Analytical determination of cefadroxil/Asian Journal of Pharmaceutical Sciences 2008, 3(5): 217-232

Recent developments in the analytical determination of cefadroxil

M. Espinosa Boscha, A.J. Ruiz Snchezb, F. Snchez Rojasc,*, C. Bosch Ojedac
a

Department of Pharmacy, General Hospital, University Hospital Virgen del Roco, Manuel Siurot s/n, 41013, Sevilla, Spain
b
Department of Organic Chemistry, Faculty of Sciences, University of Mlaga, Campus Teatinos s/n, 29071, Mlaga, Spain
c
Department of Analytical Chemistry, Faculty of Sciences, University of Mlaga, Campus Teatinos s/n, 29071 Mlaga, Spain
Received 24 April 2008; Revised 31 June 2008; Accepted 5 October 2008

_____________________________________________________________________________________________________________

Abstract
Cefadroxil is an orally active semi-synthetic -lactam antibiotic from the cephalosporin group, characterized by its prolonged action.
This compound is effective against susceptible bacteria causing infections of the urinary tract, skin and soft tissue as well as pharyngitis
and laryngitis.
A variety of analytical methods have been proposed for the determination of cefadroxil in biological fluids and pharmaceutical
samples. This review includes the most relevant analytical methodologies used for its determination in recent years.
Keywords: Cefadroxil; Antibiotics; Biological and pharmaceutical samples; Review
_____________________________________________________________________________________________________________

1. Introduction

urinary tract infections, skin, and soft tissues infections

that are often caused by sensitive bacteria. Cefadroxil
(CFL), represented in Fig. 1, is chemically designated
as 8-[2-amino-2-(4-hydroxyphenyl)-acetyl]amino-4methyl-7-oxo-2-thia-6-azabicyclo[4.2.0]oct-4-ene-5-car
boxylic acid. It has the formula C16H17N3O5S. CFL is a
first generation cephalosporin antibacterial drug that is
the para-hydroxy derivative of cefalexin, and is used in
the treatment of mild to moderate susceptible infections.
CFL is a broad spectrum antibiotic effective in Grampositive and Gram-negative bacterial infections; it is a
bactericidal antibiotic. CFL is active against many
bacteria, including Staphylococcus aureus, Streptococcus
pneumoniae, Streptococcus piogenes, Moraxella catarrhalis, Escherichia coli, Klebsiella and Proteus mirabilis.
CFL is almost completely absorbed from the gastrointestinal tract and is generally well tolerated. About
20% of CFL is reported to be bound to plasma proteins
and its plasma half-life is about 1.5 h. CFL is widely
distributed in body tissues and fluids. More than 90% of

Cephalosporins are -lactam antibiotics which are

closely related in structure and in their anti-bactericidal
mechanism of action mechanism to penicillin and cephamicin which are also -lactam antibiotics. The main
nucleus of cephalosporins is 7-amino cephalosporanic
acid (7-ACA) which is a cephem derivative and is
obtained from cephalosporin C which is formed as a
fermentation product of the Cephalosporium acremonium
type of fungus. Cephalosporins which are used for therapeutic purposes are semisynthetic products. They are
divided in four generations (Table 1), based approximately on the time of their discovery and their antimicrobial properties. In general, progression from first to
fourth generation is associated with a broadening of the
Gram-negative antibacterial spectrum, some reduction in
activity against Gram-positive organisms, and enhanced
resistance to -lactamases.
-Lactam antibiotics, i.e., penicillins and cephalosporins, are probably the most widely used class of
medicines to treat respiratory tract infections, prostatitis,

NH2

__________
*Corresponding author. Address: Department of Analytical
Chemistry, Faculty of Sciences, University of Mlaga, Campus
Teatinos s/n, 29071 Mlaga, Spain. Tel.: +34-95-2137393; Fax:
+34-95-2132000.
E-mail: fsanchezr@uma.es

HO

H
N
O

S
N

CO2H
Fig. 1. Chemical structure of cefadroxil.

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a dose of CFL may be excreted unchanged in the urine

within 24 h by glomerular filtration and tubular secretion.
The most common side effects are diarrhoea or loose
stools, nausea, abdominal pain and vomiting. Rarer side
effects include abnormal liver function tests and allergic
reactions.
A wide variety of analytical methods have been
reported for the determination of CFL in pure form, in
pharmaceutical preparations and in biological fluids.
These methods mainly involve spectrophotometry,
atomic absorption spectrophotometry (AAS), fluorometry, chemiluminescence (CL), polarography, highperformance liquid chromatography (HPLC), and capillary
electrophoresis (CE). In this work, we have summarized
the methods described in the literature for the determination of CFL alone and in combination with other
similar drugs. As far as the latter topic is concerned,
a very interesting review has been published recently
about the analysis of cephalosporin antibiotics [1].

has the disadvantage of not distinguishing between

cephalosporins and other sulphide-producing degradation products [2, 3].
Chen et al. [5] developed a method based on a
charge-transfer reaction and Sastry et al. [6] reported
three spectrophotometric methods using oxidative coupling reactions. As CFL possesses a p-substituted phenol
group, in the first method, the suitability of 3-methyl-2benzothiazolinone hydrazone hydrochloride (MBTH)
in conjunction with different oxidants (ferric chloride,
potassium dichromate, sodium metaperiodate, chloramineT, potassium hexacyanoferrate(III) and ceric ammonium
sulphate) for the determination of CFL was examined
and Ce(IV) was found to be the best with respect to
sensitivity, speed and stability of the coloured product
formed. In the second procedure, CFL was allowed to
react with 4-AP in the presence of alkaline [Fe(CN)6]3-.
Other oxidising agents, such as potassium persulphate,
sodium metaperiodate and potassium iodate, were tried
and found to be inferior with respect to sensitivity.
CFL was determined in dosage forms through the
reaction with Folin-Ciocalteu reagent to form a blue
coloured chromogen [7]. A spectrophotometric method
has been developed for the determination of CFL in
bulk powder and its pharmaceutical dosage forms
based on the reaction of the primary amine group with
acetylacetoneformaldehyde reagent, which gives a
yellow chromogen [8].
CFL was determined kinetically by measuring the
absorbance at 470 nm after hydrolysis with NaOH at
80C over the concentration range 10100 g/ml [9].
Validation of the method has been performed by assay
of CFL in commercial capsules and tablets.

2. Spectrophotometric methods
2.1. Pharmaceutical analysis
2.1.1. Cefadroxil
Various analytical procedures have been adopted for
the determination of this drug, such as colorimetric
methods [2-4], and also, fluorimetry, polarography, and
liquid chromatography. In general, the colorimetric
methods require a preliminary treatment of the samples
such as alkaline or acid hydrolysis of the antibiotic and
the use of a complexing agent. Moreover, the method
Table 1
Classification of cephalosporins.
Generation

Compounds

First

Cefadroxil, Cefacetrile, Cefalexin, Cefaloglycin, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone,

Cefazolin, Cefradine, Cefroxadine, Ceftezole

Second

Cefaclor, Cefamandole, Cefmetazole, Ceforanide, Cefotiam, Cefprozil, Cefuroxime

Third

Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefoperazone, Cefotaxime, Cefpiramide,

Cefpodoxime, Cefsulodin, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Latamoxef

Fourth

Cefepime, Cefpirome, Cefquinome

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A flow-injection spectrophotometric method was

reported for the determination of CFL in drug formulations based on hydrolysis of this drug in sodium hydroxide
solution and treatment with 1,4-phenylenediamine and
Fe(III) [10].
A sequential injection analysis (SIA) for the reaction
between CFL and 4-aminoantipyrine in the presence of
alkaline potassium hexacyanoferrate(III) to produce a
red colour has been developed recently [11]. In contrast
to other methods using sophisticated instruments, this

method is simple to implement with relatively inexpensive instrumentation and fewer operators are required
since the method is almost fully automated. The speed
of analysis and the precision make this method also
suitable for the quality controls of most industrial products, such as pharmaceuticals, food, and beverages.
Table 2 summarises some of the reported methods for
the analysis of CFL in pure forms and pharmaceutical
formulations.

Table 2
Spectrophotometric methods for the determination of cefadroxil in the pure state and in pharmaceutical dosage forms.
Reagent

Experimental conditions

Analytical characteristics

Ref.

Chloranilic acid

In a mixture of methanol and ethanol at 50C

for 15 min; at 528 nm

Linear range 20400 g/ml; RSD

1.4%; recoveries 99.0%100.8%

Diazonium salt of
sulfanilic acid

At 440 nm

Linear range 116 g/ml; recoveries

99.2%101%

12

MBTH in presence of
ceric ammonium sulphate

25 ml calibrated flasks, with aliquots of CFL, added

2 ml of MBTH and kept for 2 min at room temperature. Then 2 ml of Ce(IV) added, kept for 15 min
and diluted to the mark with water; at 410 nm

Linear range 1.06.0 g/ml;

recoveries 98.0%100.3%;
RSD 1%

4-aminophenazone in
presence of potassium
hexacyanoferrate(III)

25 ml calibrated flasks with aliquots of CFL,

0.6 ml of sodium carbonate, 1 ml of 4-AP and
1 ml of K3[Fe(CN)6] added successively and total
volume brought to 9 ml with water, solutions set
aside for 5 min and diluted with water; at 510 nm

Linear range 2.024.0 g/ml;

recoveries 98.0%100.3%;
RSD 1%

25 ml calibrated flasks with CFL, 5 ml of borate

2,6-dichloroquinone-4buffer solution (pH 9.4) and 2 ml of DCQC added
chlorimide (Gibbs reagent, successively and the total volume brought to 10 ml
DCQC)
with water, after 10 min the flasks were made up to
the mark with water; at 620 nm

Linear range 1.06.0 g/ml;

recoveries 98.0%100.3%;
RSD 1%

Folin-Ciocalteu in presence of
NaOH and stannous chloride

At 970 nm

Linear range 0.52.5 g/ml

1,4-phenylenediamine
and Fe(III)

In sulphuric acid solution;

flow injection analysis; at 600 nm

Linear range 80320 g/ml;

DL 40 g/ml; RSD 1.8%

10

4-aminoantipyrine in presence
of alkaline potassium
hexacyanoferrate(III)

SIA; at 510 nm

DL 0.17 g/ml; RSD 1.98%;

Linear ranges 110 g/ml
and 1050 g/ml

11

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2.1.2. Cefadroxil and other drugs

cefprozil anhydrous in their pure forms based on selective

oxidation of these drugs with either Ce(IV) or Fe(III) in
acid medium to give an intense yellow product [19].
CFL, cefotaxime, cephalexin and cefuroxime have
been determined in pure form and in preparations by
using persulfate in an alkaline medium as an oxidizing
reagent. Beers law is obeyed up to 60 g/ml for CFL [20].
CFL, cefalexin, cefaclor, ampicillin and amoxicillin
have been determined in pharmaceutical preparations
using a sensitive and selective colorimetric method based
on measuring the colour obtained when the alkaline
degradation products of these drugs were allowed to
react with ascorbic acid [21].
CFL and cefalexin have been determined by the
reaction of p-aminophenol with sulphide ions produced
by the alkaline hydrolysis of these drugs in the presence
of an oxidant to produce a violet colour measured at
550 nm [22].
CFL and cefotaxime have been determined by flow
injection spectrophotometry based on the hydrolysis of
the two cephalosporins with sodium hydroxide and the
produced sulphide ion was allowed to react either with
N,N-diethyl-p-phenylenediamine and Fe(III) or with
p-phenylenediamine and Fe(III) [23]; in this work, the
advantages of flow-injection analysis (FI) (simplicity,
high precision, rapidity and low reagent consumption)
are combined with the benefits of using dye formation
for the spectrophotometric determination of CFL and
cefotaxime. CFL and ceftizoxime were determined by
the addition of sodium hydroxide followed by iodine
and wool fast blue BL [24].
CFL, cefapirin, cefuroxime, cefotaxime, ceftazidime,
cefaclor, cefazolin and cefoperazone have been determined by the formation of ion-pair complexes with
ammonium reineckate [25].
CFL, cefradine and cefaclor have been determined
after treatment with glucitol and sodium hydroxide [26].
CFL and amoxycillin have been determined in dosage
forms by coupling with diazotized benzocaine [27].
Sequential injection for determination of CFL and
amoxicillin has been proposed by Feng et al. [28]. The
analysis was based on the determination of the red
product of the reaction of drugs with 4-aminoantipyrine

Four methods are proposed for CFL monohydrate,

cefoperazone sodium, and anhydrous cefprozil that
were determined either through their nitration and
subsequent complexation with a nucleophilic reagent
(method I), nitrosation and subsequent metal chelation
(method II), coupling with diazo reagent (method III),
and reaction with copper and extraction of the resulting
chelate into chloroform (method IV) [13].
Charge-transfer complexation between cephalosporins
as electron donors and certain -acceptors form the basis
of several spectrophotometric methods. Saleh et al. [14]
used -chloranilic acid (-CA) as -acceptor to determine 15 cephalosporin antibiotics, including CFL.
Reviewing the available colorimetric procedures
developed for the analysis of cephalosporins, one can
easily recognize that most of these methods involve
cleavage of the -lactam moiety of the cephalosporin
structure. Direct chemical analysis that is based on the
reactivity of the intact molecule without its cleavage is
not frequently encountered. In this sense, CFL monohydrate, cefapirin sodium, cefazolin sodium, cefalexin
monohydrate, cefotaxime sodium, cefoperazone sodium
and ceftazidime pentahydrate were determined through a
charge-transfer complexation reaction using an -acceptor,
such as iodine, and some -acceptors, such as 2,3-dichloro5,6-dicyano-p-benzoquinone (DDQ) and 7,7,8,8-tetracy
anoquinodimethane (TCNQ) [15].
Redox reactions have been used as the basis for many
spectrophotometric methods for the determination of
cephalosporin antibiotics, also including CFL. Saleh [16]
reported a method for the determination of cefadroxil
and amoxycillin with N-bromosuccinimide (NBS) and
N-chlorosuccinimide (NCS) as the oxidizing agents in
alkaline medium. Potassium iodate has also been used
for the determination of CFL, cefazolin sodium and
cefaclor [17]. Ten cephalosporins, including CFL, and
four penicillins were determined by oxidation using a
known excess of cerium(IV) in sulphuric acid; the unreacted cerium(IV) was measured at 317 nm [18].
Another method has been proposed for the determination of CFL monohydrate, cefoperazone sodium, and

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in the presence of potassium hexacyanoferrate(III) in

alkaline solution.
Table 3 summarizes the procedure and the analytical
characteristics of CFL in terms of the more important
methods proposed for the determination of CFL and
other compounds based on UV/VIS spectroscopy.

frequently used procedure to resolve binary mixtures

by spectrophotometry. The 2nd-derivative spectra of
mixtures were recorded against water and the values
of derivative were measured at 257 and 279 nm (zerocrossing wavelengths of 2nd-derivative of CFL) for the
determination of CFE and at 242 and 286 nm (zerocrossing wavelengths of 2nd-derivative of CFE) to
determine CFL. Satisfactory results were obtained with
all methods.

2.1.3. Cefadroxil in mixtures

Gortazar and Vazquez [29] describe the discrimination and direct determination of cephalosporins, including CFL, by circular dichroism (CD) and ultraviolet
spectra. There are sufficient CD spectral dissimilarities
observed to discriminate among the cephalosporin
homologues and to classify these antibiotics into five
spectroscopic groups.
Derivative spectrophotometry was applied for the
determination of CFL and cefotaxime (CFE) in binary
mixtures [30]. Cefotaxime sodium and cefadroxil
monohydrate have closely overlapping spectra, which
prevents the use of zero-order UV/VIS spectrophotometry
for their determination. Derivative spectrophotometry
is a suitable tool for overcoming this problem. Various
orders of derivative and different kinds of measurements
were investigated, i.e., ratio-spectra 1st- and 2ndderivative and zero-crossing 2nd-derivative. According
to the theory of the ratio-spectra derivative method, the
absorption spectrum of the mixture was divided,
wavelength-by-wavelength, by a standard spectrum of
CFL for determining CFE and by a standard spectrum
of CFE for determining CFL. Then, the 1st- and 2ndderivatives of the above ratio-spectra were recorded and
the values of the derivatives were measured at suitably
selected wavelengths. In particular, the concentration of
CFE was proportional to the value of the 1st-derivative
of the ratio-spectra at 239.5 and 291.5 nm and to the
value of the 2nd-derivative at 284 and 303 nm. The
concentration of CFL was proportional to the value of
the 1st-derivative of the ratio-spectra at 238 and 283
nm and to the value of the 2nd-derivative at 229.5 and
245.5 nm.
The zero-crossing technique has found practical
applications more recently. It has become the most

2.2. Biological analysis

Monitoring antibiotic concentrations in biological
fluids, such as urine, is important for pharmacokinetic
studies. Direct spectrophotometric methods are nonspecific because they are subject to strong interference
by endogenous biological components. Derivative
spectrophotometry has proved advantageous in eliminating spectral interferences. El-Gindy et al. [31] presents
two methods for determining CFL and cefuroxime
in urine using first-derivative spectrophotometry and
high performance liquid chromatography. For the 1st
derivative method, aliquots of each standard solution
were transferred to 50-ml volumetric flasks. To each
flask, 2 ml blank urine was added and the solutions
were diluted to 50 ml with 0.1 mol/l sodium hydroxide
to obtain a concentration range of 210 g/ml for both
cefuroxime and CFL. The 1D curves were scanned in
the range of 320270 nm for cefuroxime and 290250 nm
for CFL against a blank of 2 ml urine diluted to 50 ml
with 0.1 mol/l sodium hydroxide. The values of the 1D
amplitudes at 292.5 nm for cefuroxime and 267.3 nm
for CFL were measured, and the concentrations versus
their absolute first-derivative amplitudes were plotted in
order to obtain the calibration graph. The proposed 1D
and HPLC methods provide a simple, accurate, sensitive
and direct quantitative analysis for the assay of cefuroxime and CFL in urine. The methods can be used for
determination of dissolution profiles of tablets and capsules containing cefuroxime and CFL. The HPLC method
was found to be more selective than the 1D method,
while the 1D method has the advantages of speed and
low cost.

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Table 3
Spectrophotometric methods: procedure and analytical characteristics for CFL.
Linear range Detection limit
(g/ml)
(DL, g/ml)

Procedure
15 ml of stock solutions placed in 10 ml calibrated flasks. Distilled water
added to give volumes of 5 ml. Fresh ascorbic acid reagent (1 ml) was then
added to each flask before heating at 100C for 20 min. After cooling, the
volume was completed to 10 ml with water, mixed well before reading at
410 nm

315

FI manifold consists of three channels; sample (200 l) introduced via an

injection valve into an NaOH stream and pumped at a flow-rate of 1.77 ml/min,
while N,N-DPPD pumped through the other channel, then mixed. Mixture
stream passes through reaction coil 50 cm long for extra mixing and
36.34109.2
submerged in heated water bath at 70C.Then mixture cooled in ice bath
using 100 cm reaction coil; cold mixture mixed with 0.01 mol/l Fe (III).
All three reagents pumped at same flow-rate (1.77 ml/min in each channel);
monitored at 670 nm

RSD (%)

Ref.

0.6

< 1%

21

0.036

0.83

23

Aqueous solution of PPDD (0.01 mol/l) acting as carrier stream supplied

through channel 1 and aqueous iron (III) (0.01 mol/l) through channel 2;
hydrolyzed drug sample injected into PPDD stream from a 50 l Teflon
rotary valve injector; both streams propelled by a four-channel peristaltic
pump and mixed at the PTFE T-piece; mixture stream passes through
reaction coil of 50 cm. Both reagents pumped at same flow-rate (1.5 ml/min
in each channel); absorbance measured at 597.3 nm and peaks recorded at a
recorder speed of 1 cm/min.

0.5400

0.4

0.9

23

To each 25-ml graduated test tube containing aliquots of standard drug

solution 2.0 ml of 1.0 mol/l NaOH solution added and the volume made up
to 10 ml with distilled water. The contents allowed to stand for 10 min at
room temperature, then 3.0 ml of iodine (200 mg/ml) added, and diluted to
19 ml with distilled water. After 5 min, 6.0 ml of dye solution added and
absorbance measured 5 min later at 540 nm

0.84.0

0.74

24

In 10 ml volumetric flask, 2 ml of sample drug, 0.4 ml of hydrochloric acid

added, 2.5 ml of fresh saturated Reineckes salt solution added with agitation
for 5 min and complete to volume with re-distilled de-ionized water. The
formed precipitate filtered through sintered glass funnel after 1 h and
washed three times with 5 ml ice water. Then, precipitate dried in a vacuum
desiccator; formed precipitate in crucible was then dissolved with acetone
into a 25 ml volumetric flash together with successive washings of the
funnel and filtration device; volume completed quantitatively with acetone
to appropriated volume and absorbance measured at 525 nm

535

98.7100.1
(recovery)

25

Conversion of CFL to piperazine-2,5 dione derivative by heating in alkaline

sorbitol-zinc ion solution for 1025 min at 90C and subsequent treatment
of derivative with 0.1 mol/l sodium hydroxide to obtain highly absorbing
products with max at 345 nm

12.587.5

100.37
0.72 (mean
recovery)

26

Transfer 1ml of benzocaine solution (2 mg/ml) into 25 ml standard flasks,

followed by 0.1 ml of 0.5 mol/l H2SO4 and 2 ml of NaNO2 (0.1%). Allow to
stand for 15 min, then add portions of standard CFL solutions. Let stand for
5 min and finally add 1 ml of triethyl-amine solution and dilute to volume
with distilled water, leave for 20 min; at 455 nm

0.812

0.087

100.5
0.7 (mean
recovery)

27

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Table 3 (Continued)
Spectrophotometric methods: Procedure and analytical characteristics for CFL.
Linear range
(g/ml)

Procedure

Detection limit
RSD (%)
(DL, g/ml)

Ref.

25100 mg transferred to a 100 ml calibrated flask, treated with 2 ml nitric

acid, and 2 ml sulphuric acid, left to stand 10 min; then, cooled and diluted
with distilled water. 20 ml of solution diluted with distilled water to 100 ml.
0.53.0 ml of diluted solution transferred into 25 ml calibrated flasks, treated
with 23 ml acetone and 5 ml potassium hydroxide solution and diluted to
volume with distilled water; absorbance measured at 390 nm.

515

0.111

0.02

13

Into wide mouth test tubes, accurate volumes of standard solutions transferred
and the volumes adjusted to 10 ml with water. 2 ml of 3% sodium nitrite
followed by 2 ml of copper acetate added, resulting solution mixed well and
0.4 ml of 1 mol/l HCl added. Test tubes placed in a boiling water bath for
25 min. Solutions cooled and transferred quantitatively into 25 ml volumetric
flasks; absorbance measured at 520 nm.

412

0.257

0.08

13

1 ml of o-nitroaniline mixed with 2.5 ml sodium nitrite in a 25 ml calibrated

flask and mixture was left to stand 10 min. An aliquot of the drug solution
added to diazo reagent followed by 3 ml of 1 mol/l sodium hydroxide and
mixture was left to stand 5 min. Solution was then diluted to volume with
distilled water; absorbance measured at 435 nm

210

0.129

0.09

13

1 ml of o-nitroaniline and 2.5 ml of sodium nitrite mixed and left to stand

10 min. Accurately measured aliquots of standard drug added followed by
1.5 ml of sodium hydroxide. Mixture allowed to stand 5 min and then treated
with 5 ml of copper sulfate, 6 ml of 0.5 mol/l sulphuric acid and extracted
three times with a total volume of 25 ml of chloroform. Extracts collected in a
25 ml calibrated flask; absorbance measured at 415 nm

28

0.087

0.02

13

0.0412 mg transferred into 10 ml calibrated flasks. 1 ml of p-CA (4 g/l in

acetonitrile) added, reaction allowed to proceed 5 min at room temperature
and dilute to volume with acetonitrile; absorbance measured at 520 nm

20320

11.93

1.8

14

1.8

15

Standard solutions: Into a 50 ml calibrated flask, 24300 mg drug weighed

accurately and dissolved in 2 ml methanol, completed to volume with
the same solvent for DDQ, with 1,2-dichloroethane for iodine and with 640 (iodine)
1.37 (iodine)
acetonitrile for TCNQ, and diluted to obtain the suitable concentrations.
40160 (DDQ) 3.01 (DDQ)
Aliquot volumes of standard stock solutions, containing 403000 g drug
0.24 (TCNQ)
transferred to 10 ml calibrated flasks. 1 ml of the reagent added and diluted 418 (TCNQ)
to volume with 1,2-dichloroethane, methanol, or acetonitrile for iodine,
DDQ, and TCNQ procedures, respectively; absorbances of the resulting
solutions measured at 364, 460 and 843 nm, respectively
In 10 ml calibrated flasks, place 1 ml of standard drug solution followed by
1 ml of 0.1 mol/l sodium hydroxide and 1 ml of NBS or NCS solution. Mix well,
complete to volume with methanol and measure the absorbance at 395 nm

120

1.8 (NBS);
1.38 (NCS)

16

Aliquots of each sample reacted with potassium iodate in a moderately acidic

medium after hydrolysis of the -lactam ring with sodium hydroxide at 80C
for 10 min

11035103

17

Aliquot of standard solutions, containing 50300 g drug, transferred to 10 ml

calibrated flasks. 1 ml of 4 mol/l perchloric acid added followed by 2 ml
of Ce (IV) or Fe (III) solutions, mixed well and completed to volume with
distilled water, absorbances measured at 397 nm after 5 min at 25 5C

530

0.017 (Ce)
0.097 (Fe)

0.981.25

19

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3. Atomic absorption spectrometric methods

and cefradine on the basis of the reaction of the target

compounds with fluorescamine at a specific pH, ranging
from 7.8 to 8.4. The produced derivatives exhibit
maximum fluorescence intensities at 472478 nm
after excitation at 370372 nm. The method has been
applied to the determination of these drugs in biological
fluids and pharmaceutical formulations. The method is
sensitive to 5 ng/ml of each compound and can be used
for routine analysis of alpha-aminocephalosporins.
Four penicillins and ten cephalosporins, including
CFL, have been determined by a luminescence method
based on the luminescence of the produced Ce(III)
formed after oxidation of the studied drugs by Ce(IV)
at an elevated temperature; the luminescence intensity
was measured at 356 nm [18].
The simultaneous determination of multi-component
mixtures has always been an interesting question for
analysts. CFL and cefalexin have been determined
simultaneously by using the coupling technique of
synchronous fluorimetry and H-point standard addition
methods (HPSAM) [36]. Synchronous fluorimetry offers
several advantages over other fluorimetric methods,
including narrowing of the spectral band, enhancement
in selectivity by spectral simplification and the
elimination of scattered light. On the other hand,
HPSAM is based on the principle of dual wavelength
spectrophotometry and standard addition methods. The
greatest advantage of HPSAM is that it can remove the
errors resulting from the presence of an interferant and
blank reagent. The alkaline degradation products of
CFL and cephalexin are fluorescent and their excitation
peaks are at 355 and 365 nm and their emission peaks
are at 448 and 450 nm, respectively. Because their
emission spectra almost completely overlap, CFL
and cephalexin in the mixture cannot be determined
accurately by synchronous fluorimetry or HPSAM alone,
but they can be determined selectively by coupling of
synchronous fluorimetry and HPSAM.

Salem and Askal [25] have reported a method for the

determination of eight cephalosporins, including CFL,
through the formation of ion pair complexes between
these drugs and ammonium reineckate; the formed
precipitates were determined by an AAS procedure
using the chromium precipitate formed or the residual
un-reacted chromium in the filtrate. In the direct
procedure, precipitates were collected on a sintered
glass crucible and washed with five 2 ml portions of ice
water. The drug-reineckate precipitates were dissolved
in 25 ml acetone. The solution was then nebulized in an
airacetylene flame for AAS measurement of chromium
at 358.6 nm. For the indirect procedure, the filtrate and
washings from the direct procedure were collected in a
100 ml volumetric flask and made up to the markwith
acetone. The resulting solution (2 ml) was diluted to
25 ml with acetone. A blank (omitting addition of drugs)
was prepared and absorbance was measured under the
same conditions of samples. The AAS method is selective and suitable for routine quality control.

4. Spectrofluorimetric methods
CFL and other cephalosporins were determined in
pharmaceutical formulations by fluorescence quenching
after mixing each drug with fluorescein-Hg and 1 mol/l
sodium hydroxide [32].
CFL, cefradine, cefotaximum and amoxicillin were
determined by mixing with sodium hydroxide and heating at 100C to obtain the corresponding fluorescence
degradation products [33].
El-Walily et al. [34] have proposed a selective
fluorimetric method for the determination of CFL,
cefoperazone and amoxicillin through the formation of
their coumarin derivatives based on the reaction between
these drugs and ethylacetoacetate, in acidic medium,
to give yellow fluorescent products with excitation
wavelengths ranging from 401 to 467 nm and emission
wavelengths ranging from 465 to 503 nm.
Hefnawy et al. [35] have reported a fluorimetric
method for the determination of CFL, cefaclor, cefalexin

5. Chemiluminescence methods
Recently, CL analysis has attracted interest mainly
because it offers the advantages of sensitivity, a wide

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linear range and relatively simple and inexpensive

instrumentation. Reviewing the literature shows that, up
to the method proposed by Aly et al. [37], nothing has
been published concerning CL determination of CFL.
Analytical procedures applying CL measurements in FI
setups combine the advantages of instrument simplicity (no
monochromator required), rapidity in signal detection
(normally 0.110 s), sensitivity and ease of use. Since
many CL reactions are very fast, they give rise to
imprecise measurements as a result of irreproducible
mixing of sample and reagents, but the reproducibility
and selectivity of the CL analysis can be improved
by combination with an FI method. In this way, Aly
et al. [37] proposed a method for determination of
CFL in pharmaceutical samples and biological fluids
based on the FI-CL reaction of CFL with potassium
permanganate in sulphuric acid, sensitized by quinine.
The method described requires only deproteination of
plasma samples and dilution of urine samples to avoid
any potential interference.
Electrogenerated chemiluminescence (ECL) coupled
to an FI system was used for the determination
of CFL using the tris (2,2-bipyridine) ruthenium(II)
complex, the luminescence of which increased in
the presence of CFL [38]. ECL can be defined as a
luminescent chemical reaction in which light is emitted
only when an appropriate potential is applied to an
electrode in contact with a solution containing an
appropriate luminescent compound. There are a variety
of methods to obtain the active oxidizing reagent
Ru(bpy)33+, including chemical, photochemical or electrochemical oxidation. CL is observed when RRu(bpy)33+
reacts with Ru(bpy)3+ and yields the excited state
Ru(bpy)32+*, giving an orange emission centered at
610 nm. Although, many ruthenium complex-based
ECL reactions are known, Ru(bpy)32+ has been widely
studied because of its characteristics of chemical stability,
redox properties and excited state life. This study
demonstrates the applicability of the proposed ECL
method for the determination of CFL in commercial
products. This method can be successfully used for
routine quality control and offers the advantages of
speed, simplicity, and reliability. Another important

factor is the low cost of the method, compared with

liquid chromatography.
Sun et al. [39] proposed an FI-CL method for the
determination of three cephalosporin antibiotics based
on the ability of these antibiotics to enhance the CL
reaction of glyoxal and potassium permanganate in
sulphuric acid medium. Compared with previous
reported CL methods, the present method has a lower
detection limit and a wider calibration range. The
method was applied to the determination of CFL in
pharmaceutical formulations with satisfactory results.
An examination of the bibliography revealed that until
the method proposed by Thongpoon et al. [40], nothing
was published concerning the Ru(bpy)32+-potassium
permanganate CL methods for determining CFL in
pharmaceuticals. Preliminary experiments showed that
CFL and other cephalosporins produced relatively weak
CL with acidic potassium permanganate. It was found
that increasing the concentration of Ru(bpy)32+ with
an acidic permanganate system can strongly enhance
weak CL intensities, much more than in the absence
of Ru(bpy)32+. For this reason, the FI method has been
developed using tris(2,2-bipyridyl) ruthenium(II)
potassium permanganate in the presence of perchloric
acid, catalyzed by Mn(II). CL has been reported for
the determination of some cephalosporin antibiotics,
including CFL [40]. The proposed FI-CL method has
proven to be highly sensitive, selective, reproducible
and rapid for the determination of CFL, cefoxitin,
cefazolin, and cephalexin with a sample throughput
rate of 90 samples/h. The recommended method has
been successfully applied to the determination of the
drugs in commercial pharmaceutical preparations. This
method is thus suitable for routine analysis. Later, the
same authors proposed another FI-CL method based on
the CL-emtting reaction between CFL and potassium
permanganate in sulphuric acid medium, enhanced by
formaldehyde [41].
Li and Lu [42] proposed an FI-CL method for the
determination of CFL and other -lactam antibiotics
based on injection of these drugs into a stream of
potassium permanganate with alkaline luminol. In
alkaline aqueous solution, the most extensively used

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CL reagent is luminol, which can be oxidized by

many oxidants. However, the application of the
luminolKMnO4 CL system has not been extensively
investigated. It was found that strong CL was produced
when a -lactam antibiotic (CFL, amoxicillin, cefoperazone sodium, cefazolin sodium, cefradine, or
ceftriaxone sodium) was injected into the reaction
mixture containing KMnO4 and alkaline luminol. CFL,
cefalexin, cefaclor and cefradine were determined by
an FI-CL method based on enhancement of the CL
arising from the reaction of luminol with potassium
iodate in alkaline solution [43]. The method has been
applied to the determination of drugs in pharmaceutical
preparations.
CFL has been determined by an FI-CL method
based on the CL-emitting reaction between CFL and
potassium permanganate in sulphuric acid medium,
enhanced by formaldehyde [44]. The method has
been applied successfully for determining CFL in
pharmaceutical samples with a high sampling rate of
120 h-1.
Recently, a CL micro-flow system combined with
on-line solid phase extraction (SPE) has been described
for determination of CFL and other -lactam antibiotics
(penicillin, cefradine, cefalexin) in milk [45]. It is
based on the enhancement effect of -lactam antibiotics
on the luminol-K3Fe(CN)6 CL system. The microflow system was constructed from two polymethyl
methacrylate (PMMA) plates (50 mm40 mm5 mm)
with microchannels 200 m wide and 150 m deep.
C18-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 m)
to serve as an SPE device. Extraction and preconcentration of the analytes were carried out using an online SPE micro-flow system and the selectivity of CL
detection was improved. The authors proposed a CL
micro-flow system combined with on-line SPE to
improve the selectivity of the CL measurements for real
samples. The proposed on-line SPE microflow system
with CL detection has effectively solved the problem of
matrix interference and has been successfully applied in
analyzing CFL and other -lactam antibiotics in milk.
The on-line SPE micro-flow CL system is easy and
fast, reducing reagent consumption and is suitable for

developing a miniature version of the instrument for onsite analysis.

6. Voltammetric methods
Electro-oxidation of CFL monohydrate has been
investigated using a glassy carbon electrode depending
on the pH and supporting electrolyte. It was shown that
direct determination of the substance in capsules and
oral suspension could be made by differential pulse
voltammetry (DPV) [46].

7. Chromatographic methods
7.1. High performance liquid chromatography (HPLC)
In recent years, HPLC has proved to be a powerful
analytical tool for measuring many drugs in biological
fluids, because of its specificity, rapidity and sensitivity.
Parasrampuria and Das Gupta [47] have applied an
HPLC method to determine CFL in capsules, suspensions and tablets from two different manufacturers. Hsu
et al. [48] developed a reverse-phase column LC method
for the assay of CFL in bulk drugs and pharmaceutical
preparations using dimethylphthalete as internal standard (IS) and obtained a linear range of 0.020.8 mg/ml
for CFL.
Hendrix et al. [49] describe a comparative study of
two LC methods in pharmaceutical dosage forms; more
details about these methods and other HPLC methods
which appear in the literature are summarised in Table 4
and Table 5.
Gorski et al. [67] have proposed a method for the
determination of CFL, cefsulodin and cefmenoxime as
residues on surfaces. Analytes are degraded instantaneously in aqueous sodium hypochlorite, sodium
hypochlorite-detergent, or alkaline detergent solutions.
These solutions are used to clean surfaces that have
been exposed to the cephalosporins. The detection limit
for each compound is 0.1 g/ml.
Rao et al. [68] describe a solid-phase extraction system followed by liquid chromatographic-electrospray
ionization mass spectrometry (LC-ESI-MS) for determination of antibiotics including CFL.

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Table 4
HPLC methods for the simultaneous determination of CFL with other analytes.
Other analytes

Column

Mobile phase

Detection

Applications

Ref.

Twelve
cephalosporins

C18

Methanol-water-acetic acid
(30:70:0.1)

UV, 254 nm

Raw material and

dosage forms

55

Microbondapak
C18

Water/acetonitrile/phosphoric acid
(90:10:1.46), pH 3.5

UV, 254 nm

Capsules

56

Cephalexin, cefazolin, Phenomenex C18

cephradine, ampicillin
and ODS C18

Aqueous 10% acetic acid/propan-2ol/water (4:9:87)

UV, 240 nm

Solutions

57

5 m Hypersil
ODS C18

50 mmol/l mono-potassium phosphate

(pH 3.4)-acetonitrile (87.5:12.5)

UV, 254 nm

Pharmaceutical
samples

58

3.5 m
Symmetry C18

Acetonitrile-methanol-phosphate
buffer (50 mmol/l) with
1-pentanesulfonic acid sodium salt
(7 mmol/l) to pH 2.1 with phosphoric
acid (9:13:78)

UV, 254 nm

Pharmaceutical
samples

59

5 m LiChrospher
100 RP-18

0.02 mol/l potassium dihydrogen

phosphate-acetonitrile (95:5) with
0.003% hexanesulphonic acid
sodium salt, pH 3.0

UV, 260 nm

Biological
samples

31

Methanol/monobasic phosphate
buffer (20:80) pH 2.6

UV, 240 nm

Human serum

60

ABZ

Acetonitrile/10 mmol/l phosphate

buffer pH 7.0 (13:87)

UV, 260 nm

Bovine serum and

muscle tissue

61

Cephalexin, cefaclor,
cefotaxime

SpeerROD
RP-18e

Acetate buffer pH 4.0/

methanol (9:1)

UV, 265 nm

Pharmaceutical
samples

62

Cephalexin, cefaclor,
cefotaxime

5 m Spherisorb
ODS-2

Acetate buffer pH 4.0/

methanol (78:22)

UV, 265 nm

Pharmaceutical and
body fluids

63

Cephalexin,
cefaclor, isoniazid,
pyrazinamide

JASCO RP-C18

0.025 mol/l Tetra-butyl ammonium

hydrogen sulphate/methanol/
acetonitrile (32:1:1), pH 3.0

UV, 248 nm

Pharmaceutical
samples

64

Serum and urine

65

Porcine serum

66

Cephalexin

Five
cephalosporins

Seven
cephalosporins

Cefuroxime

Cephalexin, cefixime, Altex Ultrasphere

cefaclor, cephradine
Octyl C8

Amoxicillin

Eight cephalosporins
and derivatives

5 m Hypersil
ODS

Ten cephalosporins

4 m Nova-Pak
C18 Radial-Pak
cartridge

Indirect
electrochemical
0.1 mol/l acetate buffer, pH 4.1, and
using in-line
methanol (83:17) with 0.01mol/l NaBr electrochemically
generated bromine
as oxidizing agent
Acetate buffer

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Pulsed
amperometric

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Table 5
HPLC methods for the determination of CFL alone.
Column

Mobile phase

Detection

Applications

Ref.

Acetonitrile-0.01 mol/l phosphate buffer solution

pH 4.5 (60:40)

UV, 254 nm

Pharmaceutical samples

48

Acetonitrile/0.272% potassium hydrogen phosphate

UV, 230 nm

Bulk samples

49

UV, 230 nm

Bulk samples

49

Acetonitrile/0.02mol/l sodium octanesulfonate/0.2 mol/l

phosphoric acid/water (10.5:20:5:up to 100)

UV, 254 nm

Bulk samples

50

Sphere C18

Sodium acetate buffer (0.015mol/l, pH 4.0)methanol (90:10); salicylic acid as IS

UV, 272 nm

Pharmaceutical samples

51

Polaris C18

0.05 mol/l potassium dihydrogen phosphate (adjusted pH to

5.5 by 1mol/l potassium hydroxide)-acetonitrile (96:4)

UV, 230 nm

Pharmaceutical samples

52

Sodium phosphate buffer pH 2.6/methanol (17:3)

UV, 230 nm

Pharmaceutical samples

53

Phosphate buffer solution (pH 5)/acetonitrile (95:5);

paracetamol as IS

UV, 280 nm

Urine

54

10 m Bondapak
C18
Alkyl bonded
phase C18

Poly(styrenedivinylbenzene)

Poly(styrenedivinylbenzene

Ultrasphere C8
10 m C8

Acetonitrile/0.02mol/l sodium 1-octane-sulfonate/

0.2 mol/l phosphoric acid /water
Water/acetonitrile/0.02mol/l sodium octane-sulfonate/
0.2 mol/l phosphoric acid (129:21:40:10)

7.2. Thin-layer chromatography (TLC)

easy to use and is very reproducible. The low amount

of sample required and the relatively short analysis time
are the main advantages of this method.
CZE has been used for the determination of the
dissociation constants of nine cephalosporins, including
CFL, using a fused-silica tube operated at an applied
voltage of 30 kV [71]. Very recently, acidity constants
of CFL, cefalexin, cefaclor, cefotaxim, cefoperazon and
cefoxitin have been determined using CZE and pHpotentiometric titrations. Since CZE is a separation
method, it is not necessary for the samples to be of high
purity and known concentration because only mobilities
are measured. The effect on the determination of dissociation constants of different matrices (serum, 0.9%
NaCl, fermentation matrix) was also examined in this
work [72]. CZE was compared with potentiometric
titration and its advantages and limitations are described

Shabadi et al. [69] reported a method for simultaneous

determination of CFL and cephalexin in pharmaceutical
preparations using quantitative TLC. Chromatography
was carried out on silica gel, with chloroform-methanolacetonitrile-ammonia (7.5:3:2:1.2) as the mobile phase
and para-hydroxy-phenyl acetamide as the IS. The
samples are scanned at 254 nm with a Camag TLC
scanner II densitometer controlled by Cats software.
Recently another TLC method has been proposed by
Dhingra and Pandey [70].
7.3. Capillary electrophoresis
Capillary zone electrophoresis (CZE) is a relatively
new technique for CFL analysis. The CZE technique is

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in determining the pKa values in this work. According

to the results obtained, the CZE method is capable of
determining the pKa values from real samples with
complicated matrices. The direct analysis of real samples
is very important because it is not necessary to extract
and purify the analyzed compound and, so, accurate
pKa values are easy to obtain during drug discovery,
development and pharmacokinetic measurement stages.
CZE can be used for the determination of physicochemical properties under physiological conditions. CZE
does not require high purity substances because it enables
separation of impurities and decomposition products
from the main component. With the help of the high
separation resolving ability of CZE the pKa values of
several compounds can be determined at the same time.
The aim of the work presented by Mrestani et al. [73]
was the application of CZE for the separation and
identification of nine cephalosporins (CFL, cephalexin,
cefaclor, ceftazidim, cefsuladin, cefotaxim, cefamandol,
cefuroxim and cefodizim) in urine and in bile. For this
purpose, the samples were only diluted with buffer and
then injected without any further sample preparation.
The performance of the CZE approach for the determination of drugs was tested by measuring the detection
limit at 210 nm and the relative standard deviation of
the migration times and of the peak areas. The method
developed was used to measure CFL in urine and in bile
of patients and rabbits.
The use of CZE has been described for the analysis
and stability investigation of fourteen cephalosporins,
including CFL [74].
Du et al. [75] established a method for determination
of CFL and trimethoprim in plasma. The analysis was
performed in an uncoated fused silica capillary and a
buffer solution of 20 mmol/l disodium tetraborate (pH 7.1).
The applied voltage was 20 kV and the UV detection
wavelength was 267 nm. Recently, Solangi et al. [76]
proposed the quantitative analysis of eight cephalosporin
antibiotics, including CFL, in pharmaceutical products
and urine. Separation was performed in less than 11 min
with 50 mmol/l sodium tetraborate buffer (pH 9.0) and
an applied voltage of 30 kV. Detection was by UV
absorbance at 214 nm. Limits of detection were in the

range 0.55 g/ml.

Li et al. [77] reported a micellar electrokinetic capillary
chromatography (MEKC) method for the determination
of CFL using a fused-silica capillary operated at an
applied voltage of 18 kV and detection was performed
at 254 nm. The separation was performed in an acetate
buffer (50 mmol/l, pH 5.25) with sodium dodecyl sulphate (110 mmol/l).

8. Conclusions
As can be seen in this review, several methods for
analyzing CFL in pharmaceuticals and biological fluids
have been reported, including spectrophotometry, AAS,
spectrofluorometry, chemiluminescence, voltammetry,
and chromatography, principally HPLC. Compared with
other techniques, spectrophotometry is very simple,
rapid, non-destructive and less expensive. In addition,
spectrophotometers are commonly available in all
laboratories. Most of these detectors have been coupled
with flow injection analysis (FI) and sequential injection
analysis (SIA).
In recent years, CL is becoming a powerful analytical
tool for pharmaceuticals in general and also for CFL
determination because of the low detection limit and
wide linear dynamic range. The combination of FI and
CL detection has been developed rapidly bringing
together the advantages of both techniques in rapid and
simple instrumentation and allowing improvements in
sensitivity, selectivity and precision.
HPLC methods have been described for the determination of CFL in pharmaceutical preparations and
biological fluids using different stationary phases, mobile
phases with different buffer systems (mostly phosphates
or ion pairing agents), detection modes, e.g. UV and
electrochemical, and sample preparation procedures.
Speed of analysis has become of paramount importance
in many application areas of HPLC, such as in pharmaceutical and clinical analysis, in order to increase throughput and reduce costs. In this way, the recently invented
monolithic columns offer new practical possibilities for
decreasing retention times and/or increasing column
efficiencies.

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Abbreviations
AAS

Atomic absorption spectrophotometry

IS

Internal standard

ACA

Amino cephalosporanic acid

LC

Liquid chromatography

CD

Circular dichroism

MBTH

3-methyl-2-benzothiazoline hydrazone hydrochloride

CE

Capillary electrophoresis

MEKC

Micellar electrokinetic capillary chromatography

CFE

Cefotaxime

NBS

N-bromosuccinimide

CFL

Cefadroxil

NCS

N-chlorosuccinimide

CL

Chemiluminescence

N,N-DPPD

N,N-diethyl-p-phenylenediamine sulfate

CZE

Capillary zone electrophoresis

p-CA

p-chloranilic acid

DCQC

2,6-dichloroquinone-4-chlorimide

PMMA

Polymethyl methacrylate

DDQ

2,3-dichloro-5,6-dicyano-p-benzoquinone

PPDD

p-phenylenediamine dihydrocloride

DL

Detection limit

PTFE

Polytetrafluoroethylene

DPV

Differential pulse voltammetry

RSD

Relative standard deviation

ECL

Electrogenerated chemiluminescence

SIA

Sequential injection analysis

FI

Flow injection analysis

SPE

Solid phase extraction

FI-CL

Flow injection-Chemiluminescence

TCNQ

7,7,8,8-tetracyanoquinodimethane

HPLC

High performance liquid chromatography

TLC

Thin-layer chromatography

HPSAM

H-point standard addition method

UV/VIS

Ultraviolet/visible

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