Kinetic spectrophotometric determination of certain cephalosporins using iodate/iodide mixture

doi:10.4236/ns.2010.25053

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Vol.2, No.5, 432-443 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.25053

Kinetic spectrophotometric determination of certain

cephalosporins using iodate/iodide mixture

Salwa R. El-Shaboury, Fardous A. Mohamed, Gamal A. Saleh, Azza H. Rageh*

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt; [email protected];

[email protected]; [email protected]; *Corresponding Author: [email protected]

Received 10 December 2009; revised 28 January 2010; accepted 8 March 2010.

ABSTRACT

A simple, precise and accurate kinetic spectro-

photometric method for determination of ce-

fradine anhydrous, cefaclor monohydrate, ce-

fadroxil monohydrate, cefalexin anhydrous and

cefixime in bulk and in pharmaceutical formula-

tions has been developed. The method based

on a kinetic investigation of the reaction of the

free carboxylic acid group of the drug with a

mixture of potassium iodate and potassium io-

dide at room temperature to form yellow col-

oured triiodide ions. The reaction was followed

up spectrophotometrically by measuring the

increase in absorbance at 352 nm as a function

of time. The initial rate, fixed time, variable time

and rate-constant methods were adopted for

constructing the calibration curves but fixed

time method has been found to be more appli-

cable. The analytical performance of the method,

in terms of accuracy and precision, was statis-

tically validated; the results were satisfactory.

The method has been successfully applied to

the determination of the studied drugs in com-

mercial pharmaceutical formulations. Statistical

comparison of the results with a well estab-

lished reported method showed excellent ag-

reement and proved that there is no significant

difference in the accuracy and precision.

Keywords: Cephalosporins; Kinetic

Spectrophotometry; Lodate/Lodide Mixture;

Pharmaceutical Analysis

1. INTRODUCTION

Because cephalosporins are among the safest and the

most effective broad-spectrum bactericidal antimicrobial

agents available to the clinician, they have become the

most widely prescribed of all antibiotics. All of these

semi-synthetic antibiotics are derived from 7-amino-ce-

phalosporanic acid and contain a β-lactam ring fused to a

dihydrothiazine ring (Table 1) but differ in the nature of

the substituents attached at the 3 and/or 7-positions of

the cephem ring. These substitutions affect either the

pharmacokinetic properties (3-position) or the antibacte-

rial spectrum (7-position) of the cephalosporins. Cepha-

losporins operate by inhibiting bacterial cell wall bio-

synthesis which grows actively against a wide range of

both gram-positive and gram-negative bacteria. The po-

sitive results of these drugs include the resistance of

penicillinases and ability to treat infections that are re-

sistant to penicillin derivatives. The official methods for

analyzing cephalosporins are mostly chromatographic

methods [1] which are expensive. Most of the reported

methods involve the cleavage of the β-lactam moiety of

the cephalosporin structure. These methods include spe-

ctrophotometric [2-6] spectrofluorimetric [7-10]. and

electrochemical methods [11-13]. A direct chemical ana-

lysis based on the reactivity of the intact molecule is not

frequently encountered.

Kinetic spectrophotometric methods are becoming of

great interest in chemical and pharmaceutical analysis

[14]. The application of these methods offered some

specific advantages [15,16].

1) Simplicity owing to elimination of some experi-

mental steps such as filtration and extraction prior to

absorbance measurements.

2) High selectivity due to the measurement of the in-

crease or decrease of the absorbance as a function of

reaction time instead of measuring the concrete absorb-

ance value.

3) Avoiding the interference of the coloured and/or

turbidity background of the samples, and possibility of

avoiding the interference of the other active compounds

present in the commercial product if they are resisting

the established reaction conditions.

The literatures are still lacking analytical procedures

based on kinetics for determination of the investigated

drugs in commercial dosage forms. A kinetic spectro-

photometric method has been reported for determination

of cefadroxil based on its alkaline hydrolysis [17]. With

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

433

the exception of cefadroxil, this part represents the first

attempt for assaying the investigated drugs without deg-

radation in pure forms and in different pharmaceutical

dosage forms using kinetic spectrophotometric method.

The literature reveals a kinetic spectrophotometric me-

thod for determination of ramipril [18] that based on the

reaction of its carboxylic acid group with iodate/iodide

mixture in aqueous medium at room temperature to form

yellow coloured triiodide ions. The reaction was fol-

lowed up spectrophotometrically by measuring the in-

crease in absorbance at 352 nm as a function of time.

This reaction drew our attention to investigate it on

our studied drugs that contain free carboxylic acid group

(Table 1). Accordingly, this reaction was studied in

order to find out if it would lend itself applicable to

the analysis of cefradine anhydrous, cefaclor mo-

nohydrate, cefadroxil monohydrate, cefalexin anhydrous

and cefixime in pure forms and in pharmaceutical for-

mulations. As a result of these investigations; a simple,

rapid and accurate kinetic spectrophotometric method

for determination of the aforementioned cephalosporin

drugs without degradation was devised. The fixed time

method is adopted after full investigation and under-

standing of the kinetics of the reaction. The proposed

method does not require the elaboration of treatment and

procedures, which are usually associated with chroma

tographic methods.

2. EXPERIMENTAL

2.1. Apparatus

Shimadzu UV-1700 PC, UV-Visible Spectrophotometer

(Tokyo, Japan), Ultrasonic cleaner (Cole – Parmer, Chi-

cago, USA) and Sartorious handy balance – H51 (Han-

nover, Germany).

2.2. Materials and Reagents

All solvents used were of analytical-reagent grade, po-

tassium iodide (El-Nasr Chemical Co. Cairo, Egypt)

freshly prepared aqueous solution (1.5 M), potassium

iodate (El-Nasr Chemical Co. Cairo, Egypt) freshly pre-

pared aqueous solution (0.3 M), cefaclor monohydrate

and cefradine anhydrous (Sigma Chemical Co., St. Louis,

USA) cefadroxil monohydrate (Amoun Pharmaceutical

Industries Co., APIC, Cairo, Egypt), cefalexin anhydrous

(GalaxoWellcome, S.A.E., El Salam City, Cairo, Egypt)

and cefixime (El-Hekma Co., Cairo, Egypt) were ob-

tained as gifts and were used as supplied and pharma-

ceutical formulations containing the studied drugs were

purchased from local market.

Table 1. Chemical structures of the investigated cephalosporin antibiotics.

OOR

No. Name R1 R2 Generation

1. Cefalexin anhydrous

NH2

-CH3 First

2. Cefradine anhydrous

NH2

-CH3 First

3. Cefadroxil

monohydrate

NH2

-CH3 First

4. Cefaclor

monohydrate

NH2

-Cl Second

5. Cefixime

2NC

NOCH2

CO2

CH2

Third

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

434

2.3. Preparation of Standard Solutions

Stock solutions containing 1 mg mL-1 of each cepha-

losporin namely, cefradine anhydrous, cefadroxil mono-

hydrate, cefaclor monohydrate, cefalexin anhydrous and

cefixime were prepared in methanol. Working standard

solutions containing 0.1-0.5 mg mL-1 (in case of ce-

fixime, working standard solutions containing 0.05-0.25

mg mL-1) were prepared by suitable dilution of the stock

solution with methanol. The stock and working standard

solutions must be freshly prepared.

2.4. Preparation of Sample Solutions

2.4.1. Tablets and Capsules

Twenty tablets or the contents of 20 capsules were

weighed, finely powdered and mixed thoroughly. An

accurately weighed amount of the powder obtained from

tablets or capsules equivalent to 250 mg of each drug

was transferred into a 50-mL volumetric flask, dissolved

in about 25 mL methanol, sonicated for 15 min, diluted

to the mark with methanol, mixed well and filtered; the

first portion of the filtrate was rejected. Further dilutions

with methanol were made to obtain sample solution

containing 0.3 mg mL-1 (in case of cefixime, further di-

lutions with methanol were made to obtain sample solu-

tion containing 0.15 mg mL-1) and then the general pro-

cedure was followed.

2.4.2. Powder for Oral Suspension

An accurately weighed amount of powder equivalent to

250 mg of each drug was transferred into a 50 mL volu-

metric flask, then the procedure was followed as under

tablets and capsules beginning from (dissolved in about

25 mL methanol).

2.3. General Procedure

Accurately measured one millilitre aliquot volume of the

standard or sample solutions was transferred into 10- mL

volumetric flask. One millilitre of 0.3 M of potassium

iodate was added followed by 1 mL of 1.5 M of potas-

sium iodide. The content of the flask was mixed well

and diluted to volume with methanol. The increase in

absorbance was measured at 352 nm against reagent

blank treated similarly. The four kinetic methods namely,

initial rate, fixed time, variable time and rate constant

methods were used for construction of the calibration

curves and determination of the studied drugs.

3. RESULTS AND DISCUSSION

3.1. Absorption Spectra

Absorption spectrum of cefradine anhydrous which was

taken as a representative example for all studied drugs is

shown in Figure 1. This spectrum shows no absorption

at 352 nm whereas the absorbance of the reagent solu-

tion (KIO3 and KI in methanol) at 352 nm is about 0.02.

The wavelengths of maximum absorption of the interac-

tion coloured product of cefradine anhydrous with KIO3

and KI are at 298 and 352 nm. It is obvious that at 298

nm there is background absorption from the drug itself

and from the reagent blank (Figure 1). Therefore, the

absorbance measurements for the determination of the

studied drugs were made at 352 nm. The equilibrium is

attained in ~30 minutes. Therefore, a kinetically based

spectrophotometric method was developed for the quan-

titative determination of the investigated drugs by meas-

uring the increase in absorbance at 352 nm as a function

of time.

3.2. Optimization of Reaction Conditions

The experimental parameters affecting the reaction be-

tween the investigated drugs, potassium iodate and po-

tassium iodide were carefully studied and optimized.

Cefardine anhydrous (30 μg mL-1) was taken as a repre-

sentative example for this study. These factors include:

3.2.1. Effect of Potassium Iodate Concentration

The concentration of potassium iodate, for the maximum

colour development at 352 nm, was studied in the range

of 0.05-0.6 M. From Figure 2, it was found that the

Figure 1. Absorption spectra of (a) cefradine anhydrous

(30 μg mL-1); (b) reagent solution (0.3 M potassium iodate

and 1.5M potassium iodide) and (c) the interaction coloured

product of cefradine anhydrous with potassium iodate and

potassium iodide.

0.1

0.2

0.3

0.4

0.5

0.6

00.10.2 0.3 0.4 0.50.6 0.7

Potassium iodate concentration (M)

Absorbance, 352 nm

Figure 2. Effect of potassium iodate concentration on the

absorbance of the reaction coloured product at 352 nm.

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

435

absorbance of the interaction coloured product is in-

creased with increasing potassium iodate concentration.

Maximum absorbance was attained by using 0.25 M;

above this concentration and up to 0.6 M KIO3, the ab-

sorbance remains constant. Therefore, 1 mL of 0.3 M

potassium iodate was selected during subsequent work.

3.2.2. Effect of Potassium Lodide Concentration

The influence of potassium iodide concentration on

producing the maximum absorption intensity was inves-

tigated using 0.3-2.4 M potassium iodide. Maximum

absorption readings were obtained upon using 1 mL of

1.3 M potassium iodide; above this concentration the

absorbance remains constant. So, 1 mL of 1.5M of KI

was used for further work (Figure 3).

3.2.3. Effect of Diluting Solvent

Different solvents were tested in order to select the most

appropriate solvent for producing the maximum absorp-

tion intensity. The results given in Table 2 show the

slight effect on λmax while the absorption intensity was

affected. Methanol was used throughout this work be-

cause it gave the highest absorbance readings and the

most reproducible results.

3.2.4. Effect of Temperature

As expected from the Arrhenius equation [19], the reac-

tion rate is increased with increasing temperature. So,

trials have been done to carry out the reaction at higher

temperatures. It was found that the studied drugs un-

dergo degradation and iodine is unstable at higher tem-

peratures [20]. Therefore, room temperature (25 ± 5℃)

was recommended as the optimum temperature for this

study.

3.2.5. Quantitation Methods

The initial rate, fixed time, variable time and rate con-

stant methods [21,22] were tested and the most suitable

analytical approach was chosen regarding the applicability,

sensitivity, the values of the intercept and correlation coef-

ficient (r).

3.2.6. Initial Rate Method

Under the optimum experimental conditions, the assay

of cefradine anhydrous, cefadroxil monohydrate, cefa-

clor monohydrate, cefalexin anhydrous and cefixime

was performed at different concentration levels for 17

min at intervals of 2 min starting from 1 min at room

temperature (25 ± 5℃). The absorbance at 352 nm was

then recorded at each time interval. The assay was car-

ried out in presence of excess concentration of potassium

iodate and potassium iodide. Therefore, a pseudo-zero

order reaction condition was worked out with respect to

the concentration of the reagent.

The kinetic plots are all sigmoid in nature and the ini-

tial rate of reaction was obtained by measuring the

slopes (ΔA/Δt) of the initial tangent to the absorb-

ance-time curves at different concentrations of the inves-

tigated drugs. Figure 4 shows the kinetic plot for ce-

fradine anhydrous as a representative example.

The initial rate of reaction would follow a pseudo-first

order and obeyed the following rate equation:

v'



 (1)

whereas ν is the reaction rate, A is the absorbance, t is

the measuring time, k' is the pseudo-first order rate con-

stant, C is the concentration of the drug and n is the or-

der of the reaction. The logarithmic form of the above

equation is written as follows:

Cn'k

vloglogloglog 



 (2)

A calibration curve was constructed by plotting the

logarithm of the initial rate of reaction (log v) versus

logarithm of initial concentration of the investigated

drugs (log C), which showed a linear relationship over

concentration range of 2.59 × 10-5 - 1.44 × 10-4 M for

cefadroxil monohydrate, cefaclor monohydrate, ce-

falexin anhydrous and cefradine anhydrous (in case of

cefixime, 1.10 × 10-5 - 5.51 × 10-5 M). The regression

equations of log rate versus log C are given in Table 3.

0.1

0.2

0.3

0.4

0.5

0.6

0 0.30.60.91.21.51.82.12.42.7

potassium iodide concentration (M)

Absorbance, 352 nm

Figure 3. Effect of potassium iodide concentration

on the absorbance of the reaction coloured product at

352 nm.

Figure 4. Absorbance-time curve for the reaction of

cefradine anhydrous (μg mL-1) with potassium iodate

and potassium iodide.

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

436

Table 2. Effect of solvent on λmax and the absorption intensity of the reaction coloured product of the studied drugs with KIO3 and KI.

Drug

Solvent

Cefradine anhydrous

(30 μg mL-1)

Cefadroxil mono-

hydrate (30 μg mL-1)

Cefaclor monohy-

drate (30 μg mL-1)

Cefalexin anhy-

drous (30 μg mL-1)

Cefixime

(15 μg mL-1)

λmax (nm) Aa λmax (nm)Aa λmax (nm)Aa λmax (nm)Aa λmax (nm)Aa

Water 346 0.420 346 0.394 346 0.308 347 0.331 348 0.318

Ethanol 357 0.520 358 0.410 359 0.382 358 0.410 358 0.394

Methanol 352 0.544 352 0.510 352 0.400 352 0.429 352 0.413

Acetone 359 0.400 360 0.385 359 0.294 360 0.315 360 0.303

Acetonitrile 352 0.410 356 0.390 351 0.312 351 0.322 353 0.306

Propan-1-ol 358 0.390 359 0.375 360 0.296 358 0.306 357 0.280

Propan-2-ol 361 0.434 361 0.420 361 0.327 361 0.333 360 0.300

DMF 351 0.380 355 0.360 354 0.286 351 0.290 352 0.260

DMSO 350 0.375 349 0.370 350 0.282 350 0.287 350 0.260

a Average of 3 determinations.

Table 3. Relation between reaction rates and concentrations.

log ΔA/Δt log [Drug] (M)

Calibration equation log ν = log k' + n log C Correlation coefficient (r)

Cefradine anhydrous

-1.577 -4.543 log ν = 2.729 + 0.956 log C 0.9867

-1.377 -4.240

-1.164 -4.066

-1.066 -3.941

-0.893 -3.844

Cefadroxil monohydrate

-1.577 -4.581 log ν = 2.765 + 0.956 log C 0.9868

-1.377 -4.280

-1.164 -4.104

-1.066 -3.979

-0.893 -3.882

Cefaclor monohydrate

-1.699 -4.586 log ν = 3.441 + 1.122 log C 0.9971

-1.377 -4.285

-1.164 -4.109

-1.066 -3.984

-0.893 -3.887

Cefalexin anhydrous

-1.553 -4.541 log ν = 2.976 + 1.002 log C 0.9828

-1.268 -4.240

-1.155 -4.064

-1.011 -3.939

-0.801 -3.842

Cefixime

-1.523 -4.958 log ν = 4.011 + 1.126 log C 0.9856

-1.314 -4.656

-1.039 -4.480

-0.905 -4.355

-0.738 -4.259

The correlation coefficients (r) of all studied drugs rang-

ed from 0.9828 to 0.9971. The order (n) with respect to

the studied drugs was evaluated by plotting the logari-

thm of the initial rate of reaction versus logarithm of the

concentrations of the investigated drugs and was found to

be approximately one which confirms the first-order reac-

tion with respect to all investigated drug concentrations.

3.2.7. Fixed Time Method

In this method, the absorbance changes caused by effect

of drug acidity on a mixture of potassium iodate and

potassium iodide were recorded at a preselected fixed

time at intervals of 2 min. The change in absorbance (ΔA)

between the times t1 (1 min) and t2 (3, 5, 7, 9, 11,

13, 15 and 17) was computed and plotted against the

concentration of each of the studied drugs. The corre-

sponding linear regression equations with correlation co-

efficients are summarised in Table 4. It is evident from

the table that the most acceptable linearity was obtained

when the calibration graphs were plotted by considering

the change in absorbance between 1 and 11 min (i.e.

ΔA= A11 -A1). It is also clear that the slope increases with

time and the most acceptable values of r and the inter-

cept were obtained for a fixed time of 10 min, which was

therefore chosen as the most suitable time interval for

the measurement. The calibration curve was linear in the

range of 10 to 50 µg mL-1 for cefadroxil

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

437

Table 4. Calibration equations for the studied drugs of different concentrations at different time intervals using fixed time method.

Δt (min) Calibration equation ΔA = a + b C Correlation coefficient (r)

Cefradine anhydrous

2 ΔA = -0.008 + 0.005 C 0.9842

4 ΔA = 0.033 + 0.008 C 0.9989

6 ΔA = 0.063 + 0.010 C 0.9991

8 ΔA = 0.871 + 0.012 C 0.9994

10 ΔA = 0.104 + 0.015 C 0.9997

12 ΔA = 0.140 + 0.016 C 0.9988

14 ΔA = 0.163 + 0.016 C 0.9980

16 ΔA = 0.196 + 0.017 C 0.9963

Cefadroxil monohydrate

2 ΔA = -0.008 + 0.005 C 0.9842

4 ΔA = 0.009 + 0.009 C 0.9975

6 ΔA = 0.026 + 0.011 C 0.9978

8 ΔA = 0.044 + 0.013 C 0.9991

10 ΔA = 0.036 + 0.016 C 0.9997

12 ΔA = 0.048 + 0.017 C 0.9995

14 ΔA = 0.062 + 0.018 C 0.9981

16 ΔA = 0.089 + 0.019 C 0.9963

Cefaclor monohydrate

2 ΔA = -0.018 + 0.005 C 0.9898

4 ΔA = -0.019 + 0.009 C 0.9955

6 ΔA = -0.017 + 0.011 C 0.9967

8 ΔA = 0.001 + 0.012 C 0.9985

10 ΔA = 0.012 + 0.013 C 0.9996

12 ΔA = 0.006 + 0.015 C 0.9984

14 ΔA = 0.009 + 0.016 C 0.9989

16 ΔA = 0.016 + 0.016 C 0.9983

Cefalexin anhydrous

2 ΔA = -0.019 + 0.006 C 0.9666

4 ΔA = -0.074 + 0.012 C 0.9852

6 ΔA = -0.048 + 0.013 C 0.9928

8 ΔA = -0.031 + 0.014 C 0.9954

10 ΔA = -0.012 + 0.015 C 0.9991

12 ΔA = -0.018 + 0.017 C 0.9987

14 ΔA = -0.014 + 0.017 C 0.9989

16 ΔA = -0.005 + 0.018 C 0.9971

Cefixime

2 ΔA = -0.038 + 0.015 C 0.09859

4 ΔA = -0.003 + 0.021 C 0.09994

6 ΔA = 0.008 + 0.024 C 0.9988

8 ΔA = 0.005 + 0.026 C 0.9982

10 ΔA = 0.017 + 0.027 C 0.9994

12 ΔA = 0.011 + 0.029 C 0.9992

14 ΔA = 0.021 + 0.031C 0.9987

16 ΔA = 0.020 + 0.033 C 0.9974

monohydrate, cefaclor monohydrate, cefalexin anhy-

drous and cefradine anhydrous (in case of cefixime, 5-25

µg mL-1). The correlation coefficients (r) of all studied

drugs ranged from 0.9991 to 0.9997. Reasonable values

of LOD and LOQ were obtained which ranged from 0.22

to 1.10 and from 0.67 to 3.33 µg mL-1; respectively as

indicated in Table 5.

3.2.8. Variable Time Method

The general procedure was followed up for each of the

studied drugs at different concentration levels by re-

cording the time in seconds required for the absorbance

to reach 0.20. This preselected value of the absorbance

was chosen as it gives the widest calibration range. The

reciprocal of time (1/Δt) versus the initial concentration

of the studied drugs was plotted and the equations of the

calibration graphs are given in Table 6. The correlation

coefficients (r) of all studied drugs ranged from 0.9646

to 0.9873.

3.2.9. Rate Constant Method

Under the described experimental conditions, analysis

was carried out for each of the studied drugs at different

concentration levels starting from 1 min until 17 min at

regular intervals of 2 min at room temperature (25 ±

5℃). Graphs of log absorbance change at 352 nm versus

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

438

Table 5. Summary of quantitative parameters and statistical data using fixed time method.

Drug Intercept (a) ±

SDa

Slope (b) ±

SDa

Linearity range

(μg mL-1 )

Correlation

coefficient (r)

Determination

coefficient (r2)

LOD (µg

mL-1)

LOQ (µg

mL-1)

Cefradine

anhydrous 0.104 ± 0.005 0.015 ± 0.002 10-50 0.9997 0.9994 1.10 3.33

Cefadroxil

monohydrate 0.036 ± 0.002 0.016 ± 0.002 10-50 0.9997 0.9994 0.41 1.25

Cefaclor

monohydrate 0.012 ± 0.001 0.013 ± 0.001 10-50 0.9996 0.9992 0.25 0.80

Cefalexin

anhydrous -0.012 ± 0.001 0.015 ± 0.001 10-50 0.9991 0.9982 0.22 0.67

Cefixime 0.017 ± 0.002 0.027 ± 0.003 5-25 0.9994 0.9988 0.24 0.74

a Average of six determinations.

Table 6. Calibration equations and correlation coefficients using variable time method.

Δt (min) 1/ Δt (s-1) [Drug] (M) Calibration equation1/Δt = a + b C Correlation coefficient (r)

Cefradine anhydrous

7.5 2.22 × 10-3 2.86 × 10-5 1/Δt = -0.001 + 73.720 C 0.9646

5 3.33 × 10-3 5.73 × 10-5

3 5.56 × 10-3 8.59 × 10-5

2.5 6.67 × 10-3 1.15 × 10-4

1.5 11.11 × 10-3 1.43 × 10-4

Cefadro × il monohydrate

11 1.52 × 10-3 2.62 × 10-5 1/Δt = -0.001 + 85.911 C 0.9754

5 3.33 × 10-3 5.24 × 10-5

3 5.56 × 10-3 7.87 × 10-5

2.5 6.67 × 10-3 1.05 × 10-4

1.5 11.11 × 10-3 1.13 × 10-4

Cefaclor monohydrate

16 1.04 × 10-3 2.80 × 10-5 1/Δt = -0.002 + 94.370 C 0.9793

6 2.78 × 10-3 5.18 × 10-5

3.5 4.76 × 10-3 7.78 × 10-5

2.5 6.67 × 10-3 1.04 × 10-4

1.5 11.11 × 10-3 1.30 × 10-4

Cefale × in anhydrous

16 1.04 × 10-3 2.88 × 10-5 1/Δt = -0.003 + 114.352C 0.9724

6 2.78 × 10-3 5.76 × 10-5

3.5 4.76 × 10-3 8.64 × 10-5

1.5 11.11 × 10-3 1.15 × 10-4

1.25 13.33 × 10-3 14.40 × 10-4

Cefi × ime

16 1.04 × 10-3 1.10 × 10-5 1/Δt = -0.003 + 346.347C 0.9873

6 4.17 × 10-3 2.21 × 10-5

2.5 6.67 × 10-3 3.31 × 10-5

1.5 11.11 × 10-3 4.41 × 10-5

1 16.67 × 10-3 5.51 × 10-5

time in seconds for each of the studied drugs were con-

structed. Pseudo first-order rate constants (k') corre-

sponding to different investigated drugs concentrations

-2.303. Pseudo first-order rate constant (k') versus the

initial concentration of the studied drugs was then plot-

ted and the equations of the calibration graphs are given

in Table 7. The correlation coefficients (r) for all the

studied drugs ranged from 0.8742 to 0.9290. These low

values of r may be due to slight changes in temperature.

3.3. Method Validation Study

Fixed time method was chosen to carry out the valida-

tion study as it gives the highest values of correlation

coefficients. The proposed method was validated ac-

cording to ICH (International Conference on Harmoni-

zation) guidelines on the validation of analytical meth-

ods [23] and complied with USP 31 validation guidelines

[1]. All results were expressed as percentages, where n

represents the number of values. For the statistical

analysis Excel 2003 (Microsoft Office) was used. A 5%

significance level was selected.

3.3.1. Accuracy

The accuracy of the method was determined by investi-

gating the recovery of each of the studied drugs at three

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

439

concentration levels covering the specified calibration

range (six replicates of each concentration). The results

shown in Table 8 depict good accuracy and recovery

percentage ranged from 98.0 to 101.9%.

3.3.2. Precision

As indicated in Table 9, the results of SD and % RSD

can be considered to be very satisfactory which prove

the precision of the proposed method.

3.3.3. Selectivity

The selectivity of the proposed method for determination

of the studied drugs in the presence of frequently en-

countered excipients such as; starch, talc, lactose, glu-

cose, sucrose, magnesium-stearate and gum acacia was

studied. It was found that there is no interference from

these excipients and additives. So, the proposed method

can be considered a selective one.

3.3.4. Robustness

Robustness was examined by evaluating the influence of

small variation of method variables including; potassium

iodate concentration, potassium iodide concentration,

measurement time on the method suitability and sensi-

tivity. It was found that none of these variables signifi-

cantly affected the performance of the method (Table 10).

Table 7. Values of k' calculated from slopes of log A versus t graphs multiplied by -2.303 for different concentrations of the studied

drugs.

k' (s-1) [Drug] (M) Calibration equation k' = a + b C Correlation coefficient (r)

Cefradine anhydrous

-1.79 × 10-3 2.86 × 10-5 k' = -0.002 + 1.942 C 0.9256

-1.77 × 10-3 5.73 × 10-5

-1.63 × 10-3 8.59 × 10-5

-1.67 × 10-3 1.15 × 10-4

-1.57 × 10-3 1.43 × 10-4

Cefadro × il monohydrate

-1.85 × 10-3 5.24 × 10-5 k' = -0.002 + 4.112 C 0.9290

-1.71 × 10-3 7.87 × 10-5

-1.72 × 10-3 9.20 × 10-5

-1.72 × 10-3 1.05 × 10-4

-1.49 × 10-3 1.31 × 10-4

Cefaclor monohydrate

-1.61 × 10-3 2.59 × 10-5 k' = -0.002 + 3.203 C 0.8731

-1.50 × 10-3 5.18 × 10-5

-1.32 × 10-3 7.78 × 10-5

-1.43 × 10-3 1.04 × 10-4

-1.23 × 10-3 1.30 × 10-4

Cefale × in anhydrous

-1.43 × 10-3 2.88 × 10-5 k' = -0.002 + 5.458 C 0.8742

-1.26 × 10-3 5.76 × 10-5

-1.41 × 10-3 8.64 × 10-5

-0.90 × 10-3 1.15 × 10-4

-0.83 × 10-3 1.44 × 10-4

Cefi × ime

-1.17 × 10-3 1.10 × 10-5 k' = -0.001 + 7.215 C 0.9147

-1.14 × 10-3 2.21 × 10-5

-0.94 × 10-3 3.31 × 10-5

.00 × 10-3 4.41 × 10-5

0.84 × 10-3 5.51 × 10-5

Table 8. Accuracy of the proposed kinetic spectrophotmetric method for analysis of the studied drugs at three concentration levels.

Recovery (%) ± SDa

Drug 20 µg mL-1 30 µg mL-1 40 µg mL-1

Cefradine anhydrous 99.3 ± 0.72 98.0 ± 0.40 101.9 ± 1.00

Cefadroxil monohydrate 100.3 ± 1.13 101.4 ± 1.00 98.7 ± 0.54

Cefaclor monohydrate 101.1 ± 1.14 99.0 ± 0.22 99.7 ± 1.39

Cefalexin anhydrous 98.5 ± 1.16 99.1 ± 1.25 98.6 ± 0.82

Recovery (%) ± SDa

10 µg mL-1 15 µg mL-1 20 µg mL-1

Cefixime 100.3 ± 0.91 101.6 ± 1.43 100.7 ± 0.88

a Average of six replicates.

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

440

3.4. Applications to the Analysis of

Pharmaceutical Dosage Forms

The proposed method (fixed time) was applied success-

fully for determination of the studied drugs in their

pharmaceutical dosage forms. The results obtained (Ta-

ble 11) were satisfactory compared to those given by a

previously reported method [24]. Recovery studies were

also carried out by standard addition method [25]. Good

recoveries (96.3 to 102.8%) were obtained and these

values confirmed the absence of interference due to

common excipients (Table 12). The proposed method

couldn’t be applied to pharmaceutical formulations con-

taining L-arginine as it is a basic amino acid (its side

chain contains a strongly basic guanidine group, pKa =

13.2 [26]) and so, interferes with iodine liberation from

the studied drug.

3.5. Suggested Reaction Mechanism

It has been suggested that water-soluble acidic compounds

liberate iodine from a solution containing both KIO3 and

KI according to the reaction [27];

223 3IO3H6H5IIO  



Yellowing of the solution reveals the occurrence of the

reaction. The yellow colour of the solution is due to the

formation of I2, which immediately converted into triio-

dide ions (I2 + I− → I3

−) exhibiting absorption maxima at

290 nm and 360 nm [18]. The chemical structure of in-

vestigated cephalosporins contains –COOH group in its

moiety and hence possibly undergo a similar reaction

with iodide-iodate mixture resulting in the production of

iodine. The liberated iodine immediately reacts with

potassium iodide to give triiodide ions showing absorp-

tion maxima at 298 nm and 352 nm. The reaction se-

quence is shown in Formula (1).

OH3I6RCOOK5KIKIO6RCOOH 223







(1)

32 KIKII 



(2)

Formula (1) suggested reaction sequence of the proposed

method.

Table 9. Intra- and inter-day precision of the proposed kinetic spectrophotometric method.

Intra-day precision Inter-day precision

Drug Drug Conc. (µg mL-1) Mean ± SDa % RSD Mean ± SDa % RSD

20 98.5 ± 0.90 0.91 99.5 ± 0.81 0.81

30 98.6 ± 1.54 1.57 99.7 ± 1.17 1.17

Cefradine anhydrous

40 99.8 ± 1.02 1.03 99.6 ± 1.48 1.48

20 99.4 ± 0.99 1.00 100.6 ± 1.63 1.62

30 98.9 ± 1.12 1.13 101.0 ± 1.27 1.26

Cefadroxil monohydrate

40 99.7 ± 0.67 0.67 100.8 ± 1.15 1.14

20 101.0 ± 1.27 1.26 100.6 ± 1.63 1.62

30 100.6 ± 1.36 1.35 100.5 ± 1.15 1.14

Cefaclor monohydrate

40 99.8 ± 1.02 1.03 100.9 ± 0.99 0.98

20 100.7 ± 1.12 1.12 99.8 ± 1.65 1.65

30 98.6 ± 0.52 0.53 101.1 ± 1.20 1.19

Cefalexin anhydrous

40 100.0 ± 1.56 1.56 98.6 ± 0.94 0.95

10 100.0 ± 1.15 1.15 100.7 ± 1.12 1.12

15 100.7 ± 0.87 0.87 99.0 ± 0.97 0.98

Cefixime

20 99.9 ± 1.65 1.66 99.8 ± 1.85 1.85

a Average of six determinations.

Table 10. Robustness of the proposed kinetic spectrophotometric method.

Recovery (%) ± SDa

Experimental parameter variationCefradine anhy-

drous (30 μg mL-1)

Cefadroxil

monohydrate

(30 μg mL-1)

Cefaclor mono-

hydrate (30 μg

mL-1)

Cefalexin an-

hydrous (30 μg

mL-1)

Cefixime (15 μg

mL-1)

No variationb 97.9 ± 1.20 100.5 ± 1.23 101.5 ± 1.32 99.5 ± 0.47 99.4 ± 1.31

1 - Potassiium iodate concentration

0.28M

0.32M

98.0 ± 1.35

97.7 ± 1.65

101.9 ± 0.79

102.4 ± 0.85

99.8 ± 1.37

100.9 ± 0.99

99.4 ± 1.29

98.5 ± 0.90

101.9 ± 1.45

100.8 ± 0.88

2 - Potassium iodide concentration

1.45M

1.55M

101.4 ± 0.99

102.1 ± 1.13

98.7 ± 1.45

97.8 ± 0.64

98.7 ± 1.21

102.3 ± 1.56

99.0 ± 1.36

98.7 ± 1.23

102.1 ± 0.73

100.7 ± 0.92

3 - Measurement time

8 min

12 min

98.7 ± 1.53

101.6 ± 1.45

99.0 ± 1.75

100.0 ± 0.47

98.6 ± 0.84

99.9 ± 1.38

98.6 ± 0.64

97.6 ± 0.88

100.7 ± 0.61

97.8 ± 0.81

a Average of three determinations.

b Following the general assay procedure conditions.

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

441

Table 11. Determination of the studied drugs in their pharmaceutical dosage forms using fixed time method.

Recovery % ± SD

Drug Pharmaceutical product

Proposed method (n = 6) Reported methoda (n = 6)

Ceclor® suspensionc

99.2 ± 0.60

t = 1.225b

F = 1.778b

98.7 ± 0.80

Cefaclo monohydrate

Bacticlor® suspensiond

101.2 ± 0.60

t = 1.569

F = 1.440

100.7 ± 0.50

Duricef® tabletse

98.9 ± 0.60

t = 2.038

F = 2.250

99.8 ± 0.90

Duricef® suspensione

102.2 ± 1.60

t = 0.630

F = 1.129

101.6 ± 1.70

Duricef® capsulese

98.9 ± 1.30

t = 0.831

F = 1.173

99.5 ± 1.20

Biodroxil® capsulesf

101.4 ± 1.00

t = 1.275

F = 1.234

100.7 ± 0.90

Cefadroxil monohydrate

Biodroxil® suspensionf

99.1 ± 0.90

t = 0.906

F = 2.250

98.7 ± 0.60

Ceporex® tabletsg

97.8 ± 1.10

t = 1.054

F = 1.860

98.6 ± 1.50

Ceporex® suspensiong

100.5 ± 1.20

t = 1.470

F = 1.778

99.6 ± 0.90

Cefalexin anhydrous

Ospexin® suspensionh

99.7 ± 1.50

t = 1.153

F = 3.516

100.5 ± 0.80

Ve lo s ef ® capsulese

101.2 ± 1.50

t = 0.432b

F = 3.516b

100.9 ± 0.80

Cefradine anhydrous

Ve lo s ef ® tabletse

99.9 ± 1.20

t = 1.307

F = 1.778

100.7 ± 0.90

Cefixime Xi macef® capsulesi

98.7 ± 0.40

t = 1.644

F = 4.000

99.0 ± 0.20

a Reference 24.

b Theoretical value for t and F at 95% confidence limit, t = 2.228 and F = 5.053.

c Egyptian Pharmaceuticals and chemicals industries Co., S.A.E., Bayad El-Arab, Beni Suef, Egypt.

d Pharco Pharmaceuticals, Alexandria under license from Ranbaxy UK.

e Bristol-Myers Squibb Pharmaceutical Co., Cairo, Egypt.

f Kahira Pharm. & Chem. Ind. Co. under license from Novartis Pharma S.A.E., Cairo, Egypt.

g GlaxoSmithKline, S.A.E., El Salam City, Cairo, Egypt.

h Pharco Pharmaceuticals, Alexandria under license from Biochemie GmbH., Vienna, Austria.

i Sigma pharmaceutical industries, S.A.E., Egypt.

The confirmatory test for the presence of iodine in the

final solution of the drug is established by the blue col-

our, which appears on addition of starch solution. In case

of cefixime, it may be suggested that 3 mole of cefixime

instead of six react with iodate/ioide mixture as it con-

tains 2 carboxylic acid groups.

S. R. El-Shaboury et al. / Natural Science 2 (2010) 432-443

442

Table 12. Standard addition method for the assay of the studied drugs in their pharmaceutical dosage forms using fixed time method.

Drug Pharmaceutical formulation

Authentic drug added

(μg mL-1)

Authentic drug found

(μg mL-1) Recovery (%) ± SDa

10.00 9.95 99.5 ± 1.40

15.00 15.15 101.0 ± 1.10

Ceclor® suspension

20.00 19.80 99.0 ± 1.70

10.00 10.07 100.7 ± 1.10

15.00 14.95 99.7 ± 1.00

Cefaclor monohydrate

Bacticlor® suspension

20.00 20.19 100.9 ± 1.50

10.00 9.87 98.7 ± 1.20

15.00 15.25 101.7 ± 1.50 Duricef® tablets

20.00 19.60 98.0 ± 1.70

10.00 9.75 97.5 ± 1.60

15.00 14.90 99.3 ± 1.40 Duricef® suspension

20.00 20.40 102.0 ± 1.50

10.00 9.75 97.5 ± 1.20

15.00 14.85 99.0 ± 0.90

Duricef® capsules

20.00 20.30 101.5 ± 1.00

10.00 9.87 98.7 ± 1.10

15.00 14.85 99.0 ± 0.80 Biodroxil® capsules

20.00 20.21 101.1 ± 0.70

10.00 10.23 102.3 ± 1.30

15.00 15.30 101.0 ± 1.20

Cefadroxil monohydrate

Biodroxil® suspension

20.00 19.85 99.3 ± 0.80

10.00 10.13 101.3 ± 0.40

15.00 14.63 97.5 ± 0.60

Ceporex® tablets

20.00 20.16 100.8 ± 1.70

10.00 9.85 98.5 ± 1.30

15.00 15.09 100.6 ± 0.90 Ceporex® suspension

20.00 19.86 99.3 ± 1.80

10.00 10.22 102.3 ± 0.70

15.00 15.11 100.7 ± 1.90

Cefalexin anhydrous

Ospexin® suspension

20.00 20.18 100.9 ± 1.50

10.00 9.90 99.0 ± 0.90

15.00 14.67 97.8 ± 1.10 Ve lo s ef ® capsules

20.00 20.19 101.0 ± 0.80

10.00 10.14 101.4 ± 1.30

15.00 14.73 98.2 ± 0.60

Cefradine anhydrous

Ve lo s ef ® tablets

20.00 20.26 101.3 ± 0.90

10.00 9.89 98.9 ± 0.90

12.50 12.25 98.0 ± 1.40

Cefixime Ximacef® capsules

15.00 20.15 100.8 ± 0.70

aAverage of six determination.

4. CONCLUSIONS

The developed kinetic spectrophptometric technique is

precise, selective and accurate. The proposed method is

applicable in aqueous medium at room temperature and

thus there is no fear of decomposition of the drug due to

heat, acid or base. Statistical analysis proves that the me-

thod is repeatable and selective for the analysis of ce-

fadroxil monohydrate, cefaclor monohydrate, cefalexin

anhydrous, cefradine anhydrous and cefixime in bulk

drug and in pharmaceutical formulations and can be used

for routine quality control analyses of active drug in the

laboratories of hospitals, pharmaceutical industries and

research institutions.

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