Vol.2, No.8, 828-840 (2010) Natural Science
doi:10.4236/ns.2010.28104
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/NS/
Spectophotometric method for determination of certain
cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1,
3-diazole (NBD-Cl)
Azza H. Rageh*, Salwa R. El-Shaboury, Gamal A. Saleh, Fardous A. Mohamed
Department of Pharmaceutical Analytical Chemistry, Facutly of Pharmacy, Assiut University, Assiut, Egypt; *Corresponding Author:
azhesham@yahoo.com
Received 11 February 2010; revised 23 March 2010; accepted 30 March 2010.
ABSTRACT
A simple, accurate and precise spectropho-
tometric method has been proposed for the de-
termination of eleven cephalosporins, namely;
cefaclor monohydrate, cefadroxil monohydrate,
cefalexin anhydrous, cefradine anhydrous, ce-
fotaxime sodium, cefoperazone sodium, ceftri-
axone sodium, ceftazidime penthydrate, cefa-
zolin sodium, cefixime and cefpodoxime pro-
xetil in bulk drug and in pharmaceutical formu-
lations. The method depends on hydrolysis of
the studied drugs using 0.5M NaOH at 100°C
and subsequent reaction of the formed sulfide
ions with NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,
3-diazole) to form a yellow-colored chromogen
measured at 390 nm. Different variables affect-
ing the reaction (e.g. NaOH concentration, hy-
drolysis time, NBD-Cl concentration and dilut-
ing solvent) were studied and optimized. Under
the optimum conditions, linear relationships
with good correlation coefficients (0.9990-
0.9999) were found in the range of 5-160 μg mL-1
for all studied drugs. The limits of assay detec-
tion and quantitiation ranged from 0.289 to 5.867
and from 0.878 to 17.778 μg mL-1; respectively.
The accuracy and precision of the proposed
method were satisfactory. The method was suc-
cessfully applied for analysis of the studied
drugs in their pharmaceutical formulations and
the recovery percentages ranged from 96.6 to
103.5%.
Keywords: Spectrophotometry; Cephalo spo rins;
NBD-Cl; Pharmaceutical Analysis
1. INTRODUCTION
Cephalosporins have been used since 1948. These anti-
biotics have assumed a prominent role in modern antim-
icrobial therapy due to enhanced intrinsic microbiologi-
cal activities and favorable safety profile. Chemical
structures of cephalosporins drive from the 7-aminoce-
phalosporanic acid (7-ACA) composed of a β-lactam
ring fused with a dihydrothaizine ring (Figure 1), but
differ in the nature of substituents at the 3- and/or
7-positions of the cephem ring. These substituents affect
either the pharmacokinetic properties (3-position) or the
antibacterial spectrum (7-position) of the cephalosporins
[1,2]. Traditionally, cephalosporins are divided into first-,
second-, third-, and fourth-generation agents. Table 1
shows cephalosporins studied in this work. Several
methods have been reported for cephalosporins deter-
mination. The official procedures in pharmaceutical
preparations utilize high-performance liquid chromatog-
raphy (HPLC) [3] which is expensive. Other reported
procedures include spectrophotometric [4-9], spectro-
fluorimetric [10-13], chemiluminescence [14-16], chro-
matographic [17-20] and electrochemical methods [21-24]
and most of them are lengthy and/or tedious.
The hydrolytic degradation of cephalosporins was
very often used as a preliminary step in the analytical
procedure used for their determinations [25-32]. The
literature reveals that many spectrophotometric methods
were developed for cephalosporins determinations that
based on hydrolysis of these drugs using alkaline degra-
Figure 1. Chemical structure of 7-aminocephalosporanic acid.
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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829
Table 1. Chemical structures of the investigated cephalosporin antibiotics.
S
NR2
C
OOR
3
O
NC
O
R1
H
No. Name R1 R
2 R
3 Generation
1. Cefalexin anhydrous
H
C
NH2
-CH3 -H First
2. Cefradine anhydrous
H
C
NH2
-CH3 -H First
3. Cefadroxil monohy-
drate
H
C
NH2
HO
-CH3 -H First
4. Cefazolin sodium NN
NNCH
2
NN
SCH3
SC
H2
-Na First
5. Cefaclor monohy-
drate
H
C
NH2
-Cl -H Second
6. Cefpodoxime
proxetil
S
N
H2N
NOCH3
C
CH2OCH3 CHOCOCH(CH 3)2
CH3
O
Third
7. Cefixime
S
N
H2NC
NOCH2CO2
H
C
HCH2 -H Third
8. Cefoperazone so-
dium
HO H
C
NH
CO
N
NO
O
C2H5
NN
N
NSC
H2
CH3
-Na Third
9. Cefotaxime sodium
S
NC
NOCH3
H2NCH2OCCH3
O
-Na Third
10. Ceftazidime penta-
hydrate
S
NC
N
H2N
O
CCO
2HH3C
CH3
NC
H2
+
-H Third
11. Ceftriaxone sodium
S
NC
NOCH3
H2N
N
N
N
O
H3C
SC
H2
OH
-Na Third
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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830
dation and subsequent reaction of the formed sulfide
ions with chromogenic reagents [26,27].
NBD-Cl (Figure 2) has been reported as fluorogenic
reagent for determination of amines [33] and for spec-
trophotometric determination of many compounds
[34-41]. Thiocompounds have been reported to form
intensely colored products in an alkaline medium with
NBD-Cl which could be used for their colorimetric de-
termination [42]. It is always required to develop ana-
lytical methods using low cost techniques. UV-Vis spec-
trophotometry is still considered a convenient and eco-
nomical technique for routine analysis of drugs in phar-
maceutical formulations. On the basis of the aforemen-
tioned reasons, it was decided to develop a quantitative
method for the determination of the studied cepha-
losporins based on their alkaline hydrolysis and subse-
quent reaction of the resulting hydrolysates with
NBD-Cl, which may be used for their analysis either in
pure forms or in pharmaceutical formulations. This
method is selective for cephalosporins, since other
β-lactam antibiotics such as penicillins do not give sul-
fide ions under the degradation conditions employed
[27,43-45].
2. EXPERIMENTAL
2.1. Apparatus
Shimadzu UV-1700 PC, UV-Visible Spectrophotometer
(Tokyo, Japan), ultrasonic cleaner (Cole-Parmer, Chi-
cago, USA), sartorious handy balance-H51 (Hannover,
Germany) and MLV type thermostatically controlled
water bath (Salvis AG Emmenbruck, Luzern, Germany).
2.2. Materials and Reagents
All solvents used were of analytical-reagent grade, so-
dium hydroxide (El-Nasr Chemical Co. Cairo, Egypt)
0.5 M aqueous solution, hydrochloric acid (El-Nasr
Chemical Co. Cairo, Egypt), 4-cholor-7-nitrobenzo-
furazan [NBD-Cl] (Fluka Chemie AG, Switzerland)
freshly prepared (3 × 10-3 M) equivalent to 0.060% w/v
in acetone, samples of cephalosporins were generously
supplied by their respective manufacturers and were
used as supplied: cefaclor monohydrate and cefradine
H
N
N
H
O
Cl
NO2
Figure 2. Chemical structure of NBD-Cl.
anhydrous (Sigma Chemical Co., St. Louis, USA), ce-
fadroxil monohydrate (Amoun Pharmaceutical Industries
Co., APIC, Cairo, Egypt), cefalexin anhydrous (Gal-
axoWellcome, S.A.E., El Salam City, Cairo, Egypt), ce-
fotaxime sodium (CID, Cairo, Egypt), cefoperazone so-
dium (Pfizer Co., Egypt), ceftazidime pentahydrate and
ceftriaxone sodium (T3A Pharma Group, Assiut, Egypt),
cefpodoxime proxetil (Hoechst Marion Roussel, S. A. E.,
Cairo, Egypt), cefixime (El-Hekma Co., Cairo, Egypt)
and cefazolin sodium (Bristol-Myers Squibb Pharma-
ceutical Co., Cairo, Egypt) and pharmaceutical formula-
tions containing the studied drugs were purchased from
local market.
2.3. Preparation of Standard Solutions
Stock solutions containing 100 mg mL-1 of each cepha-
losporin were prepared in double distilled water
(methanol was used in case of cefadroxil monohydrate,
cefalexin anhydrous, cefaclor monohydrate, cefradine
anhydrous, cepodoxime proxetil and cefixime). Working
standard solutions containing 0.5-2.5 mg mL-1 (in case
of cefadroxil monohydrate and cefalexin anhydrous), 1-
6 mg mL-1 (in case of cefradine anhydrous), 2-8 mg mL-1
(in case of cefaclor monohydrate, cefazolin sodium, ce-
fotaxime sodium, ceftriaxone sodium and cefpodoxime
proxetil), 2-10 mg mL-1 (in case of cefixime) and 2-
16 mg mL-1 (in case of cefoperazone sodium and cef-
tazidime pentahydrate) were prepared by suitable dilu-
tion of the stock solution with double distilled water (in
case of cefadroxil monohydrate, cefalexin anhydrous,
cefaclor monohydrate, cepodoxime proxetil and ce-
fixime, dilution was made using methanol). The stock
and working standard solutions must be freshly pre-
pared.
2.4. Preparation of Sample Solutions
Tablets and capsules. Twenty tablets or the contents of
20 capsules were weighed, finely powdered and mixed
thoroughly. An accurately weighed amount of the pow-
der obtained from tablets or capsules equivalent to 250 mg
of each drug was transferred into a 25-mL volumetric
flask, dissolved in about 10 mL double distilled water
(10 mL methanol was used in case of cefadroxil mono-
hydrate, cefalexin anhydrous, cefaclor monohydrate,
cefradine anhydrous, cepodoxime proxetil and cefixime),
sonicated for 15 min, diluted to the mark with double
distilled water (in case of cefadroxil monohydrate, ce-
falexin anhydrous, cefaclor monohydrate, cefradine an-
hydrous, cefpodoxime proxetil and cefixime, dilution
was made using methanol), mixed well and filtered; the
first portion of the filtrate was rejected. Further dilutions
with the same solvent were made to obtain sample solu-
tion containing the specified concentration for each drug
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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831
as mentioned under the preparation of standard solutions
and then the general procedure was followed.
Vials and powder for oral suspension. An accurately
weighed amount of powder equivalent to 250 mg of each
drug was transferred into a 25-mL volumetric flask, then
the procedure was followed as under tablets and capsules
beginning from (dissolved in about 10 mL double dis-
tilled water……….).
2.5. General Procedure
Accurately measured one milliliter aliquot volume of the
standard or sample solutions was transferred into 10-mL
volumetric flask. 5 mL of 0.5 M NaOH were added and
the flask was heated in a boiling water-bath for 30 min,
cooled to room temperature and completed to volume
with double distilled water. One milliliter of the resulting
drug hydrolysate was pipetted into 10-mL volumetric
flask, 1.0 mL of 3 × 10-3 M NBD-Cl was added followed
by 1 mL of concentrated HCl. The resulting solution was
mixed well and the flask was completed to volume with
ethanol. The absorbance was measured at 390 nm against
reagent blank treated similarly.
3. RESULTS AND DISCUSSION
3.1. Absorption Spectra
As shown in Figure 3, the absorption spectrum of
NBD-Cl in acetone shows a maximum absorption at
340 nm. All the investigated drugs after alkaline hy-
drolysis give a very weak absorption taking cefalexin
anhydrous hydrolysate as a representative example
which gives a very broad absorption maximum at 350 nm.
The interaction colored product of cefalexin anhydrous
hydrolysate with NBD-Cl shows absorption maximum at
390 nm (Figure 3).
Figure 3. Absorption spectra of (a) NBD-Cl (3 ×
10-3 M), (b) cefalexin anhydrous hydrolysate alone
(20 µg mL-1) and (c) the reaction colored product
between NBD-Cl and cefalexin anhydrous hydro-
lysate.
3.2. Optimization of Reaction Variables
Since the developed method depends on the formation of
colored product by the interaction of NBD-Cl with sul-
fide ions resulted from the alkaline degradation of
cephalosporins so, optimization studies were carried out
extensively to find the optimum conditions for the alka-
line degradation and subsequently the optimum yield of
sulfide ions and the maximum stability of the chromogen
formed taking cefalexin anhydrous (15 μg mL-1) as a
representative example for these studies. These variables
include:
Effect of NaOH concentration. The influence of so-
dium hydroxide concentration on producing the maxi-
mum absorption intensity was investigated using 0.1-
1.0 M NaOH keeping other factors constant. Maximum
absorption readings were obtained upon using 0.5 M
NaOH; above this concentration and up to 1 M NaOH,
the absorbance remains constant. So, this concentration
was selected for further work (Figure 4).
Effect of hydrolysis time. The effect of hydrolysis time
on the absorption intensity was studied using different
heating times in a boiling water bath (at 100°C) starting
from 10 min until 2 hours and the reaction was carried
out as usual. The obtained absorbance readings were
plotted against hydrolysis time. The maximum absorp-
tion intensity was attained after 20 min and remained
stable for at least 100 min. Thirty minutes hydrolysis
time was used in all subsequent experiments as shown in
Figure 5.
Effect of NBD-Cl concentration. The concentration of
NBD-Cl, for the maximum color development was var-
ied in the range of 0.75 × 10-3-4 × 10-3 M. It was found
that 1 mL of 3 × 10-3 M NBD-Cl was the most suitable con-
0
0.1
0.2
0.3
0.4
0.5
0.6
00.1 0.20.3 0.4 0.5 0.6 0.7 0.80.911.1
NaOH concentration (M)
Absorbance, 390 nm
Figure 4. Effect of NaOH concentration on the absorbance
of the reaction colored product at 390 nm.
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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832
0
0.1
0.2
0.3
0.4
0.5
0.6
0 20406080100120140
Hydrolysis time (min)
Absorbance, 390 nm
Figure 5. Effect of hydrolysis time on the absorbance of the
reaction colored product at 390 nm.
Figure 6. Effect of NBD-Cl concentration on the ab-
sorbance of the reaction colored product at 390 nm.
centration for determination of the studied drugs as
shown in Figure 6. Owing to the presence of labile chlo-
ride, a daily fresh solution is recommended.
Effect of type and concentration of acid. Different ac-
ids such as sulfuric, hydrochloric, perchloric, nitric and
acetic acids were tested to determine the most suitable
acid for the reaction. One milliliter of concentrated hy-
drochloric acid was selected in this study as it gave the
highest absorbance readings taking cefalexin anhydrous
(15 μg mL-1) as a representative example (Table 2).
Further investigations were carried out in order to find
the most suitable concentration of hydrochloric acid. It
was observed that higher absorbance readings and more
reproducible results were obtained upon increasing hy-
drochloric acid concentration. As a result of these inves-
tigations, 1 mL of concentrated hydrochloric acid was
used for subsequent work.
Effect of reaction time. The reaction between the in-
vestigated drugs hydrolysates and NBD-Cl was very
Table 2. Effect of different acids on the absorbance readings of
the reaction colored product of cefalexin anhydrousa with
NBD-Cl.
Acid (1mL) Absorbanceb
Hydrochloric acid 0.460
Sulfuric acid 0.400
Perchloric acid 0.413
Acetic acid 0.210
Nitric acid 0.315
aCefalexin anhydrous concentration used is 15 μg mL-1; bAverage
of three determinations.
rapid and the interaction colored product can survive
before dilution unchanged for at least 1 hour. However,
measurements were achieved instantaneously.
Effect of diluting solvent. Different solvents were
tested in order to select the most appropriate solvent for
optimum color development. The results given in Table
3 show small shifts in the position of the maximum ab-
sorption peak. The absorption intensities were slightly
influenced. Ethanol was used throughout this work be-
cause it gave the highest absorbance readings and the
most reproducible results.
Stability of the reaction colored product. Stability time
was obtained by following the absorbance readings of
the developed reaction product for 24 hours at room
temperature (25 ± 5°C). It was found that the produced
color was stable for 24 hours for all studied drugs.
3.3. Calibration Curves
Linear relationship was obtained for all studied drugs by
applying the developed method (Table 4). Good linear-
ity of the calibration curves were clearly evident by ex-
cellent correlation coefficients which ranged from
0.9990 to 0.9999 and coefficients of determination
ranged from 0.9978 to 0.9998. This wide variation in the
linearity range may be attributed to the different yields
Table 3. Effect of solvent on λ max and the absorbance of the
formed chromogen between cefalexin anhydrousa and NBD-Cl.
Solvent λmax(nm) Ab
Water 404 0.404
Ethanol 390 0.470
Methanol 391 0.401
Acetone 392 0.427
Acetonitrile 398 0.456
Propan-1-ol 390 0.467
Propan-2-ol 389 0.463
Dimethylformamaide 393 0.425
Dimehtylsulfoxide 401 0.458
aCefalexin anhydrous concentration is 15 μg mL-1; bAverage of 3
determinations.
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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833
Table 4. Summary of quantitative parameters and statistical data using the proposed procedure.
Drug Intercept
(a) ± SDa
Slope
(b) ± SDa
Linear-
ity
Range
(µg
mL-1)
Correlation
coefficient
(r)
Determina-
tion coeffi-
cient
(r2)
LODb
(µg
mL-1)
LOQc
(µg
mL-1)
Cefadroxil monhy-
drate -0.013 ± (3.6 × 10-3) 0.041 ± (0.2 × 10-3)5-25 0.9999 0.9998 0.29 0.88
Cefalexin anhydrous -0.126 ± (5.0 × 10-2) 0.398 ± (1.2 × 10-3)5-25 0.9999 0.9998 0.42 1.26
Cefradine anhydrous 0.076 ± (7.5 × 10-3) 0.013 ± (1.5 × 10-4)10-60 0.9999 0.9998 1.90 5.77
Cefaclor monohy-
drate 0.055 ± (1.3 × 10-2) 0.010 ± (2.1 × 10-4)20-80 0.9996 0.9992 4.29 13.00
Cefazolin sodium 0.016 ± (4.3 × 10-3) 0.009 ± (1.9 × 10-4)20-80 0.9994 0.9988 1.58 4.78
Ceftriaxone sodium 0.033 ± (5.3 × 10-3) 0.010 ± (0.5 ×10-4) 20-80 0.9996 0.9992 1.75 5.30
Cefotaxime sodium 0.056 ± (1.2 × 10-2) 0.010 ± (1.5 × 10-4)20-80 0.9990 0.9980 3.96 12.00
Cefpodoxime proxetil 0.046 ± (1.6 × 10-2) 0.009 ± (1.3 × 10-4)20-80 0.9990 0.9980 5.87 17.78
Cefixime 0.095 ± (3.2 × 10-3) 0.007 ± (0.2 × 10-4)20-100 0.9989 0.9978 1.51 4.57
Cefoperazone sodium 0.019 ± (8.3 × 10-3) 0.005 ± (0.6 × 10-4)20-160 0.9998 0.9996 5.48 16.60
Ceftazidime penta-
hydrate 0.048 ± (7.4 × 10-3) 0.005 ± (0.7 × 10-4)20-160 0.9994 0.9988 4.88 14.80
aAverage of six determinations; b Limit of detection; c Limit of quantitation.
of sulfide ions from the studied cephalosporins [45].
3.4. Method Validation Study
The method was validated according ICH guidelines on
the validation of analytical methods [46] and complied
with USP 31 validation guidelines [3]. All results were
expressed as percentages, where n represents the number
of values. For the statistical analysis Excel 2003 (Mi-
crosoft Office) was used. A 5% significance level was
selected.
LOD and LOQ. The limits of detection and quantita-
tion for all studied drugs ranged from 0.29 to 5.87 and
from 0.88 to 17.78 μg mL-1; respectively which indicate
high sensitivity of the proposed method (Table 4).
Accuracy. The accuracy of the method was deter-
mined by investigating the recovery of each of the stud-
ied drugs at three concentration levels covering the spe-
cified range (six replicates of each concentration). The
results shown in Table 5 depict good accuracy and re-
covery percentage ranged from 98.0 to 102.3%.
Precision. As shown in Table 6, the small values of
SD and % RSD point to high precision of the proposed
method.
Selectivity. The effect of the presence of common ex-
cipients such as; starch, talc, lactose, glucose, sucrose,
magnesium-stearate and gum acacia was studied. It was
found that no interference was introduced by any of
them.
Robustness. Robustness was examined by evaluating
the influence of small variation in the experimental pa-
rameters on the analytical performance of the proposed
method [47]. The studied parameters were: NaOH con-
centration, NBD-Cl concentration, heating temperature
and heating time on the method suitability and sensitivity.
It was found that none of these variables significantly
affects the performance of the method (Table 7) which
indicates the robustness of the proposed method.
3.5. Applications to the Analysis of
Pharmaceutical Dosage Forms
The proposed method was applied successfully for de-
termination of the studied drugs in their pharmaceutical
dosage forms. Six replicate measurements were made in
each case, the results obtained were validated by com-
parison with a previously reported method [48]. No sig-
nificant difference was found by applying t- and F-tests
at 95% confidence level indicating good accuracy and
precision (Table 8). Recovery studies were also carried
out by standard addition method [49]. The results in Ta-
ble 9 indicate good recoveries (96.0 to 103.8%) and
confirm that there is no interference from frequently
encountered excipients or additives.
3.6. Suggested Reaction Mechanism
Cephalosporins were previously reported to produce
sulfide ions upon alkaline degradation and it was found
to be one of their major degradation products [43-45,
50-55]. NBD-Cl is an active halide derivative, which
was considered as a likely target for good nuclophiles,
under alkaline conditions, such as amines, amino acids
and thiocompounds [40-42].
In the proposed method, sulfide ions were allowed to
react with NBD-Cl via SN2 mechanism. The high nu-
cleophilicity of sulfide ions, the presence of Cl anion as
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834
Table 5. Accuracy of the proposed method for analysis of the studied drugs at three concentration levels.
Recovery (%) ± SD
a
Drug 25 µg mL-1 50 µg mL-1 75 µg mL-1
Cefaclor monohydrate 100.6 ± 0.93 101.4 ± 0.75 102.1 ± 0.30
Ceftriaxone sodium 99.3 ± 0.52 100.6 ± 0.96 100.2 ± 0.51
Cefotaxime sodium 99.7 ± 1.35 101.5 ± 0.83 101.3 ± 1.16
Cefixime 98.3 ± 1.24 98.7 ± 0.58 98.6 ± 0.73
Cefazolin sodium 101.1 ± 1.08 98.9 ± 0.60 102.3 ± 0.68
Cefpodoxime proxetil 99.4 ± 0.35 99.4 ± 0.47 99.0 ± 0.29
Recovery (%) ± SD
a
Drug 10 µg mL-1 15 µg mL-1 20 µg mL-1
Cefadroxil monohydrate 99.9 ± 1.31 100.4 ± 0.83 99.1 ± 0.90
Cefalexin anhydrous 98.6 ± 0.26 102.2 ± 1.29 98.0 ± 0.41
Recovery (%) ± SD
a
Drug 40 µg mL-1 80 µg mL-1 120 µg mL-1
Ceftazidime pentahydrate 102.3 ± 0.86 98.9 ± 1.25 99.6 ± 0.82
Cefoperazone sodium 98.0 ± 0.70 100.3 ± 1.11 101.4 ± 1.03
Recovery (%) ± SD
a
Drug 15 µg mL-1 30 µg mL-1 45 µg mL-1
Cefradine anhydrous 98.3 ± 0.51 100.8 ± 0.66 100.7 ± 0.87
aAverage of six replicates.
Table 6. Intra- and inter-day precision of the proposed spectrophotometric method.
Intra-day precision Inter-day precision
Drug Drug Conc. (µg
mL-1) Mean ± SDa % RSD Mean ± SDa % RSD
Cefaclor monohydrate 25 99.6 ± 0.93 0.93 99.4 ± 1.29 1.30
50 99.9 ± 1.65 1.66 98.5 ± 0.90 0.91
75 100.1 ± 1.40 1.40 100.7 ± 1.12 1.12
Cefalexin anhydrous 10 100.3 ± 1.17 1.16 98.9 ± 1.23 1.25
15 100.4 ± 1.53 1.52 99.0 ± 0.97 0.98
20 100.1 ± 1.16 1.16 100.5 ± 0.88 0.88
Cefadroxil monohydrate 10 99.4 ± 0.99 1.00 101.0 ± 1.09 1.08
15 99.9 ± 0.85 0.85 100.5 ± 0.77 0.76
20 100.2 ± 1.37 1.37 98.7 ± 0.68 0.69
Cefradine anhydrous 15 100.1 ± 1.03 1.02 99.5 ± 1.13 1.14
30 100.0 ± 1.15 1.15 98.4 ± 0.85 0.86
45 100.6 ± 1.16 1.15 100.4 ± 1.54 1.54
Cefoperazone sodium 40 99.6 ± 0.93 0.93 101.3 ± 1.43 1.41
80 99.7 ± 0.67 0.67 101.7 ± 1.63 1.61
120 100.3 ± 1.35 1.34 99.1 ± 1.40 1.41
Ceftazidime pentahydrate 40 100.0 ± 1.39 1.39 100.9 ± 0.99 0.98
80 99.6 ± 1.30 1.31 101.3 ± 1.27 1.25
120 99.9 ± 1.04 1.05 98.9 ± 1.12 1.13
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
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835
Table 6. (Continued).
Intra-day precision Inter-day precision
Drug Drug Conc. (µg
mL-1) Mean ± SDa % RSD Mean ± SDa % RSD
Ceftriaxone sodium 25 100.4 ± 1.57 1.56 99.2 ± 0.99 1.00
50 101.0 ± 1.27 1.26 100.8 ± 1.15 1.14
75 99.8 ± 1.38 1.39 98.6 ± 1.54 1.57
Cefotaxime sodium 25 99.3 ± 1.05 1.06 99.0 ± 0.77 0.78
50 98.8 ± 0.78 0.79 101.4 ± 1.46 1.44
75 99.4 ± 1.29 1.30 98.5 ± 0.91 0.93
Cefixime 25 99.5 ± 0.81 0.81 100.9 ± 0.99 0.98
50 99.8 ± 1.02 1.03 99.7 ± 1.17 1.17
75 99.6 ± 1.48 1.48 98.3 ± 1.65 1.67
Cefazolin sodium 25 100.5 ± 1.15 1.14 100.6 ± 0.68 0.67
50 100.6 ± 1.36 1.35 99.6 ± 1.35 1.35
75 101.4 ± 0.74 0.73 98.8 ± 1.12 1.14
Cefpodoxime proxetil 25 100.4 ± 0.89 0.89 101.3 ± 0.77 0.76
50 100.5 ± 1.15 1.15 99.7 ± 1.55 1.55
75 100.0 ± 1.70 1.70 100.6 ± 1.63 1.62
aAverage of six determinations.
Table 7. Robustness of the proposed spectrophotometric method.
Recovery (%) ± SDa
Experimental parameter variationCefadroxil
monohydrate
(20 µg mL-1)
Cefalexin
anhydrous
(20 µg mL-1)
Cefradine
anhydrous
(40 µg mL-1)
Cefaclor
monohydrate
(60 µg mL-1)
Cefopera-
zone sodium
(80 µg mL-1)
Ceftazidime
pentahydrate
(80 µg mL-1)
No variationb 99.4 ± 1.21 99.8 ± 0.31 99.5 ± 1.82 99.5 ± 1.13 99.2 ± 0.56 99.5 ± 0.47
1- NaOH concentration
0.45 M
0.55 M
98.3 ± 0.85
98.6 ± 1.19
97.9 ± 1.20
100.9 ± 1.15
101.5 ± 1.32
98.2 ± 0.52
97.5 ± 0.54
101.3 ± 0.77
101.8 ± 1.11
99.4 ± 1.31
97.4 ± 1.12
98.3 ± 1.34
2- NBD-Cl concentration
2.8 × 10-3M
3.2 × 10-3M
102.0 ± 0.25
98.5 ± 1.31
100.3 ± 1.35
100.9 ± 0.92
98.2 ± 1.56
98.0 ± 1.15
102.1 ± 0.35
98.5 ± 0.91
100.9 ± 1.58
98.5 ± 0.83
100.9 ± 1.15
102.0 ± 0.88
3- Heating temperature
95°C
100°C
98.8 ± 0.78
99.6 ± 1.35
97.9 ± 1.20
100.8 ± 1.60
102.4 ± 156
100.4 ± 0.90
102.7 ± 2.21
99.8 ± 1.02
101.0 ± 1.27
101.3 ± 1.27
101.4 ± 2.04
98.0 ± 2.15
4- Heating time
25 min
35 min
100.5 ± 1.23
100.1 ± 1.40
99. 2 ± 0.99
100.4 ± 1.53
98.5 ± 1.57
98.7 ± 0.68
97.5 ± 1.98
99.6 ± 0.93
99.1 ± 1.40
100.9 ± 0.99
99.4 ± 1.29
98.5 ± 0.90
Table 7. (Continued).
Recovery (%) ± SDa
Experimental parameter variationCeftriaxone so-
dium
(60 µg mL-1)
Cefotaxime
sodium
(60 µg mL-1)
Cefixime
(60 µg mL-1)
Cefazolin
sodium
(60 µg mL-1)
Cefpodoxime
proxetil
(60 µg mL-1)
No variationb 99.5 ± 0.66 97.6 ± 1.55 100.7 ± 0.98 99.5 ± 1.23 99.5 ± 1.01
1- NaOH concentration
0.45 M
0.55 M
100.2 ± 1.35
98.7 ± 0.98
99.5 ± 0.67
98.3 ± 0.49
99.6 ± 1.27
98.4 ± 0.73
98.6 ± 0.88
102.4 ± 1.145
98.1 ± 0.60
99.5 ± 1.27
2- NBD-Cl concentration
2.8 × 10-3M
3.2 × 10-3M
99.6 ± 1.15
98.0 ± 0.75
102.0 ± 0.71
102.3 ± 0.58
102.3 ± 1.30
99.5 ± 0.69
99.7 ± 1.35
101.5 ± 0.70
98.4 ± 1.40
99.9 ± 0.49
3- Heating temperature
95°C
100°C
99.0 ± 1.30
97.5 ± 0.68
99.4 ± 0.66
98.4 ± 1.13
98.7 ± 0.66
101.9 ± 1.35
100.6 ± 0.48
97.5 ± 1.44
100.8 ± 0.72
99.1 ± 0.65
4- Heating time
25 min
35 min
101.1 ± 1.44
98.1 ± 0.81
97.8 ± 0.90
101.4 ± 1.41
99.6 ± 0.56
100.8 ± 1.27
99.7 ± 0.70
101.9 ± 0.72
97.8 ± 0.49
98.0 ± 1.34
aAverage of three determinations;
bFollowing the general assay procedure conditions.
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/NS/
836
Table 8. Determination of the studied drugs in their pharmaceutical dosage forms.
Recovery % ± SD
Drug Pharmaceutical product
Proposed method
(n = 6)
Reported methodb
(n = 6)
Cefaclor
monohydrate
Ceclor® suspensionc
250 mg of cefaclor monohydrate/5 mL
97.8 ± 0.5, t = 0.382a
F = 1.562a 97.7 ± 0.40
Bacticlor® suspensiond
250 mg of cefaclor anhydrous/5 mL
97.2 ± 0.5, t = 1.913
F = 1.562 96.7 ± 0.40
Cefadroxil
monohydrate
Duricef® tabletse
1 g of cefadroxil monohydrate/tablet
98.7 ± 0.3, t = 2.038
F = 2.250 99.0 ± 0.20
Duricef® suspensione
250 mg of cefadroxil monohydrate/5 mL
96.6 ± 1.3, t = 1.605
F = 2.641 97.6 ± 0.80
Duricef® capsulese
500 mg of cefadroxil monohydrate/capsule
97.9 ± 1.3, t = 1.332
F = 1.032 98.9 ± 1.30
Biodroxil® capsulesf
500 mg of cefadroxil monohydrate/capsule
102.4 ± 1.4, t = 0.930
F = 1.361 101.7 ± 1.20
Biodroxil® suspensionf
250 mg of cefadroxil monohydrate/5 mL
103.1 ± 0.6, t = 1.359
F = 2.250 102.7 ± 0.40
Cefalexin
anhydrous
Ceporex® tabletsg
500 mg of cefalexin anhydrous/tablet
99.3 ± 1.6, t = 0.646
F = 1.778 98.7 ± 1.20
Ceporex® suspensiong
250 mg of cefalexin anhydrous/5 mL
99.0 ± 1.5, t = 0.735
F = 3.516 98.5 ± 0.80
Ospexin® suspensionh
250 mg of cefalexin anhydrous/5 mL
103.5 ± 1.5, t = 0.576
F = 3.516 103.1 ± 0.80
Cefradine
anhydrous
Velosef® capsulese
250 mg of cefradine anhydrous/capsule
97.7 ± 0.5, t = 1.530
F = 1.562 97.3 ± 0.40
Velosef® tabletse
1 g of cefradine anhydrous/tablet
103.3 ± 1.2, t = 1.019
F = 2.250 102.7 ± 0.80
Velosef® suspensione
205 mg of cefradine anhydrous/5mL
99.0 ± 1.5, t = 0.588
F = 3.516 98.5 ± 0.80
Velosef® vialse
1 g of cefradine anhydrous/vial
97.9 ± 1.2, t = 0.267
F = 1.778 97.7 ± 0.90
Cefotax® vialsi
500 mg of cefotaxime sodium/vial
98.2 ± 1.8, t = 0.365,
F = 3.932; 97.9 ± 0.90
Cefotaxime
sodium Claforan® vialsj
500 mg of cefotaxime sodium/vial
97.9 ± 1.2, t = 0.326,
F = 1.778; 97.7 ± 0.90
Ceftazidime
pentahydrate
Fortum® vialsk
500 mg of ceftazidime pentahydrate/vial
98.9 ± 0.6, t = 1.460,
F = 4.001; 98.5 ± 0.30
Cefoperazone
sodium
Cefozon® vialsl
500 mg of cefoperazone sodium/vial
102.3 ± 1.4, t = 1.721,
F = 4.003; 101.2 ± 0.70
Ceftriaxone
sodium
Ceftriaxone® vialsm
500 mg of ceftriaxone sodium/vial
97.7 ± 0.4, t = 1.643,
F = 4.002; 98.0 ± 0.20
Cefixime Xi macef® capsulesn
400 mg of cefixime/capsule
102.1 ± 1.4, t = 0.797
F = 1.361 101.5 ± 1.20
Cefazolin
sodium
Zinol® vialso
500 mg of cefazolin sodium/vial
98.9 ± 1.3, t = 1.493
F = 3.449 98.0 ± 0.70
Cefpodoxime
proxetil
Orelox® tabletsq
100 mg of cefpodoxime proxetil/tablet
98.2 ± 1.8, t = 0.298
F = 3.932 97.9 ± 0.90
a Theoretical value for t and F at 95% confidence limit, t = 2.228 and F = 5.053; b Reference 48; cEgyptian Pharmaceuticals and chemicals in-
dustries 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;
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/NS/
837
g GlaxoSmithKline, S.A.E., El Salam City, Cairo, Egypt; h Pharco Pharmaceuticals, Alexandria under license from Biochemie GmbH., Vienna,
Austria; i T3A Pharma Group, Assiut, Egypt; j Hoechst Orient, S.A.E., Cairo, Egypt ; k GalaxoWellcome, S.A.E., El Salam City, Cairo, Egypt;
lEgyptian International Pharmaceutical Industries Co., El Asher Ramadan City, Cairo, Egypt; m Kahira Pharm. & Chem. Ind. Co. under licence
from Novartis Pharma S.A.E., Cairo, Egypt; n Sigma pharmaceutical industries, S.A.E., Egypt; oPharco Pharmaceuticals, Alexandria,
Egypt; q Aventis, Zeitoun, Cairo, Egypt.
Table 9. Standard addition method for the assay of the studied drugs in their pharmaceutical dosage forms by the proposed method.
Drug Pharmaceutical
formulation
Authentic drug added
(μg mL-1)
Authentic drug found
(μg mL-1)
Recovery (%) ± SDa
10.00 9.97 99.7 ± 1.21
20.00 19.40 97.0 ± 1.80
Ceclor® suspension
30.00 28.83 96.1 ± 1.52
10.00 10.06 100.6 ± 1.10
20.00 19.26 96.3 ± 0.81
Cefaclor
monohydrate
Bacticlor® suspension
30.00 29.40 98.0 ± 0.90
5.00 5.11 102.2 ± 1.41
10.00 9.95 99.5 ± 1.72
Duricef®
tablets 15.00 14.85 99.0 ± 0.50
5.00 4.90 98.0 ± 1.81
10.00 10.02 100.2 ± 1.63
Duricef® suspension
15.00 14.64 97.6 ± 1.12
5.00 4.83 96.6 ± 1.50
10.00 9.60 96.0 ± 1.30
Duricef® capsules
15.00 14.78 98.5 ± 0.71
5.00 4.89 97.8 ± 1.54
10.00 10.36 103.6 ± 0.91
Biodroxil® capsules
15.00 14.98 99.9 ± 0.60
5.00 4.87 97.4 ± 1.91
10.00 9.84 98.4 ± 0.42
Cefadroxil
monohydrate
Biodroxil® suspension
15.00 15.38 102.5 ± 1.20
5.00 4.98 99.6 ± 1.41
10.00 10.07 100.7 ± 1.73
Ceporex® tablets
15.00 14.82 98.8 ± 0.62
5.00 5.05 101.0 ± 1.00
10.00 9.79 97.9 ± 1.81
Ceporex® suspension
15.00 15.06 100.4 ± 0.91
5.00 4.95 99.0 ± 1.30
10.00 9.92 99.2 ± 1.81
Cefalexin anhydrous
Ospexin® suspension
15.00 14.68 97.9 ± 1.94
10.00 9.85 98.5 ± 0.85
20.00 20.09 100.5 ± 0.63 Velosef® capsules
30.00 28.86 96.2 ± 1.11
10.00 9.69 96.9 ± 0.80
20.00 19.85 99.3 ± 0.50
Velosef®
tablets 30.00 30.10 100.3 ± 0.71
10.00 9.72 97.2 ± 0.92
20.00 19.94 99.7 ± 1.50 Velosef® suspension
30.00 31.14 103.8 ± 1.93
10.00 10.16 101.6 ± 1.10
20.00 19.62 98.1 ± 1.63
Cefradine
anhydrous
Velosef®
vials 30.00 29.85 99.5 ± 1.94
20.00 20.34 101.7 ± 1.33
40.00 38.76 96.6 ± 1.63
Ceftazidime
pentahydrate
Fortum®
vial 60.00 61.32 102.2 ± 1.32
10.00 9.66 96.6 ± 0.7
20.00 20.20 101.0 ± 0.9
Cefotax®
vials 30.00 29.30 97.6 ± 1.5
10.00 10.35 103.5 ± 1.8
20.00 19.60 98.0 ± 1.6
Cefotaxime
Sodium Claforan®
vials 30.00 29.19 97.3 ± 0.7
20.00 19.50 97.5 ± 1.8
40.00 38.88 97.2 ± 0.9
Cefoperazone
Sodium
Cefozon®
vials 60.00 59.46 99.1 ± 0.7
10.00 10.08 100.8 ± 1.4
20.00 19.36 96.8 ± 1.5
Ceftriaxone
Sodium Ceftriaxone vials
30.00 30.98 103.3 ± 1.3
A. H. Rageh et al. / Natural Science 2 (2010) 828-840
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/NS/
838
Table 9 (Continued)
Drug Pharmaceutical
formulation
Authentic drug added
(μg mL-1)
Authentic drug found
(μg mL-1)
Recovery (%) ± SDa
15.00 14.76
98.4 ± 0.5
30.00 29.82
99.4 ± 1.1
Cefixime Ximacef® capsules
45.00 44.91
99.8 ± 1.4
10.00 9.78
97.8 ± 0.8
20.00 19.87
99.4 ± 0.7
Cefazolin
Sodium
Zinol®
vials 30.00 30.27
100.9 ± 1.7
10.00 9.84
98.4 ± 0.9
20.00 19.83
99.2 ± 1.5
Cefpodoxime
proxetil
Orelox®
tablets 30.00 29.60
98.6 ± 1.6
a Average of six determinations.
a good leaving group at position 4 in addition to the
presence of nitro group as an electron withdrawing
group at position 7 of the aromatic ring in NBD-Cl result
in replacement of Cl anion with the attacking sulfide
ions which in turn lead to the formation of a yellow-
colored chromophore (λmax at 390 nm). The reaction
product is stable in strong acidic medium, moreover
acidification could minimize possible competition be-
tween the generated sulfide nucloephile and excess OH
which may lead to decrease in the chromogen formed.
The proposed reaction mechanism is given in the fol-
lowing scheme:
N
H
O
H
N
Cl
NO2
0.5M NaOH
100°C/ 30 min
Conc. HCl
N
H
O
H
N
SH
NO2
S
N
R2
C
OR
3
O
HNC
O
R1
Sulfide ions
Scheme 1 Suggested reaction mechanism between sul-
fide ions and NBD-Cl
The production of sulfide ions was confirmed by car-
rying out specific qualitative tests such as dilute hydro-
chloric acid, cadmium acetate, sodium nitroprusside and
methyelene blue tests [56] or by comparing λmax of the
formed chromogen with that obtained after applying the
developed method to sodium sulfide and the same results
were obtained.
4. Conclusions
The developed spectrophotometric method is precise,
accurate and sensitive. No interference from the fre-
quently encountered excipients and additives. Statistical
analysis proves that the method could be applied for the
analysis of the studied drugs in their pure forms and in
pharmaceutical formulations.
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