Development and validation of colorimetric method for the quantitative analysis of kanamycin in bulk and pharmaceutical formulation

Article history: Received on: 05/02/2017 Accepted on: 19/03/2017 Available online: 30/04/2017 The aim of this study was to develop a simple spectrophotometric method for the determination of Kanamycin (KM) in pure bulk form and in its pharmaceutical formulations. Being an amino group containing molecule, KM reacted with ascorbic acid to form a water soluble, purple-pink, 1:1 complex that showed two wavelengths maxima (λmax) at 390 nm and 530 nm. The color was developed after heating for 40 minutes at 100 ̊ C and remained stable for at least 48 hours. The validity of developed method was tested by analyzing KM under the optimum experimental conditions. Beer ’ s law was found valid over the concentration range (40-200μg /ml) with an excellent correlation coefficient (less than 0.999). The repeatability and reproducibility results showed a low relative standard deviation values (RSD % < 2), which reflected the precision of the developed method. The good percentages added recovery (100.09 ± 0.28 % and 99.98 ± 0.88 %, n = 3) at 390nm and 530nm, respectively, reflected the method freedom from interferences.


INTRODUCTION
Kanamycin (Fig. 1) is an aminoglycoside antibiotic obtained from the soil bacterium Streptomyces kanamyceticus, used parenterally in the treatment of various infections, especially those caused by gram-negative bacteria (Pestka, 1975).The coupling reagent (ascorbic acid) was reported to be used for determination of many primary amines containing drugs (Krishna and Sanker, 2007;Adam et al., 2015), penicillins and cephalosporins having α-aminoacyl functionality (Gadkariem et al., 2009;EL-Obeid et al., 1999).Although several methods were reported for the analysis of KM in bulk form, pharmaceutical dosage form, and also in biological fluids (Mahmoud et al., 2013;Mirela et al., 2007, Ahmed et al., 2007;Sekkat et al., 1989;Kim et al., 2001), these methods are either expensive, require many chemical reagents or sophisticated instruments.
Therefore, the aim of the present work was to develop simple and accurate colorimetric method for the determination of KM in bulk and pharmaceutical forms using ascorbic acid.

Reference and sample
KM sulphate RS was obtained from Aladdin Industrial Corporation, Shanghai, China.Kanamycin injection contains kanamycin acid sulphate B.P. equivalent to 1g kanamycin base, Shanghai Medicines & Healthproducts, China.

METHODOLOGY Preparation of Stock Solutions
KM standard stock solution 0.04g of KM standard was accurately weighed and transferred into 10 ml volumetric flask.The volume was then completed to mark with distilled water (solution A; 4000μg /ml or 0.4% w/v).

KM sample stock solution
A quantity of KM powder for injection equivalent to 0.04g of KM was accurately weighed and transferred into 10 ml volumetric flask.The volume was then completed to mark with distilled water (solution B; 4000 μg/ml or 0.4% w/v).

Coupling reagent solutions (Ascorbic acid)
Ascorbic acid solution (0.2%w/v) was prepared using different solvents (DMSO and DMF; solution C and D, respectively).

Blank reagent
2 ml of freshly prepared solution C was added to 0.5 ml distilled water in stoppered glass tube.The volume was then completed to 10 ml with DMSO.

Effect of heating time
Serial volumes from solution A (0.1˗ 0.5ml) were transferred into five stoppered glass tubes.The volumes were completed to 0.5ml with distilled water. 2 ml of freshly prepared solution C and 7.5 ml of DMSO were added to each tube.The above dilutions were repeated four times and heated in a boiling water bath for a time ranged between 20-50 minutes.After cooling to room temperature the absorbance values were measured against the blank reagent.

Effect of different solvents
Serial dilutions were made from solution A by transferring 0.1 ml, 0.3 ml and 0.5 ml into three stoppered glass tubes.The volume was then completed to 0.5ml with distilled water. 2 ml of freshly prepared solution C and 7.5 ml of DMSO were added to each tube.The solutions were heated for 40 minutes in a boiling water bath.After cooling, the absorbance values were measured against blank at 390 and 530nm.The above procedure was repeated using solution D instead of solution C and the volumes were completed to 10 ml using DMF.

Effect of ascorbic acid concentration
Two ml of 0.1%, 0.2% or 0.3% w/v ascorbic acid solution in DMSO were added separately to three stoppered glass tubes containing 0.5 ml of solution A. 7.5 ml of DMSO was added to each tube and the solutions were heated in a boiling water bath for 40 minutes.After cooling at room temperature, the absorbance values were measured against the blank reagent.

Construction of Calibration Curve:
Serial aliquots of solution A (0.1˗ 0.5ml) were transferred into five stoperred glass tubes.The volumes were completed to 0.5ml with distilled water. 2 ml of freshly prepared solution C were added to each flask.The volumes were then completed to 10 ml with DMSO.The mixture solutions were heated for 40 minutes in a boiling water bath.After cooling, the solutions were scanned at 350-600nm against the blank.The measured absorbance values at 390nm and 530nm were plotted against the corresponding concentrations to obtain the calibration curve.
Solution B was also treated as under calibration curve.The injection content was determined by the slope ratio method and direct sample/ standard comparison.

Method precision
The precision of the developed method was evaluated by the repeatability and reproducibility results.Different concentrations within the linearity range were analysed three times in the same day and between-days.The relative standard deviation (RSD) was then calculated.

Percentage Added recovery
0.2 ml of each solution A and B was transferred into separate stoppered glass tubes.0.2ml of solution A was mixed with 0.2ml of solution B in a third tube.These solutions were treated as under calibration curve and the percentage recovery was calculated (Adam et al., 2016).

Molar ratio method for determination of the stoichiometry
In a volumetric flask (10 ml), 0.034g of KM standard was dissolved in distilled water (concentration 5.0 x 10 -3 M).Then 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml & 1 ml of this solution were transferred into nine stoppered glass tubes.Distilled water was added to adjust the volumes to 1.0 ml.0.4 ml of freshly prepared ascorbic acid solution (5.0 x 10 -3 M ) was added to each tube and the volumes were then completed to 10 ml with DMSO.The above solutions were heated for 40 minutes in a boiling water bath at 100 0 C.After cooling, the absorbance values were measured at 530 nm and 390 nm against the blank.

RESULTS AND DISCUSSION
KM is composed of 2-deoxystreptamine (aminocyclitol moiety) glycosidically linked to amino sugars.It exhibits weak UV-absorption, thus a suitable chromogen is needed to obtain a more UV/VIS light absorbing chromophore that can be useful as a sensitive spectrophotometric method for its determination in bulk and dosage forms.
Ascorbic acid, naturally occurring cheap organic compound (sugar acid), was found to react with KM in presence of DMSO to produce pink ˗purple colored complex absorbing at 390nm and 530nm.The different experimental factors affecting the color development, intensity and stability were studied.
These factors include the solvent, the reagent concentration, the reaction time and temperature.During the study of the effect of different solvents on the color formation and stability, solutions of variable color intensities were obtained.In an attempt to reach an explanation for this observation on the reaction process, the effect of two solvents of different dielectric constants (D.E.) were studied (Table 1).The results obtained reflected that a hyperchromic effect was observed with the solvent with higher D.E. (DMSO).An assumption was drawn that DMSO with its medium polarity (DE 47) possibly enhances the reactivity of the ascorbic acid and stabilizes the formed π to π* and n to π* transitions.
The optimal volume and concentration for ascorbic acid to give satisfactory results were found to be 1.0 ml of 0.2% w/v in DMSO.The results obtained for heating effect are shown in figure 2. A fixed time of 40 minutes was established as the most suitable time (best r-value and color intensity) to give reproducible absorbance values with low standard deviations.

Fig. 2 :
Fig.2: Effect of heating time on colored product formation.

Table 1 :
Effect of different solvents on the formation of kanamycin-ascorbic acid complex.