STUDIA UNIVERSITATIS BABES-BOLYAI, PHYSICA, SPECIAL ISUUE, 2003
HPLC DETERMINATION OF SOME ANTIBIOTICS
Virginia Coman1, Veronica Avram1, Loredana Soran1, Rodica Grecu1,
Z. Moldovan2 and Hajnalka Farkas3
“Raluca Ripan” Institute for Research in Chemistry,
Department of Chromatography, 30 Fântânele Street,
RO-3400 Cluj-Napoca, Romania,
E-mail: v.coman@icrr.cj.edu.ro
2
National Institute for Research & Development of
Isotopic and Molecular Technology, 65-103 Donath
Street, RO-3400 Cluj-Napoca, Romania
3
“Babeş-Bolyai” University, Faculty of Chemistry and
Chemical Engineering, 11 Arany Janos Street,
RO-3400 Cluj-Napoca, Romania
1
ABSTRACT
The determination of antibiotics presents some difficulties because of their
thermal instability. The HPLC method at room temperature is preferred in the
last decade. This work presents a simple HPLC method for the
determination of some antibiotics (ampicillin, cefuroxime, lincomicin,
penicillin, gentamicin, oxacillin and chloramphenicol). The registering of
chromatograms was performed with a high performance liquid
chromatograph JASCO – 980 (JASCO, Japan) equipped with an UV-VIS
detector (190–400 nm). The BORWIN Soft has processed the HPLC data.
For all antibiotics the calibration curves were registered in the
concentration range of 0.2–1.0 mg/ml.
INTRODUCTION
The antibiotics are considered to be a group of chemical substances
obtained from the cellular metabolism or through partial synthesis, some of them
reproduced by synthesis, which in highly diluted solutions have the property to
stop the development and even to destroy some microorganisms [1]. Traditionally,
microbiological methods have been used for analysis of antibiotics; these methods
are time-consuming (entailing bacterial culture) and normally non-specific. More
recently, other methods have been described, including GC with different
detectors, HPLC and TLC; these have been shown to be fast, accurate, and specific
for many antibiotics. Most of these methods are used for the evaluation of raw
material in the pharmaceutical industry, where the moiety to be evaluated is almost
always free from interference [2].
At present, high performance liquid chromatography (HPLC) is the most
important separation method and the most important analytical technique as well
[3]. HPLC is an excellent tool for the determination of antibiotics [4].
VIRGINIA COMAN ET ALL
A HPLC system, using a strong cation exchanger and isocratic elution, was
developed for the separation of the main components of gentamicin (C1, C1a, C2,
C2a) and C2b (sagamicin) in less than 20 minutes. The detection was performed by
postcolumn derivatization with o-phthalaldehyde and a fluorescence detector. The
detection limit was 10 ng for gentamicin C1. Some commercial gentamicin samples
were analyzed. The stationary phase was two columns Nucleosil 100-10 SA, 250 x
4.6 mm ID each; eluent: 1.1 M KCl solution, adjusted to pH 3.0 with HCl, flow
rate 2 mL/min; detection: fluorescence, excitation 338 nm and emission 455 nm [5].
A reversed phase liquid chromatographic method for the analysis of some
penicillins and cephalosporins of closely related structure is described. The
influence of eluent pH and NaCl concentration on the resolution of ampicillin and
epicillin is discussed. The method can be applied to the selective analysis of
synthetically produced antibiotics and their pharmaceutical preparations. Because
of the high sensitivity (10–7g/cm3) the method can also be used for the analysis of
penicillins in physiological fluids. The stationary phase was column Nucleosil 1005 CN, 250 x 3 mm ID; eluent: 0.05 M phosphate buffer pH 7 + CH3OH (7:3.6,
v/v); flow rate: 3.2 mL/min; detection: UV 220 nm [6].
EXPERIMENTAL
This work presents a simple HPLC method for the determination of some
antibiotics, such as: ampicillin, chloramphenicol, oxacillin and penicillin
(ANTIBIOTICE, Iaşi), cefuroxime (SCHEIN, USA), lincomicin (LEK, Slovenia),
gentamicin (KRKA, Slovenia).
The chromatograms were performed using a high performance liquid
chromatograph (Jasco, Japan) equipped with a HPLC pump (Model PU-980), a low
pressure gradient unit (Model LG-980-02), an in-line degasser (Model DG-980-50)
and an UV-VIS detector (Model UV-970/975).
The solutions of studied antibiotics were prepared in HPLC grade water.
The samples were introduced by manual injection with Hamilton
Rheodyne Syringe (50 L) in a valve of 20 L loop.
To find the optimum UV wavelength for the detection of mentioned
antibiotics, the UV-VIS spectra were registered on UNICAM UV4 spectrometer.
For the determination of these antibiotics, the following experimental
HPLC parameters were established: wave length 220 nm; column NUCLEOSIL
120–C18, 5 , 250.4 cm; mobile phase acetonitrile – water with ammonium
acetate buffer 80 mM, in the ratio 20:80 (v/v); flow rate of 1 mL/min.
Some antibiotics were detected also at 270 nm (cefuroxime,
chloramphenicol) and 250 nm (gentamicin) in order to improve the HPLC
detection limit.
The BORWIN Soft has processed the HPLC data. For all antibiotics,
the calibration curves were registered in the concentration range of 0.2–1.0
mg/ml.
HPLC DETERMINATION OF SOME ANTIBIOTICS
RESULTS AND DISCUSSION
The ampicillin, cefuroxime, lincomicin, penicillin, gentamicin, oxacillin,
chloramphenicol can been separated by this method because they have different
retention times in the same HPLC conditions. The chromatogram is given in Figure
presented below.
Figure. HPLC chromatogram of studied antibiotics:
1-ampicillin; 2-cefuroxime; 3-lincomicin; 4-penicillin;
5-gentamicin; 6-oxacillin; 7-chloramphenicol.
The calibration curves were performed by plotting peak area versus known
injected amount of compounds. These are straight lines with the correlation factors
of r  0.995.
The retention times, detection limits and correlation factors for studied
antibiotics are given in the table below.
VIRGINIA COMAN ET ALL
Table 1
Retention times (RT), detection limits (DT) and correlation factors (r) for
studied antibiotics.
No.
1
2
3
4
5
6
7
Antibiotic
Ampicillin
Cefuroxime
Lincomicin
Penicillin
Gentamicin
Oxacillin
Chloramphenicol
RT [min]
2.70
3.58
5.12
7.00
7.95
11.01
12.46
DL [g/ml]
50.0
12.5
50.0
3.1
50.0
0.8
12.5
r
0.99567
0.99972
0.99960
0.99895
0.98335
0.99573
0.99925
CONCLUSIONS
A HPLC method was performed for the determination of seven antibiotics,
namely: ampicillin, chloramphenicol, oxacillin, penicillin, cefuroxime, lincomicin,
gentamicin.
The HPLC conditions of this method are: column NUCLEOSIL 120–C18,
5 , 250.4 cm; mobile phase acetonitrile – water with ammonium acetate buffer
80 mM (20:80, v/v); flow rate of 1 mL/min; detection at 220 nm.
The quantitative determination of mentioned antibiotics are possible using
the calibration curves.
This HPLC method can be used for the identification of antibiotics from
different matrices (biological media, food products etc), for the verifying of
antibiotic identity and to put in evidence the possible modifications during the
storage.
ACKNOWLEDGEMENTS
This work was financial supported by VIASAN Program of the National Plan for
Research-Development and Innovation (2001-2003).
REFERENCES
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