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;
[e-mail]; [e-mail]; *Corresponding Author:
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

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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.
S
NR
2
C
OOR
3
O
NC
O
R
1
H
12
3
4
6
7
85
No. Name R1 R2 Generation
1. Cefalexin anhydrous
H
C
NH2
-CH3 First
2. Cefradine anhydrous
H
C
NH2
-CH3 First
3. Cefadroxil
monohydrate
H
C
NH2
HO
-CH3 First
4. Cefaclor
monohydrate
H
C
NH2
-Cl Second
5. Cefixime
S
N
H
2NC
NOCH2
CO2
H
C
H
CH2
Third

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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
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.

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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:
n
Ck
t
A
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
t
A
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
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

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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
(C) were calculated from the slopes, multiplied by
-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

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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.

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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.

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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.

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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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