Aminoglycoside antibiotics have similar chemical and biological properties as well as mechanism of action. Neomycin (Scheme 1), belongs to a group of broad spectrum aminoglycoside antibiotics which are widely used in clinical therapy of serious infections. It inhibits the growth of both gram-positive and gram-negative bacteria.1 Streptomycin (Scheme 2), is a human antibiotic drug which also is used as a pesticide, to control fungi and algae, it is active against numerous gram-negative and grampositive bacteria. One of the greatest virtues of streptomycin is its effectiveness against tubercle bacillus. In itself it is not a cure, but it is valuable adjunct to the standard treatment of tuberculosis. Streptomycin remains one of the agents of choice for the treatment of certain ‘occupational’ bacterial infections, such as brucellosis, tularemia, bubonic plague, it is used rather widely in the treatment of infections of the intestinal tract.2
Many analytical techniques have been employed for the detection and determination of aminoglycoside antibiotics include potentiometric,3 voltammetric,4 amperometric,5 fluorometric,6-10 immunoassay,11 spectropolarimetric,12 electrophoresis,13 colorimetric,14 spectrophotometric,15-20 gas chromatographic,21 thin layer chromatographic,22-24 liquid chromatographic,25-31 and high performance liquid chromatographic.32-34. To the best of our knowledge, there is no work in the literature reported about the application of potassium permanganate for the determination of neomycin and streptomycin.
The present communication describes four visual spectrophotometric methods (A-D) for the assaying of the cited drugs in bulk form and in commercial pharmaceutical formulations. Methods A-D are indirect procedures, involving the addition of an excess KMnO4 and the determination of unreacted oxidant by the decrease in absorbance of the different dyes.
Apparatus. Absorbance measurements were carried out using Biotech (UV-VIS) spectrophotometer (Cyprus), with scanning speed 400 nm/min and band width 2.0 nm, equipped with quartz cells of 10 mm path length.
Reagents and materials. All chemical and reagents used were of analytical or pharmaceutical grade and doubly distilled water were used throughout.
Pure neomycin and streptomycin were obtained from the Egyptian International Pharmaceutical Industries Company (EIPICO). Stock solutions of the studied drugs were freshly prepared daily by dissolving 20 mg of the drug in distilled water and then, completed to the mark in a 100 mL calibrated flask with distilled water. Working standard solutions were prepared by suitable dilution of the stock.
An aqueous solution of amaranth (AM) (Merck; 5.0×10-4 M), acid orange II (AO) (Merck; 5.0×10-4 M), indigocarmine (Aldrich; 5.0×10-4 M), and methylene blue (MB) (Merck; 1.0×10-4 M) were prepared by dissolving an accurate weight of dye in least amount of water and completed to the mark in a 100 mL calibrated flask. The stock solutions of dyes were allowed to stand at room temperature for a few weeks without any significant decay.
A stock solution of 5.0×10-3 M KMnO4 (Aldrich) was prepared by dissolving an accurate weight in 10 mL of warm distilled water, then completed to the mark in a 100 mL calibrated flask. Standardized using sodium oxalate and kept in a dark bottle. A 5.0×10-4 M solution of KMnO4 was prepared by diluting the previously stock solution with water and 2.0 M H2SO4 was prepared.
General procedure and calibration
Pipette a 1.0 mL aliquot of the examined drugs solution in a series of 10 mL calibrated flasks, followed by acidification by adding 0.5 mL of 2.0 M H2SO4. A 2.5 mL and 1.2 mL of 5.0×10-4 M KMnO4 were added and heated in a boiling water bath for 25 min and 20 min with neomycin and streptomycin, respectively. The mixture was cooled to laboratory temperature, then 1.3 mL of 5.0×10-4 M of AM was used for method A, 1.8 mL of 5.0× 10-4 M of AO for method B, 1.7 mL of 5.0×10-4 M Indigo for method C, and 2.5 mL of 5.0×10-4 M of MB for method D, with neomycin and streptomycin, respectively. The volume was completed to 10 mL with water. The decrease in color intensities in A, B, C and D, were measured spectrophotometrically at their corresponding maximum wavelengths. The concentration of each drug was found from a calibration graph constructed under the same conditions.
Procedure for pharmaceutical formulations: Procedure for tablet
The contents of twenty tablets of neomycin sulphate, were crushed powdered, weighed out and the average weight of one tablet was determined. An accurate weight equivalent to 20 mg of pure drug was dissolved in 20 mL distilled water and then filtered. The filtrate was diluted to 100 mL with distilled water in a 100 mL calibrated flask. This solution was further diluted stepwise to the request concentration with water and then analyzed by the recommended procedure.
Procedure for vials
Mix the content of ten streptomycin vials and weigh an accurate amount of the powder equivalent to 20 mg in a 100 mL calibrated flask. The above stated procedures described were applied to determine drug concentrations.
RESULTS AND DISCUSSION
The absorption spectra of the reaction products from the reduced drugs with amaranth dye (method A), acid orange II (method B), indigocarmine (method C) and methylene blue (method D) show characteristics λmax values at 521, 485, 610 and 664 nm, respectively, (Fig. 1). The calibration graph are linear over a concentration range of 5 - 11 μg mL-1 with neomycin sulphate and 2-7 μg mL-1 with strepto-mycin sulphate, (Table 1).
Fig. 1.Absorption spectra of the oxidation product between KMnO4 and, a- AM, b- AO, c- Indigo and d- MB
Table 1.*A = a + b C, where C is the concentration in μg mL-1.
Effect of reaction temperature
In order to obtain the highest and most stable absorbance, the effect of heating time on the oxidation reaction was studied. The reactions were performed on a boiling water bath at 100 ± 2 ℃ for the periods ranging from 5.0-35 min. Maximum and constant absorbance was obtained after 25 min for neomycin, where as 20 min for streptomycin. The results are shown in Fig. 2.
Fig. 2.Effect of heating time on the oxidation of: (■) 8.0 μg mL-1 neomycin-MB and (▲) 6.0 μg mL-1 streptomycin-AO.
Effect of the concentration of KMnO4
The influence of the volume of 5.0×10-4 M KMnO4 on the reaction has been studied. It is apparent from Fig. 3, that the absorbance increased with increasing volume of 5.0×10-4 M KMnO4 solution and reached maximum when 2.5 mL and 1.2 mL of KMnO4 solution were added to the total volume of 10 mL for neomycin and streptomycin, respectively. The color intensity decreased above the upper limits. Therefore, 2.5 mL and 1.2 mL of KMnO4 were recommended for all measurements (Fig. 3).
Fig. 3.Effect of volume of 5.0×10-4 M KMnO4 on the development of the reaction product: (▲) 8.0 μg mL-1 neomycin with MB and (■) 6.0 μg mL-1 streptomycin with AO.
Effect of acid concentration
To study the effect of sulphuric acid concentration the reaction was performed in a series of 10 mL volumetric flask containing 6.0 μg mL-1 of the cited drugs, different volumes (0.1 - 3.0 mL) of 2.0 M H2SO4 and 2.5, 1.2 mL of KMnO4 with neomycin and streptomycin, respectively. It was found that the maximum absorbance was obtained at 0.5 mL of 2.0 M H2SO4, beyond which the absorbance decreases. Thus 0.5 mL of 2.0 M H2SO4 was used through out the experiment.
Effect of dye concentration
In order to ascertain the linear relationship between the volume of added KMnO4 and the decrease in absorbance of AM, AO, Indigo and MB, experiments were performed in 0.5 mL of 2.0 M H2SO4 with varying volumes of KMnO4. The decrease in absorbance was found to be linear up to 2.5 mL and 1.2 mL of 5.0×10-4 M KMnO4 with 1.3 mL of AM, 1.8 mL of AO, 1.7 mL of indigo and 2.5 mL of MB with neomycin and streptomycin, respectively. The color was found to be stable up to 24 h.
Job’s method of continuous variation,35 was employed to determined the stoichiometry of neomycin, oxidant and dyes. Keeping the sum of the molar concentration of both fixed, the ratio of the concentrations of each two in the mixture was varied and the absorbances of the mixture were recorded at the suitable wavelength against reagent blank. The maximum absorbance, as well as known, corresponds to the stoichiometric ratio. Stoichiometric was found to be 1 : 4 for neomycin to oxidant; neomycin to dyes and 1 : 1 for oxidant to dyes (Table 1).
The stoichiometry between streptomycin, oxidant and dyes were determined by continuous variation of potassium permanganate concentration and the concentration of streptomycin being constant. The plot obtained by the molar ratio method,36 indicated that the reaction proceed by molar ratio of 1 : 20. Additionally, the stoichiometric ratio between drugs, dyes and between dyes and oxidant were examined as shown in Table 1, 2.
Table 2.*A = a + b C, where C is the concentration in μg mL-1.
Beer’s law limits, molar absorptivity, Sandell’s sensitivity, regression equations and correlation coefficients obtained by linear square treatment of the results are given in Table 1, 2. The standard deviation of the absorbance measurements was obtained from a series of 13 blank solutions. The detection (k = 3) and quantification (K = 10) limits of the methods were established according to the IUPAC definitions (C1= K S0 / s) where C1 is the detection limit, S0 is the standard error of blank determination, s is the slope of the standard curve, and K is the constant related to the confidence interval.37 In order to study the accuracy and precision of the proposed methods, three concentration levels of the studied drugs within the linearity range were selected and analyzed in five replicates. The measured standard deviation (SD), relative standard deviation (RSD) and confidence limit were, summarized in (Table 3, 4) and can be considered satisfactory, at least for the levels of concentrations examined.
Table 3.aRelative standard deviation for five determinations. bRelative Error. c95% confidence limits and five degree of freedom.
Table 4.aRelative standard deviation for five determinations. bRelative Error. c95% confidence limits and five degree of freedom.
The effects of various excipients associated with the drugs were investigated on the determination of neomycin and streptomycin in dosage forms. The results indicated that the talc, starch, glucose, alginate, gelatin and magnesium stearate.
The results indicated that the talc, starch, glucose, alginate, gelatin and magnesium stearate do not interfere with the assay of the drugs mentioned above even though they exist in excess amount. This is clear from the results obtained for the pharmaceutical preparations (Table 5, 6).
Table 5.aThe average of six determinations. bTheoretical values for t- and F-values for five degree of freedom and 95% confidence limits are 2.57 and 5.05, respectively.
Table 6.aThe average of six determinations. bTheoretical values for t- and F-values for five degree of freedom and 95% confidence limits are 2.57 and 5.05, respectively.
The proposed methods were, successfully applied to determination of the studied drugs in their pharmaceutical dosage forms (Table 5, 6). The performance of the proposed methods were assessed by calculation of t- and F- values compared with the official method.38,39 At 95% confidence level, the calculated t- and F- test,40 were less than the critical value, indicating that the proposed and official methods are equally accurate and precise. The results demonstrate the suitability of the proposed method for routine analysis of pharmaceutical preparations containing the studied drugs.
Chemistry of Colored Species
The proposed methods are based on the oxidation of the cited drugs by excess of KMnO4 to form oxidation products besides unreacted KMnO4 (step 1), and followed by the estimation of unreacted KMnO4 using AM (method A), AO (method B), Indigo (method C) and MB (method D), (step 2). The possible sequence of reactions are presented in Scheme 3.
The order of λmax values among the proposed methods in the determination of the cited drugs is D > C > A > B. The higher λmax of the visible spectrophotometric methods over reported UV and visible spectrophotometric methods is a decisive and advantage since the interference from the excipients should far less at higher wavelengths. The proposed methods are simple sensitive and can be used for routine analysis and in quality control laboratories for quantitative determination of the cited drugs in the pure or in pharmaceutical formulations.