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HPLC determination of cefotaxime and cephalexine residues in milk and

cephalexine in veterinary formulation

Article  in  Microchimica Acta · April 2008

DOI: 10.1007/s00604-007-0820-1 · Source: OAI

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Microchim Acta (2008) 160: 471–475
DOI 10.1007/s00604-007-0820-1
Printed in The Netherlands
Original Paper
HPLC determination of cefotaxime and cephalexine residues
in milk and cephalexine in veterinary formulation
Victoria F. Samanidou, Emmanouil D. Tsochatzis, Ioannis N. Papadoyannis
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece
Received 15 January 2007; Accepted 31 May 2007; Published online 31 July 2007
# Springer-Verlag 2007
Abstract. An HPLC method was developed and va-
lidated for the determination of the cephalosporins
cefotaxime and cephalexine in skimmed bovine
milk. The analytical column, Kromasil C18 (250 mm

4.0 mm, 5 mm) was operated at ambient tempera-
ture. Mobile phase consisted of CH3OH-acetate buff-
er (pH ¼ 4.0) and it was delivered isocratically at a
flow rate of 1.0 mL  min1 . Total analysis time was
less than 5 min. Caffeine was used as internal stan-
dard (5 ng  mL1 ). UV detection was performed at
265 nm. Method validation was performed by means
of intra-day (n ¼ 5) and inter-day accuracy and precision
(n ¼ 8), sensitivity and linearity. Limits of detection
(LOD) and limits of quantification (LOQ) were 0.1
and 0.3 ng  mL1 , respectively. The method was ap-
plied to the analysis of a veterinary drug (CEPOREX)
containing cephalexine. The results were quite ac-
curate with the relative error varying from 8.0 to
3.5%. Solid-phase extraction was applied to re-
move all matrix interference from milk samples.
High extraction recoveries (average 84–121%) were
achieved by using Abselut NEXUS cartridges with
acetonitrile as eluent and a rinsing step with water and
n-butanol. A pre-concentration step was necessary in
Correspondence: Ioannis N. Papadoyannis, Laboratory of Ana-
lytical Chemistry, Department of Chemistry, Aristotle University of
Thessaloniki, GR-54124 Thessaloniki, Greece
e-mail: papadoya@chem.auth.gr
a 1=10 level to reach the EU MRL concentration level
(100 mg  kg1 ). RSD values were less than 7% for
both cephalosporins.
Keywords: HPLC; cephalosporins; cefotaxime; cephalexine; sol-
id-phase extraction; milk; veterinary formulation
Cephalosporins are broad-spectrum antimicrobial
agents which exhibit activity against Gram () and
Gram (þ) bacteria. They belong to the b-lactam fam-
ily antibiotics and they are classified into four genera-
tions based on their synthesis (natural, semi-synthetic,
synthetic). Their activity is based on the reduction of
bacterial peptidoglycans and thereupon they block
their growth [1].
Cephalosporins have a widespread use in human
medicine, as well as in veterinary medicine. However
the abundant and in some cases improper use of the
cephalosporins may cause the existence of residues in
foods especially those with animal origin such as milk
and animal tissues. Their existence may cause serious
allergic reactions and they could be, in some cases,
dangerous for human health. Another negative effect
of the existence of cephalosporins in the food chain is
the generation of novel micro organisms resistant to
antibiotics [1].
Cephalexine is a 1st generation cephalosporin with a
widespread medical and veterinary use against urino-
genital, dermal and gastrointestinal infections. Cefotax-
472
ime is a 3rd generation cephalosporin with medicinal
but with not a very widespread veterinary use. The EU
regulation 2377=90 has established Maximum Residue
Limits in various foodstuffs only for cephalexine
(100 mg  kg1 ) [2].
Cephalosporins have been determined so far, by a
variety of techniques. Among them the most widely
applied are the chromatographic techniques mainly
HPLC, which provides rapid, sensitive and accurate
methods and has the advantages of solvent econo-
my and easy coupling with other techniques [3–9].
Other techniques reported for the quantification of
cephalosporins in milk include microbiolical test
kits, only as screening Maximum Residue Limits
(MRL) methods for cephalosporins residues in milk
[10, 11], flow-injection [12], ELISA [13], and bio-
sensors [14].
In the present work a simple, fast and accurate
simultaneous determination of two cephalosporins:
cefotaxime and cephalexine in skimmed bovine milk
samples is proposed.
Table 1. Linearity and sensitivity data of examined cephalosporins in spiked milk samples after SPE
Analyte Slope (ng1 ) Intercept
Cefotaxime 0.1019  0.0045 0.0069  0.0149
Cephalexine 0.0630  0.0015 0.0046  0.0051
V. F. Samanidou et al.
Experimental
Instrumentation and chemicals
The chromatographic system was consisted of a Shimadzu
LC-10 pump (www.shimadzu.com) (Kyoto, Japan), an SSI 500
detector Variable UV-Vis (SSI, State College, PA, USA) with a
Rheodyne valve (20 mL) (www.rheodyne.com) (Cotati, CA, USA)
and PC software. HPLC grade (>99%) methanol and acetonitrile
was obtained by Carlo Erba (www.carloerbareagenti.com) (Milan,
Italy). Cefotaxime, cephalexine and caffeine, the internal standard
used were all of analytical grade and were supplied by Sigma (www.
sigmaaldrich.com) (Sigma-Aldrich, Steinheim, Germany). The SPE
columns used, Abselut NEXUS (SPE), were obtained from Varian
(www.varianinc.com) (Harbor City, USA).
Milk samples were purchased from local market. Ceporex Injec-
tion solution was supplied by Schering-Plough (Bray, Ireland).
Chromatographic conditions
A Kromasil 100, C18, 5 mm (250 mm
4.0 mm) column supplied
from MZ Analysentechnik (www.mz-at.de) (Mainz, Germany) was
used for the separation of the cephalosporins at room temperature.
The mobile phase consisted of methanol-acetate buffer (CH3COOH
0.2M-CH3COONa 0.2M, 80:20 v=v, pH ¼ 4.0) in 40:60 v=v ratio.
Flow rate was 1.0 mL  min1 and injection volume was 20 mL. The
chromatographic conditions were chosen in terms of peak shape,
(R) LOD LOQ Upper limit MRL
(ng  mL1 ) (ng  mL1 ) (ng  mL1 ) (mg  kg1 )
0.994 0.2 0.6 5 –
0.998 0.2 0.6 5 100
Fig. 1. Method application to veter-
inary formulation. 1 Cephalexine
(2 ngmL1 ); 2 caffeine (5 ngmL1 )
HPLC determination of cefotaxime and cephalexine residues in milk 473

column efficiency, chromatographic analysis time, selectivity and was reconstituted in 100 mL aqueous solution of caffeine (internal
resolution. Resolution factors were 1.2 between the two antibiotics standard. 5 ng  mL1 ) and 20 mL of the solution were injected in the
and 1.5 between cephalexine and internal standard. HPLC system.

Table 2. Results of method accuracy in pharmaceuticals (n ¼ 8)

Preparation of standard solutions
Injected (ng) Found (ng) Recovery (%)
Aqueous stock solutions (100 ng  mL1 , for each cephalosporin,
were prepared every month. The working standards were prepared Cephalexine 10 9.2  0.9 92.0
every week by appropriate dilution of the stock solutions in water. 40 38.6  1.6 96.5
All solutions were stored at 4  C. 100 94.9  4.6 94.9
Labelled amount 180 170.1  3.7 94.5
(mg  mL1 )
Method validation
The developed method has been validated with respect to intra-day
Table 3. Recoveries for cefotaxime and cephalexine for intra-day
repeatability, inter-day precision, linearity and accuracy. Linearity
repeatability (n ¼ 5) and inter-day precision (n ¼ 8) in milk samples
was obtained in the range of standard concentration for each ceph-
alosporin. A series of mixed standard solutions were prepared to Cephalosporin Added Found  SD RSD Recovery
cover the entire working range and each solution was injected three (ng) (ng) (%) (%)
times.
The limits of detection (LOD) were estimated as the quantities Intra-day repeatability (n ¼ 5)
producing a signal of peak height three times and the limit of Cefotaxime 20 24.1  1.4 5.6 120.7
quantification (LOQ) ten times, the size of the background noise. 60 54.0  1.2 2.2 90.9
100 116.7  4.7 4.0 116.7
Cephalexine 20 16.8  0.9 5.6 84.1
Sample preparation 60 53.9  1.4 2.5 89.8
100 93.1  3.7 4.0 93.1
Different SPE cartridges were tested, with different rinsing and
elution solvents. The selected Abselut NEXUS cartridges were con- Inter-day precision (n ¼ 8)
ditioned prior to use with 1 mL methanol and 1 mL phosphate buffer Cefotaxime 20 23.4  1.6 7.0 116.9
solution 0.1 M (pH ¼ 8.0). The sample was applied to the cartridge 60 52.7  2.1 4.0 87.9
after protein precipitation. A rinsing step was necessary with 1 mL 100 118.7  5.5 4.6 118.7
deionised water and 0.4 mL n-butanol to remove matrix interfer- Cephalexine 20 18.0  1.1 6.2 90.1
ences. The cephalosporins were eluted by applying 1 mL acetoni- 60 50.5  2.2 4.4 84.2
trile, subsequently evaporated to dryness under a gentle stream of 100 89.9  3.8 4.2 89.9
nitrogen (45–50  C). Finally the dry residue of the cephalosporins

Fig. 2. HPLC chromatograms of

cephalosporins: cefotaxime and ceph-
alexine in blank skimmed bovine
milk sample in the presence of caf-
feine (IS)
474
Aliquots of 1 mL milk samples were spiked with 100 mL of aque-
ous standard solutions of cephalosporins at concentration levels
of 0.3, 1, 2, 3, 4 and 6 ng  mL1 and treated with 3 mL acetonitrile
to precipitate milk proteins. After vortex mixing for 1 min the sam-
ple was centrifuged at 1500 g for 15 min and then it was applied to
the solid-phase extraction cartridge.
Results and discussion
The quantification of the cephalosporins was carried
out using the internal standard method. The peaks, in
milk samples, were identified by comparing the rela-
tive retention times. Regression analysis results are
presented in Table 1. The limits of detection in milk
samples were 0.2 ng  mL1 and limits of quantification
were 0.6 ng  mL1 , for cefotaxime and cephalexine
respectively, while linearity held up to 5 ng  mL1 .
The method was applied to a veterinary drug contain-
ing cephalexine as active ingredient 180 mg  mL1
after dilution. A typical chromatogram is shown in
Fig. 1. The results were accurate with the relative error
varying from 8.0 to 3.5% (Table 2). Accuracy and
precision from spiked skimmed bovine milk samples
are found satisfactory with recovery varying from 84.1
to 120.7% and the relative standard deviation (RSD%)
values were lower than 7% as shown in Table 3.
Selectivity was tested by applying the validated
method to four different samples of commercial
skimmed bovine milk. No interferences were ob-
served neither to the cephalosporins peaks nor the in-
ternal standard peak. Typical chromatograms of blank
V. F. Samanidou et al.
Fig. 3. HPLC chromatograms of
cephalosporins: cefotaxime and ceph-
alexine in spiked skimmed milk
sample in the presence of caffeine
(IS). 1 Cefotaxime (2 ng  mL1 ) –
tR ¼ 2.514 min; 2 cephalexine
(5 ng  mL1 ) – tR ¼ 2.967 min; 3
caffeine (5 ng  mL1 ) – tR ¼ 3.614 min
and spiked bovine milk samples are presented in
Figs. 2 and 3, respectively.
Conclusion
In the present work a new method for the separation,
determination and quantification of two cephalospor-
ins, in skimmed bovine milk, is presented. Solid-
phase extraction was used as the most effective and
adequate pre-treatment of the sample. The accuracy of
the method was tested with recoveries studies where
in skimmed milk samples varied from 84 to 121%.
As a conclusion the short time of analysis, the good
resolution, the simple and fast pre-treatment, the se-
lectivity and the simplicity of the procedure make this
method a good tool for routine analysis of the studied
cephalosporins in skimmed milk samples.
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