BIOMEDICAL CHROMATOGRAPHY, VOL.

12, 133–135 (1998)

HPTLC Assay of Cephalexin and Cefaclor in

Pharmaceuticals

D. Agbaba*, S. Eric, D. Zivanov Stakic and S. Vladimirov

Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Vojvode Stepe 450, P.O. Box 146, 11000 Belgrade, Serbia,
Yugoslavia.

A simple and reliable HPTLC method for the simultaneous determination of cephalexin and cefaclor is
developed and validated. The methanol–ethyl acetate–acetone–water (5:2.5:2.5:1.5 v/v/v/v) solvent system is
used for the quantitative evaluation of chromatograms. The chromatographic zones, corresponding to the
spots of cephalexin and cefaclor on the silica gel plates, are scanned in the reflectance/absorbance mode at
265 nm. The method is found to be reproducible and convenient for the quantitative analysis of cephalexin
and cefaclor in its dosage forms. # 1998 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 12, 133–135 (1998)

INTRODUCTION Therefore this paper focuses on the development of a

b-lactam antibiotics are effective antibacterial agents
because they inhibit the enzymes involved in the final
stages of bacterial cell wall biosynthesis (Vilanova et al.,
1994). Cephalosporins are a series of antibiotics contain-
ing a b-lactam ring fused to a six-membered-ring with a
sulphur atom. Cephalexin and cefaclor belong to first and
second generation of cephalosporins, respectively, and
they have similar chemical structure with only difference
at position 3.
Several methods for the separation and detection of
natural and semisynthetic cephalosporins and their
degradation products by thin layer chromatography
(TLC) have been reported. Different visualizing agents
such as: sulphuric acid, (Hoogmartens et al., 1981),
chloroplatinic acid (Pokorny et al., 1976), and fluoresca-
mine (Fabre et al., 1985), have been proposed for their
detection using conventional TLC.
In pharmaceutical analysis conventional TLC was used
for identification, purity control and stability studies of
cephalosporins, but, in recent years, mostly the methods
using HPLC mostly have been reported (Blanchin, et al.,
1987, Das Gupta et al., 1987, Hayward, et al., 1989, Lee
et al., 1990; Moore et al., 1991).
In generally, experiences in screening test of the
cephalosporin derivatives illustrated that separation by
conventional TLC would be a problem, because the
polarity difference between these substances was small
(Karch, 1979).
HPTLC plates are preferred for quantitative analysis
because they maximize resolution and minimize light
scatter. The advantage of instrumental planar chromato-
graphy such as the ability to utilize low volume of mobile
phase as well as to utilize solvents unsuitable for HPLC,
speed of separation, and low cost have long been
recognized.
simple accurate and rapid quantitative HPTLC method
for simultaneous determination of cephalexin and
cefaclor in pharmaceuticals.
EXPERIMENTAL
Apparatus. A TLC Scanner II with a computer system and
Cats Software (V.3.15) were provided by Camag, Muttenz,
Switzerland. The radiation source was a deuterium lamp.
Nanomat III was used as an application device (Camag,
Muttenz, Switzerland)
Reagents and materials. Pre-coated silica gel HPTLC plates
with concentration zones (2.5  10 cm) (Merck, Darmstadt,
Germany), was used without any pretreatment. The mobile
phase was methanol–ethyl acetate–acetone–water
(5:2.5:2.5:1.5). All chemicals and solvents were of analytical
grade. Cephalexin monohydrate [7-(D-aminophenylacetamido)
desacetoxycephalosporanic acid] and cefaclor [7-(D-2-amino-
2-phenylacetamido)-3-chloro-3-cephem-4-carboxilic acid]
were obtained from ICN Yugoslavia (Belgrade). A formula-
tions based on cephalexin and cefaclor, were gifts as follows:
Cefaleksin capsules, containing 500 mg of cephalexin mono-
hydrate per capsule (Panfarma, Belgrade); Palitrex syrup,
containing 250 mg of cephalexin monohydrate per 5 mL,
Alfacet capsules (500 mg cefaclor per capsule) and Alfacet
syrup (125 mg cefaclor per 5 mL) (ICN Yugoslavia, Belgrade).
Standard solutions. Stock standard methanol-aqueous solution
containing 1 mg/mL of cephalexin monohydrate and cefaclor
were freshly prepared in mixture of methanol–water (9:1) For
an assay of cephalexin and cefaclor, calibration curves were
prepared by diluting the stock solution to furnish solutions with
final concentrations of 0.125, 0.250, 0.350, 0.425 and 0.5 mg/
mL for both of cephalosporins.

*Correspondence to: D. Agbaba, Faculty of Pharmacy, Department Sample solutions. Sample solution of cephalexin: a quantity of
of Pharmaceutical Chemistry, Vojvode Stepe 450, P.O. Box 146, CefaleksinR capsule containing 500 mg of cephalexin mono-
11000 Belgrade, Serbia, Yugoslavia. hydrate was transferred to 50 mL calibrated flask and dissolved

CCC 0269–3879/98/030133–03 $17.50 Received 12 June 1997

# 1998 John Wiley & Sons, Ltd. Accepted 19 June 1997
134 D. AGBABA ET AL.
Table 1. Determination of cephalexin monohydrate and
cefaclor in its dosage forms by HPTLC
Found (mg),
Dosage form Taken (mg) (n = 5) KV (%)
Cefaleksin capsule 500 506.8 1.95
Palitrex syrup 250 257.6 1.7
Alfacet capsule 500 508.2 2.03
Alfacet syrup 125 129.2 1.18
up to the mark with methanol. One millilitre of this solution
was then diluted to the mark with methanol in 10-mL calibrated
flask. PalitrexR syrup were freshly prepared before use and than
1 mL of this solution was transferred to 50-mL calibrated flask
and dissolved up to the mark with methanol.
Sample solution of cefaclor: a quantity of AlfacetR capsule
containing 500 mg of cefaclor was transferred to 50-mL
calibrated flask and dissolved up to the mark with water. One
millilitre of this solution was than diluted with methanol to the
mark in 10-mL calibrated flask. AlfacetR syrup were freshly
prepared before use, and than 1 mL of this solution was
transferred to 50-mL calibrated flask and dissolved up to the
mark with methanol.
Chromatography. A 1-mL loading of each standard and
sample solution was spotted on the HPTLC plate with
concentration zones by means of a Nanomat III. The
chromatogram was allowed to develop to a height of about
90 mm in a twin - trough chamber previously saturated with the
mobile phase. Each plate could accommodate six sample spots
and five standards. The measurement of each spot was carried
out in situ at 265 nm using absorbance/reflectance mode.
RESULTS AND DISCUSSION
TLC as an official method prescribed in the cephalospor-
ium monograms of the European Pharmacopoeia (1997),
in which different mobile phases are used for their
separation have been presented (Hoogmartens,1997). It
was observed that R f values of cephalexin and cefaclor in
different systems were very close. To achieve appropriate
mobile phase, we used different modificators and
observed that a little change in concentration of methanol
induced a improvement on separation of cephalosporins
mentioned above. A mixture methanol-ethyl acetate-
aceton-water (5:2.5:2.5:1.5) gave a good separation with
minimum tailing. Migration distances ( standard
deviation, n = number of determination) were 55.08 
0.72 (n = 10) and 68.38  0.61 (n = 8) for cephalexin and
cefaclor, respectively, which provided accurate densito-
metric reading.
A typical chromatogram of the separation of cephalex-
in and cefaclor obtained by HPTLC method under above
REFERENCES
Blanchin, M. D., Kok, W. and Fabre, H. (1987). Chromato-
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Das Gupta, V. and Parasrampuria, J. (1987). Drug Dev. Ind.
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# 1998 John Wiley & Sons, Ltd.
Figure 1. Typical chromatogram of cephalexin (1) and
cefaclor (2) obtained by HPTLC.
mentioned chromatographic conditions is shown in Fig.
1. The calibration functions were constructed for
concentration ratio 125–500 ng/per spot, for both of
cephalosporins; a 2nd order polinomial function (peak
area vs amount applied) was found to be most suitable
one. The regression equations were: Y = 61.69 ‡ 1.5
Xÿ9.34 Eÿ4 X2 for cephalexin and Y = 120.59 ‡ 0.97
Xÿ5.42Eÿ4 X2 for cefaclor. The correlation coefficient
were >0.997. The standard deviations of calibration
curves were 7.78% and 5.02% for cephalexin and
cefaclor, respectively.
The limit of detection (LOD) and quantification (LOQ)
were determined by fitting the interday, back-calculated
standard deviations of each calibration standard. The Y-
intercept was then equal to SDo (the estimated standard
deviation at a concentration of zero). The LOD was
defined as 3SDo and LOQ as 10 SDo. The LOD for
cephalexin and cefaclor was found to be 17.07 and
19.37 ng, and LOQ 56.9 and 64.5 ng.
Suitability of HPTLC method for quantitative deter-
mination of cephalexin and cefaclor was further approved
through next validation specifications: precision and
accuracy. The precision of the method was determined by
running replicate samples, each containing 125 and
350 ng per spot of cephalexin and cefaclor; the relative
standard deviation were 3.1% and 0.8% for cefalexin and
3.8% and 1.3% for cefaclor, respectively.
The accuracy of the densitometric method was proved
by the determination of cephalexin and cefaclor from the
laboratory made dosage forms (syrups), spiked with
250 mg cephalexin and 125 mg cefaclor; mean recovery
were 98.7% (n = 6) and 101.3% (n = 8) for cephalexin
and cefaclor, respectively.
The results of quantitative determination of cephalexin
monohydrate and cefaclor in its dosage forms are
presented in the Table 1. The application of HPTLC
with UV scanning densitometry provides a simple, rapid
and reliable system for the assay of cephalosporins.
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Biomed. Chromatogr. 12, 133–135 (1998)