ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 2, с. 210-215
ОРИГИНАЛЬНЫЕ СТАТЬИ
y%K 543.23+543.42:615.33
KINETIC-SPECTROPHOTOMETRIC DETERMINATION OF NEOMYCIN © 2015 I. D. RaSiC MiSiC1, G. Z. MiletiC, S. S. MitiC, D. A. KostiC, A. S. DjordjeviC
University of NiS, Department of Chemistry, Faculty of Science and Mathematics ViSegradska 33, P.O. Box 22418000NiS, Serbia 1E-mail: ivana.rasic@pmf.edu.rs Received 07.03.2013; in final form 14.02.2014
A new kinetically-based spectrophotometric method for the determination of micro quantities of neomycin is described in this paper. It is an adaptation of the modified Berthelot method which is a widely used colori-metric procedure for urea determination. The method was developed due to neomycin effect on 2,2'-dicar-boxyindophenol formation. The experimental conditions for successful determination of neomycin were optimized. The relative standard deviation (RSD) for the neomycin concentration interval of 77—154 p.g/mL, ranges from 0.7 to 1%. The method was directly applied to determining neomycin in ophthalmic drops. The obtained results in analyzed pharmaceutical samples were in accordance with disc-diffusion microbiological method results.
Keywords: kinetics, UV—Vis spectrophotometry, modified Berthelot reaction, neomycin, ophthalmic drops.
DOI: 10.7868/S0044450215020152
Neomycin is a water-soluble complex of aminoglycoside antibiotics produced from the fermentation of the actinomycete Streptomyces fradiae. Neomycin B (also known as framycetin) is the principal component of the purified complex and has the highest antibiotic activity. The free base is coupled with sulfate counter-ions, which is then used in variety of pharmaceutical products labeled as containing neomycin sulfate. It is administrated in the form of powder, aerosols, drops, creams, etc. alone or in combination with other drugs (dexamethasone, hydrocortisone, etc.). These product applications include ophthalmic, topical, oral and intravenous administrations. Neomycin sulfate has a narrow therapeutic range due to oto- and nephrotox-icity [1, 2].
Neomycin sulfate is mainly composed of mixture of neomycin B and its stereoisomer neomycin C (antimicrobial potency lower than that of B), as well as of small amounts of other constituents (A, D, E and F) that show nearly no antimicrobial activity. All substances present in neomycin sulfate that are not neo-mycin B are termed as "impurities." The official method for the assay of neomycin in pharmaceuticals is microbiological method [3, 4]. No purity information can be obtained using the microbial assay, but antibiotics impurities can produce errors in the measured activity, thereby compromising method accuracy with respect to the measurement ofjust neomycin B activity [5, 6]. Neomycin has been determined by a variety of analytical techniques, such as UV—Vis spectrophotometry [7—11], kinetic—spectrophotometry [12],
spectrofluorimetry [13, 14], HPLC [15-17], liquid chromatography [18], gas liquid chromatography [19], microbiological diffusion [20], HPLC with evaporative light scattering detection [21], capillary electrophoresis with UV-detection [22], colorimetry [23, 24] etc.
The aim of this research was to study the impact of neomycin on the two-step catalyzed modified Berthelot colorimetric procedure: urea hydrolysis by urease producing ammonia that reacts with hypochlorite producing chloramine. This compound in its further reaction with sodium salicylate forms a green coloured indophenol complex in the presence of sodium nitro-prusside as a catalyst [25, 26]:
Urea + HO
-2NH3 + CO2,
NH3 + OCl_
292 nm
slow
H2NCl,
243 nm
[Fe(CN)5NO] + 2OH_ ^
398 nm
4=4 Fe( CN)5NO2 ]4_ + H2O,
398 nm
[Fe(CN)5NO2]4_ + H2O ^ ^ [Fe(CN)5H2O]3_ + NO-,
415 nm
3 _ slow
[ Fe( CN)5H2O ]3 + H2NCl --
— Fe(CN)5H2O - H2NCl,
392 nm
(1) (2)
(3)
(4)
Salicylate + Fe(CN)5H2O - H2NCl ■ —" 2,2'-Dicarboxylindophenol*,
precursor
2,2'-Dicarboxylindofenol* + Salicylate —►2,2'-Dicarboxylindophenol.
fast
(6)
(7)
Limitations of this method are interferences of some drugs [27] and its inapplicability on automatic analyzers [28]. Literature survey has shown that there is no data for neomycin impact on this reaction. Experimental results given in this paper showed measurable inhibitory effect of neomycin. The rate of the reaction was inversely proportional to the concentration of this antibiotic. This fact was used for the development of the new method for determining micro quantities of neomycin. The proposed method has been validated for linearity, precision, accuracy and recovery.
EXPERIMENTAL
Apparatus and reagents. Deionized water was obtained using MicroMed high purity water system, TKA Wasseraufbereitungssysteme GmbH. The solutions were refrigerated, but thermo-stated at 25 ± 0.1°C prior to use in Julabo MP 5A Open Bath Circulations. Measurements of pH were carried out using Hanna Instruments pH meter. The readings of the absorbance were recorded on Perkin-Elmer Lambda 15 UV/Vis spectrophotometer, connected to a thermo-circulating bath.
The stock solution ofneomycin sulfate (1.00 x 10-3 M) was prepared by dissolving the required amount of neomycin trisulfate (Sigma-Aldrich) in deionized water. Urea solution (0.833 x 10-3 M) was made by dissolving the appropriate amount of urea (Merck). Urease (350 kU/L) and solution R4 (0.5 M sodium hydroxide and 24.8 x 10-3 M sodium hypochlorite) were taken from the Urea-Kit S 180 (bioMerieux®sa, France, ref. 61913, Lot No. 792053201). Working solution of urease (1 : 100) was prepared daily. Solution R3 (62 x 10-3 M sodium salicylate, 3.35 x 10-3 M sodium nitroprusside and 1 x 10-3 M EDTA ) was prepared by dissolving Merck chemicals, in accordance to bioMerieux R3 colour reagent, but without phosphate buffer, which was prepared as a separate solution by mixing required volumes of Na2HPO4 and KH2PO4 solutions.
Procedure. All the glassware used was cleaned in aqueous solution of HCl (1 : 1) and then thoroughly rinsed with tap, distilled and finally with deionized water. The selected volumes of reagents were transferred into the test tube in the following order: R3, phosphate buffer, urea, urease and R4 for the catalytic reaction (1), and R3, phosphate buffer, urea, neomycin, urease and R4 for the inhibited reaction (2). Water was added to the predetermined volume of 4 mL that
A 1.0 0.8 0.6 0.4 0.2 0
190
332 474
616
758
900 X, nm
Fig. 1. Changes in the absorption spectra of the reaction products with time after addition of R4 1 min (a, a'), 4.6 min (b, b') and 8.2 min (c, c'); 1 — catalytic reaction and 2 — inhibited reaction. Initial reactants concentrations
in solution: 1.0 mL R3 (CNa-salicylate =
1.55 x 10-2 M,
CNa-nitroprusside = 8.38 x 10-4 M, CedTA = 2.5 x 10-4 M),
4.16 x 10-5 M, c,,
(CNaOCl = cneomycin =
25.0 ± 0.1°C.
1.24 x 10-3 M,
175 U/L, 0.2 mL R4 CNaOH = 2.5 x 10-2 M),
123 p,g/mL, phosphate buffer with pH 7.5,
was held constant during the experiment. The cell of the spectrophotometer (optical path 2 cm) was rinsed well and filled with the reaction solution. Absorbance (A) was measured at 700 nm every 30 s, during 5 min, starting 2 min after the addition of the enzyme (R4 is added 1 min after urease was added).
RESULTS AND DISCUSSION
Absorption spectra. Figure 1 showes the changes of absorption spectra of modified Berthelot reaction product without (curves 1) and with neomycin (curves 2). Absorption maximum at 700 nm comes from 2,2'-dicarbox-ylindophenol, and its production is slower in the presence of neomycin. Intensive overlapping absorption maxima in the interval of 386—405 nm come from nitri-topentacyanoferrate, aquapentanocyanoferrate (AqF) and AqF—H2NCl, equations (3)—(5). The position of these maxima was confirmed by the absorption spectrum of the product formed between Na-nitroprusside and reagent R4 (Fig. 2). Values of the mentioned absorbance maxima were lower in the presence of neomycin. This fact points out the neomycin inhibitory effect. In order to analyze this effect, individual spectra of neomycin with R3 and R4 in phosphate buffer were recorded. Neomycin and reagent R3 did not react in phosphate buffer, while in reaction of neomycin with R4 the product absorbing at 252 nm was formed (Fig. 3a).
The reaction system neomycin—R4—NH+ (ammonium instead of urea and urease) showed the same position of absorbance maximum (Fig. 3b). This maximum probably represents organic chloramine formed between neomycin and hypochlorite from reagent R4. This way, the amount of OCl- is reduced which leads to the de-
c
urea
A
1.0 0.8 0.6 0.4 0.2
0 190
b
Ш/ж ^ a
-
- I т1— | |
332
474
616
758
900 X, nm
Fig. 2. Changes in the absorption spectra of the product formed between Na-nitroprusside and reagent R4 with time, min, after: 1 (a), 4.6 (b) and 8.2 (c). Initial reactants concentrations: cNa-nitropmsside = 4.19 x 10-4 M, 0.2 mL R4 (CNaOCl = 1.24 X 10 " phosphate buffer with pH 7.5, 25.0 ± 0.1 °C.
3 M, cNaoH = 2.5 x 10_2 M),
creased amount ofinorganic chloramine H2NCl, Fig. 3c, equation (2), which is needed as the primary intermediate product for producing complex AqF-H2NCl, which in the reaction with salicylate gives final green colored product in the modified Berthelot reaction, equations (5)-(7).
Kinetic studies. A differential variant of the tangent method was used for the processing of the kinetic data. The reaction rate was obtained by measuring the slope of the linear part of the kinetic curve (absorbance-time plot, Slope = dA/dt). Preliminary experiments were performed in order to optimize conditions for the determination of lowest possible neomycin concentration. The impact of concentration of each reactant on the reaction rate was studied at 25 ± 0.1°C. The chosen concentration values of the variables were maintained constant throughout the experiment.
The effect of pH on both catalytic and inhibited reaction was studied in phosphate buffer in the interval of 6.8-8.0. The choic
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