Flow-Injection Chemiluminescence Determination of Aminoglycoside Antibiotics Using N-BromosuccinimideFluorescein System
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Chem. Anal. (Warsaw), 49, 643 (2004) Flow-Injection Chemiluminescence Determination of Aminoglycoside Antibiotics Using N-bromosuccinimideFluorescein System by Zhifeng Fu1, Zhujun Zhang1*, Zhouping Wang1, Wanfen Luo1 and Luqiu Fang2 1 Institute of Analytical Science, Southwest Normal University, Beibei, Chongqing 400715, P.R. China 2 Department of Chemistry, Fuling Teachers College, Fuling, Chongqing 408003, P.R. China Key words: chemiluminescence, flow-injection analysis, aminoglycoside antibiotic, N-bromosuccinimide, fluorescein A flow-injection chemiluminescence method for the determination of tobramycin, micro- nomicin, amikacin and gentamycin has been described. The method utilises chemilumines- cence of the above analytes emitted during their oxidation with N-bromosuccinimide (NBS) in alkaline medium in the presence of fluorescein. The obtained detection limits (3σ) are 0.07 µg mL-1 for tobramycin and 0.08 µg mL-1 for micronomicin, amikacin and gentamycin. The evaluated method is recommended for automated and continuous analysis. In the auto- mated mode the samples can be analysed at the rate of 100 per h with RSD of about 2.0% for the determination of 5 µg mL-1 of each aminoglycoside antibiotic (n = 11). The proposed method has been successfully used for the analysis of commercial pharmaceutical formula- tions without any sample pretreatment. Opisano przep³ywow¹, chemiluminescencyjn¹ metodê oznaczania tobramycyny, mikrono- mycyny, amikacyny i gentamycyny. W metodzie tej wykorzystano chemiluminescencjê pow- staj¹c¹ podczas utleniania analitów NBS w rodowisku alkalicznym, wobec fluoresceiny. Otrzymane progi wykrywalnoci (3σ) wynosi³y 0,07 µg mL-1 dla tobramycyny i 0,08 µg mL-1 dla mikronomycyny, amikacyny i gentamycyny. Opracowana metoda jest polecana do analiz zautomatyzowanych i ci¹g³ych. W systemie zautomatyzowanym próbki mog¹ byæ analizowane z szybkoci¹ 100 na godzinê z RSD ok. 2% dla 5 µg mL-1 ka¿dego antybiotyku aminoglikozydowego (n = 11). Proponowana metoda zosta³a z powodzeniem zastosowana do analizy formulacji farmaceutycznych bez wstêpnej obróbki próbki. * Corresponding author. E-mail: [email protected] 644 Z. Fu, Z. Zhang, Z. Wang, W. Luo and L. Fang Tobramycin, micronomicin, amikacin and gentamycin represent a group of amino- glycoside antibiotics, which are widely used in both human and veterinary medicine against Gram-positive and Gram-negative bacterial infections [1]. Determination of these antibiotics in pharmaceutical preparations and biological fluids appears to be very challenging. Microbiological procedure [2] is very slow and time consuming, and affected by many foreign ions or biologically active impurities present in a drug. A number of alternative chemical methods have been also described, e.g. liquid chroma- tography [35], capillary electrophoresis [68], fluorimetry [9] and thin-layer chromato- graphy [10]. These approaches often suffer from various disadvantages: complexity, time consuming, requiring expensive instrumentation, or may not be suited for the determination of analytes at lower levers. Halvatzis et al. [11] were the first to apply N-bromosuccinimide (NBS) in the chemiluminescent determination of isoniazid. Later numerous other analytes were determined using NBS, for instance: rifampicin [12], pyrogallol [13], amiloride [14], humic acid [15], sulfide [16], amino acids [17], cationic surfactants [18], epinephrine [19], tetracyclines [20] and urea [21]. Oxidising properties of NBS are attributed to hypobromous acid, which is released during the hydrolysis of NBS [22]. A great advantage of replacing hypobromite with NBS is relatively good stability of the latter. Another approach is electrogeneration [2325] of hypobromite instead using NBS. In this paper, a flow-injection chemiluminescence method for the determination of the exemplary aminoglycoside antibiotics has been proposed. Our approach utilises chemiluminescence reaction of the antibiotic with NBS in the presence of fluores- cein. Compared with the official and other methods, the proposed procedure is highly sensitive, fast and simple. EXPERIMENTAL Reagents All solutions were prepared from the analytical grade components using deionised and distilled water. 0.05 mol L-1 NBS stock solution was prepared daily by dissolving 2.225 g of NBS in 250 mL of water. Fluorescein stock solution (5 × 10-3 mol L-1) was prepared by dissolving 0.166 g of the compound in 100 mL of 0.1 mol L-1 NaOH. 200 µg mL-1 stock solutions of tobramycin, micronomicin, amikacin and gentamycin sulfates (The Institute of Pharmaceutical and Biomaterial Authentication, China) were prepared by dissol- ving 0.02 g of the compound in 100 mL of water. Pharmaceutical formulations of the same aminoglycoside antibiotics (Shanghai Hefeng Pharmaceutical Co. Ltd., China) were purchased from the local hospital. Other chemicals were of the highest available purity and used without further purification. Determination of aminoglycoside antibiotics 645 Apparatus The flow-injection system (Fig. 1) consisted of two ND15 peristaltic pumps (Shanghai Instrument and Electromotor Plant, China) and a FFQY8/16PD injection valve (Wenzhou Longfang Instrument Accessory Plant, China). PTFE tube (0.8 mm I.D.) was used to connect all elements of the flow system. A mixing glass coil used as a flow cell was positioned in front of the photomultiplier tube (PMT). The distance from the flow cell to the valve was about 25 cm. The chemiluminescence emission was recorded with an IFFM flow-injection Chemiluminescence Analyser (Remax, Xian, China) controlled via IFFM software. The chemiluminescence spectrum was examined by a RF540 fluorimeter (Shimadzu, Japan). P1 R1 P2 V HV R2 F R3 PMT PC R4 Waste Figure 1. The scheme of the FIA-chemiluminescence system used for the determination of aminoglycoside antibiotics. R1 sample solution; R2 carrier (H O); R3 fluorescein + NaOH solutions; 2 R4 NBS solution; P1, P2 peristaltic pump; V injection valve; F flow cell; PMT photo- multiplier tube; HV negative high-voltage supply; PC personal computer; Waste waste water Procedure The solutions were pumped at a rate of 1.2 mL min-1. The sample solution was introduced with the aid of the injection valve with a 100 µL loop. Chemiluminescence emission was detected with PMT (opera- ted at 700V) without wavelength discrimination. The signals from PMT were integrated and finally sent to the computer. Freshly prepared concentrated standard solutions of the studied antibiotics were appropriately diluted in the range of 0100 µg mL-1 to obtain working standard solutions. The intensities of the emitted chemilu- minescence were measured versus the standard samples and used for the calibrations. Pharmaceutical formulations of antibiotics were appropriately diluted with water to obtain the final concentrations of the analytes within the working range. These concentrations were determined by the pro- posed method and the results were compared with those obtained by the official microbiological reference method utilising Bacillus subtilis and Bacillus pumilus (concentration of a given aminoglycoside antibiotic is determined from the diameters of the inhibition zones). 646 Z. Fu, Z. Zhang, Z. Wang, W. Luo and L. Fang RESULTS AND DISCUSSION Optimisation of experimental conditions A series of experiments was conducted to establish optimum experimental condi- tions, such as a flow rate and concentrations of reagents, applying one-at-a time ap- proach. Optimisation was performed using 2 µg mL-1 solutions of aminoglycoside antibiotics. It occurred that each analyte required different optimum conditions for its determination. Effect of fluorescent compounds The applicability of various fluorescent compounds, such as fluorescein, dichloro- fluorescein, rhodamine B, rhodamine 6G and 8-hydroxyquinoline was investigated. Fluorescein was found to give the strongest signal and thus was chosen for further studies. The fluorescein concentration was varied within the range: 1 × 10-6 mol L-1 1 × 10-3 mol L-1 in order to estimate the value providing the most intense chemilumi- nescence of particular analytes. The concentration of 4 × 10-5 mol L-1 was appropriate for the determination of tobramycin and amikacin. Relative chemiluminescence peak heights of micronomicin and gentamycin increased with the fluorescein concentra- tion. However, above 5 × 10-4 mol L-1 the noise dramatically increased, too. There- fore, the concentration of 5 × 10-4 mol L-1 was finally chosen for these two analytes. During the optimisation studies NBS and NaOH were present in the solutions at the levels of 0.04 mol L-1 and 1.0 mol L-1, respectively. Effect of NaOH concentration Optimisation of NaOH concentration was a key problem since it affects both: the fluorescence intensity and the course of redox reaction. Chemiluminescence was ob- served only in alkaline media. NaOH concentration was varied within the range of 0.05 mol L-15.0 mol L-1 and the optimum values were: 1.0 mol L-1 for tobramycin, 0.12 mol L-1 for amikacin, and 1.5 mol L-1 for micronomicin and gentamycin. These concentrations of NaOH provided maximum relative peak heights of the above analytes. In this part of optimisation procedure concentration of NBS was kept at 0.04 mol L-1. Concentration of fluorescein was as described in the previous section. Effect of NBS concentration Having established the optimum concentrations of NaOH and fluoresceine, one dealt with that of NBS providing the most intense chemiluminescence. NBS concen- Determination of aminoglycoside antibiotics 647 tration was investigated