HPLC determination of some antibiotics
HPLC determination of some antibiotics
Virginia Coman1, Veronica Avram1, Loredana Soran1, Rodica Grecu1,
Z. Moldovan2 and Hajnalka Farkas3
1 “Raluca Ripan” Institute for Research in Chemistry, Department of Chromatography, 30 Fântânele Street, RO-3400 Cluj-Napoca, Romania,
E-mail:
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
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].
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 100-5 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 mL) in a valve of 20 mL 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 m, 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.
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.
Table 1
Retention times (RT), detection limits (DT) and correlation factors (r) for
studied antibiotics.
No. / Antibiotic / RT [min] / DL [mg/ml] / r1 / Ampicillin / 2.70 / 50.0 / 0.99567
2 / Cefuroxime / 3.58 / 12.5 / 0.99972
3 / Lincomicin / 5.12 / 50.0 / 0.99960
4 / Penicillin / 7.00 / 3.1 / 0.99895
5 / Gentamicin / 7.95 / 50.0 / 0.98335
6 / Oxacillin / 11.01 / 0.8 / 0.99573
7 / Chloramphenicol / 12.46 / 12.5 / 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 m, 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
[1] M. Balş, Clinical Laboratory in Infections (Laboratorul clinic în infecţii), Medical Publishing House, Bucharest, 1982, 259–310.
[2] M. Vega, G. Garcia, R. Saelzer, R. Villegas, HPTLC Analysis of Antibiotics in Fish Feed, J. Planar Chromatogr., 1994, 7, 159–161.
[3] V.R. Meyer, Practical High-Performance Liquid Chromatography, IIIth Edition, John Wiley & Sons Ltd., Chichester, 1998.
[4] ***, LC Applications, Macherey-Nagel,1996, 90-101.
[5] G. Seidl, H.P. Nerad, Gentamicin C: Separation C1, C1a, C2, C2a and C2b Components by HPLC Using Isocratic Ion-Exchange Chromatography and Postcolumn Derivatization, Chromatographia, 1988, 25, 169–171.
[6] V. Hartmann, M. Rödiger, Application of HPLC to the Analysis of Penicillins and Cephalosporins, Chromatographia, 1976, 9, 266–272.