstic acid was found to be 10 -2 M to give a constant and stable conductance for all drugs. Concentrations less than 10 -2 M give unstable readings.

3.5.2.Composition of the complex:
In 50 mL volumetric flask , 6 millilitres of 10 -2 M drug solution were transferred , completed to 50 mL with bidistilled water , transferred to a beaker and titrated by 10 -2 M PTA. The conductance was measured after 2 minutes stirring from each addition of reagent solution. A graph of corrected conductivity versus PTA volume was constructed indicating curve break at a molar ratio of 3 : 2 (drug-reagent) except for ceftazidime pentahydrate which has a curve break at drug-reagent molar ratio of 1:1.

3.6.1.For the methods (A , B, C and D)
Standard calibration curves for the cited drugs were constructed by plotting absorbances against concentrations. Beer's law limits, linear regression equations, molar absorptivity and sandell sensitivity were estimated for each method ( Table 1). The correlation coefficients were found to be 0.9999 indicating excellent linearity over beer's law limits for all methods ( Table 1). The detection limit (LOD) and limits of quantitation for the proposed methods were estimated according to ICH O c t o b e r 1 8 , 2 0 1 3 [20] and listed in (Table 1). Their values indicate the high sensitivity of the proposed methods. Accuracy and precision were determined by analyzing one concentration of each drug in seven replicates. The relative standard deviation (RSD%) and percentage relative error (Er%) were estimated at 95% confidence levels ( Table 2). The results showed that the proposed methods have good reproducibility.

For Method E:
Recovery study of the cited drugs content in their commercial preparations was performed using the proposed method. (Table 3) showed that the proposed method is accurate and reproducible over a concentration range of 3 -30 mg. Table 4 showed comparison of the results obtained using method D with the reported method [21] using Student t-test and Variance ratio F-test at 95% confidence level . O c t o b e r 1 8 , 2 0 1 3

Analytical applications
The proposed methods were successfully applied to determine the cited drugs in their commercial dosage forms. Recovery studies were performed (Tables 3 and 5). The results validation was determined by comparison with the reported method [21] using Student t-test and Variance ratio F-test at 95% confidence level (Table 4).It was found that no significant differences between the proposed methods and reported method [21]. O c t o b e r 1 8 , 2 0 1 3

Human urine (for method C)
Method C was successfully applied to determine cefdinir in spiked human urine samples with excellent precision and accuracy (Table 6) .No interference was found from the biological urine matrix. Moreover, the linearity of the proposed method was checked over a concentration range of 4 -26 μg.mL -1 . Excellent linearity was observed. The regression equation was found to be y = 0.0347x + 0.0726 .The correlation coefficient (r 2 ) was found to be 0.9993. The detection (LOD) and quantitation limits (LOQ) were found to be 1.64 and 4.97 μg . mL -1 respectively. Their values confirm the sensitivity of the proposed method in human urine.
So, The proposed methods are simple, green, accurate and precise in determining the cited drugs in its pharmaceutical formulations and human urine without interference from common excipients or biological matrix. The proposed methods have higher sensitivity than many of the reported methods. All methods are green analytical methods so, they are inexpensive and ecofriendly. Moreover, they are less time-consuming and do not require difficult extraction procedures. So, the proposed methods are suitable for routine analysis of the cited drugs in control laboratories.