Chem. Anal. (Warsaw), 49, 643 (2004)

Flow-Injection Chemiluminescence Determination of Aminoglycoside Antibiotics Using N-bromosuccinimide–Fluorescein 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 œrodowisku alkalicznym, wobec fluoresceiny. Otrzymane progi wykrywalnoœci (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 szybkoœci¹ 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 [3–5], capillary electrophoresis [6–8], 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 [23–25] 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 ND–15 peristaltic pumps (Shanghai Instrument and Electromotor Plant, China) and a FFQY–8/16PD 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, Xi’an, China) controlled via IFFM software. The chemiluminescence spectrum was examined by a RF–540 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 0–100 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-1–5.0 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 over the range of 0.01 mol L-1–0.05 mol L-1 (Fig. 2). It was impossible to extend this range due to the limited solubility of NBS in water. Finally 0.02 mol L-1 NBS was used for amikacin, and 0.04 mol L-1 for other analytes.

60 50 40 30 20 10 Chemiluminescence 0 0 0.02 0.04 0.06 C, mol L-1

Figure 2. The effect of NBS concentration on the chemiluminescence signal intensity of: ( ) amikacin: 4 × 10-5 mol L-1 fluorescein and 0.12 mol L-1 NaOH; (p) micronomicin: 5 × 10-4 mol L-1 fluores- cein and 1.5 mol L-1 NaOH; (l) gentamycin: 5 × 10-4 mol L-1 fluorescein and 1.5 mol L-1 NaOH (n) tobramycin: 4 × 10-5 mol L-1 fluorescein and 1.0 mol L-1 NaOH

Effect of the flow rate

The flow rate was being changed in the range from 0.5 mL min-1 to 2.5 mL min-1 to investigate its effect on chemiluminescence emission. For this study the optimum concentrations of the reagents were used. Chemiluminescence was found to increase with the increasing flow rate. However, above 1.2 mL min-1 the noise dramatically increased. Moreover, high flow rate led to a greater consumption of the reagents and improved the sensitivity only slightly. Therefore, one stuck to the rate of 1.2 mL min-1 for each reagent.

Chemiluminescence mechanism In aqueous solution, the oxidising properties of NBS are attributed to hypobro- mous acid released during the hydrolysis of NBS [22]. NBS reveals similar oxidising properties to hypobromite, however it is more stable. Possibly amino and hydroxyl groups present in the molecules of aminoglycoside antibiotics are oxidised by hypo- bromous acid generated from the hydrolysed NBS, yet the product of the oxidation is unknown. The energy released during the oxidation reaction induces chemilumines- cence of fluorescein. In order to prove this hypothesis, 5 µg mL-1 solutions of each analyte were investi- gated under the optimum conditions in the absence of the fluorescein, and no chemi- 648 Z. Fu, Z. Zhang, Z. Wang, W. Luo and L. Fang luminescence signal was detected. In contrast, in the presence of fluorescein strong chemiluminescence was observed. In the recorded chemiluminescence spectrum (Fig. 3 b) only one peak appeared at 515 nm, which overlaps with the maximum emission of fluorescein (Fig. 3 a). Thus the observed chemiluminescence can be assig- ned to the energy transfer in the examined systems.

0 2500 (a)

20000

15000

10000 Fluorescence

0 500Figure 3. 0 450 500 550 600 65

Wavelength, nm

600 (b) 500

400

300

200

Chemiluminescence 100

0 400 450 500 550 600 650

Wavelength, nm

Figure 3. a) Emission spectrum of fluorescein (4 × 10-5 mol L-1 fluorescein and 1.0 mol L-1 NaOH); b) chemiluminescence spectrum of aminoglycoside antibiotics (4 × 10-5 mol L-1 fluorescein, 1.0 mol L-1 NaOH, 0.04 mol L-1 NBS; each analyte’s concentration: 100 µg mL-1; flow rate: 1.2 mL min-1) Determination of aminoglycoside antibiotics 649

On the other hand, fluorescein itself undergoes oxidation to produce chemilumines- cence, on which the aminoglycoside antibiotics may have a catalytic effect. Another explanation is, that chemiluminescence originates from the reaction between fluores- cein and the oxidised analytes. However, we have no appropriate instruments and methods on our disposal to prove this hypothesis.

Interference studies Our next aim was to assess the proposed FI chemiluminescence method with respect to its applicability to commercial pharmaceutical formulations. The effect of some common excipients and accompanying compounds present in pharmaceuticals was studied. For this purpose one determined 5 µg mL-1 of the given antibiotic added to the synthetic sample solutions containing various amounts of each excipient or accompanying compound. The latter was considered not to interfere if the relative error was less than 5% for the determination of 5 µg mL-1 aminoglycoside antibiotic. The results (Table 1) indicate that no serious interferences were observed. Thus, the analysis of commercial pharmaceutical formulations applying our method is possible without any pre-treatment or separation.

Table 1. Foreign substance (F) - to - aminoglycoside antibiotic (A) tolerance ratio (antibiotic concentra- tion: 5 µg mL-1)

Tobramycin Micronomicin Amikacin Gentamycin F A

Starch 20 15 8 8

Sucrose 10 10 8 10

Glucose 12 2 8 20

Lactose 15 15 8 10

â-Cyclodextrin 40 30 20 25

Ascorbic acid 0.8 0.8 0.8 0.8

Sodium bitartrate 50 80 40 40

Na2C2O4 8 10 20 40

NaAC 100 90 40 40

Na2SO3 6 5 4 5

Ca(NO3)2 80 15 30 10

MgSO4 12 8 8 8

650 Z. Fu, Z. Zhang, Z. Wang, W. Luo and L. Fang

Response to aminoglycoside antibiotics The investigated aminoglycoside antibiotics arouse chemiluminescence under the optimum experimental conditions. Statistical parameters referring to the determina- tion results of the above analytes are given in Table 2.

Table 2. Statistical analysis of the determination results of aminoglycoside antibiotics using the proposed chemiluminescence method

Detection Linear range Regression equation Correlation Species limit RSD*, % ìg mL-1 C in ì g mL-1 coefficient ìg mL-1 0.2–7 I = 12.2C + 5.28 0.9997 Tobramycin 0.07 2.1 30–100 I = 1.2C + 142.78 0.9948

0.5–10 I = 10.8C + 19.40 0.9996 Micronomicin 0.08 1.8 30–100 I = 1.4C + 198.99 0.9936

0.5–7 I = 18.8C + 14.89 0.9961 Amikacin 0.08 2.0 10–70 I = 5.2C + 164.75 0.9990

0.5–7 I = 6.037C + 14.12 0.9989 Gentamycin 0.08 2.5 30–100 I = 0.267C + 70.67 0.9948 * Calculated for concentration = 5 µg mL-1, n =11.

Analysis of real samples The proposed method was applied to the determination of aminoglycoside antibio- tics in commercial pharmaceutical formulations. The results of the determination are given in Table 3. They differ favourably from those obtained using the officially appro- ved method. The accuracy of our approach was examined in recovery experiments performed with the commercial injection solutions and satisfactory results were obtai- ned (Table 4).

Table 3. Determination results of aminoglycoside antibiotics in commercial formulations

Proposed method RSD, % Official method Claimed Commercial formulation mg (n = 5) mg mg Tobramycin injection 79.2 2.3 80.2 80.0

Micronomicin injection 60.5 2.1 61.9 60.0

Amikacin injection 203.0 2.3 198.5 200.0

Gentamycin injection 82.0 2.5 79.2 80.0

Determination of aminoglycoside antibiotics 651

Table 4. The results of recovery studies of aminoglycoside antibiotics added to the samples of their pharma- ceutical formulations (for injections)

Analyte Initially present Added Recovered Recovery formulation ìg mL-1 ìg mL-1 ìg mL-1 % 1.50 4.38 94.0 Tobramycin 2.97 3.00 5.91 98.0

1.50 4.47 96.7 Micronomicin 3.02 3.00 5.99 99.0

Amikacin 1.50 4.50 97.3 3.04 3.00 6.30 108.7

1.50 4.56 98.7 Gentamycin 3.08 3.00 6.06 99.3

CONCLUSION

The proposed flow-injection chemiluminescence method for the determination of aminoglycoside antibiotics in commercial pharmaceuticals is fast, simple and easily automated compared to the standard microbiological assay. In comparison with other recent methods, it is advantageous with respect to time and reagents’ consumption, sample manipulation, and costs of equipment. It provides comparable detection limit, sample throughput and linear calibration range. It can be used for the determination of aminoglycoside antibiotics in commercial pharmaceuticals without any pre-treat- ment or separation.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 20175039).

REFERENCES

1. Chen X. and Jin Y., in: Pharmacology, 14 Edn, People’s Medical Publishing House, Beijing 2000, p. 67. 2. Society of Pharmacopoeia of Hygiene Department, in: The Pharmacopoeia of P.R. China (Part 2), China Chemical Industry Press, Beijing 2000, p. 312, 331, 866, 872. 3. Feng C., Lin S., Wu H. and Chen S., J. Liq. Chromatogr. Relat. Technol., 24, 381 (2001). 4. Benjamin S., Ma L.S., Aranzazu Z. and Jose M.L., J. Liq. Chromatogr. Relat. Technol., 25, 463 (2002). 652 Z. Fu, Z. Zhang, Z. Wang, W. Luo and L. Fang

5. Szunyog J., Adams E., Roets E. and Hoogmartens J., J. Pharm. Biomed. Anal., 23, 891 (2000). 6. Yang W., Yu A. and Chen H., J. Chromatogr., A, 905, 309 (2001). 7. Oguri S. and Miki Y., J. Chromatogr., B: Biomed. Appl., 686, 205 (1996). 8. Kaale E.L.S., Van S.A., Roets E. and Hoogmartens J., J. Chromatogr., A, 895, 67 (2000). 9. El-Shabrawy Y., Spec. Lett., 35, 99 (2002). 10. Argekar A.P., Raj S.V. and Kapadia S.U., J. Planar Chromatogr. Mod. TLC, 9, 459 (1996). 11. Halvatzis S.A., Timotheou-Potamia M.M. and Calokerinos A.C., Analyst, 115, 1229 (1990). 12. Halvatzis S.A., Timotheou-Potamia M.M. and Hadjiioannou T.P., Anal. Chim. Acta, 272, 251 (1993). 13. Safavi A. and Baezzat M.R., Anal. Chim. Acta, 368, 113 (1998). 14. Halvatzis S.A., Mihalatos A.M., Palilis L.P. and Calokerinos A.C., Anal. Chim. Acta, 290, 172 (1994). 15. Michalowski J., Halaburda P. and Kojlo A., Anal. Chim. Acta, 438, 143 (2001). 16. Safavi A. and Karimi M.A., Talanta, 57, 491 (2002). 17. Zhou G. and Chen H., Anal. Sci., 18, 693 (2002). 18. Safavi A. and Karimi M.A., Anal. Chim. Acta, 468, 53 (2002). 19. Michalowski J., Kojlo A. and Estrela O.A., Chem. Anal. (Warsaw), 47, 267 (2002). 20. Halvatzis S.A., Timotheou-Potamia M.M. and Calokerinos A.C., Analyst, 118, 633 (1993). 21. Li B., Zhang Z. and Jin Y., Anal. Lett., 34, 2141 (2001). 22. Halvatzis S.A., Timotheou-Potamia M.M. and Efstathiou C.E., Talanta, 40, 1213 (1993). 23. Zheng X..and Zhang Z., Analyst, 124, 763 (1999). 24. Li B., Zhang Z., Zheng X,.and Xu C., Microchem. J., 63, 374 (1999). 25. Li B., Zhang Z., Zheng X..and Xu C., Chem. Anal. (Warsaw), 45, 709 (2000).

Received October 2003 Accepted March 2004