Uv Spectroscopic Method Derivative Method Biology Essay

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Literature survey reveals no analytical methods like UV, colorimetric method and HPTLC for the determination of deferasirox from tablet dosage form. Hence, an attempt was made to develop the following validated methods,

An attempt was also made for the determination of deferasirox from human plasma by HPTLC method and also to study it's interaction with aluminium hydroxide by equilibrium dialysis method.

UV SPECTROSCOPIC METHOD

For the determination of deferasirox by UV spectroscopy, the stock solution of the drug was prepared in methanol as the drug was freely soluble in methanol and it showed maximum absorbance at 248 nm. Different concentrations of deferasirox were prepared from the stock solution and the absorbances were noted at the selected wavelength. The calibration curve was obtained for deferasirox in the range 3-12 μg/mL. The slope, intercept and correlation coefficient values were found to be 0.0725, 0.0023 and 0.999, respectively. The results show that within the concentration range tested, there was an excellent correlation between response factor and concentration of drug. Precision of the developed method was studied under intraday precision, inter day precision. Low % RSD values show that the developed method is precise. The recovery study was carried out at 80, 100 and 120% levels. Good recovery values illustrate that the method is free from interference present in formulation. Formulation analysis was also done.

DERIVATIVE SPECTROSCOPIC METHOD

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A second derivative UV spectroscopic method was used for the determination of deferasirox in tablet dosage form. Here the normal spectrum of deferasirox was recorded in methanol and it was converted into second derivative spectrum. From the spectra, amplitude was determined and calibration graph was plotted between amplitude and concentration in the range of 3-12 μg/mL. The slope, intercept and correlation coefficient values were found to be 0.0002, 0 and 0.999, respectively. The formulation was also subjected to same methodology. The amount of deferasirox present in the formulation was calculated. When compared with the UV method, the 2nd derivative spectroscopic method eliminates the interference from UV absorbing excipients.

VISIBLE SPECTROSCOPIC METHOD

For the determination of deferasirox by visible spectroscopy, 1% ferric chloride reagent was used to produce purple coloured chromogen. The chromogen showed maximium absorbance at 527 nm. Linearity was found in the concentration range of 20-90 µg/mL. Slope, intercept and correlation coefficient values were found to be 0.01, -0.0037 and 0.9999 respectively. The developed chromogen was stable about 30 hours at room temperature. Low % RSD values show that the developed method is precise. The recovery study was carried out at 80, 100 and 120% levels. This method was successfully used for the determination of deferasirox from tablet dosage form.

HPTLC METHOD

For the determination of deferasirox by HPTLC method different mobile phase systems were tried. It was found that a system comprising of methanol: n-butyl acetate (3.5: 6.5, v/v) was selected because this system gave good separation with symmetric peaks, (Rf value: 0.50± 0.03) at the selected wavelength of 248 nm. Calibration curves were plotted with peak areas of standard drug versus concentration. Linearity was found in the concentration range of 300 to 800 ng/spot (r=0.9992). After the development, the plate was stable up to 7 hours. Low relative standard deviation value shows that the developed method is precise. Limit of detection was found to be 20 ng/spot and Limit of quantification was found to be 50 ng/spot. Recovery study was carried out at 80%, 100% and 120% levels. Good recovery values show that the method is free from interferences. This method was successfully used for the determination of deferasirox from tablet dosage form.

RP-HPLC METHOD

In RP-HPLC method, optimizations of different chromatographic parameters like selection of chromatographic method, detection wavelength, selection of mobile phase, ionic strength of mobile phase, mobile phase ratio, flow rate etc. were done. A wavelength of 248 nm was selected for the study. A mobile phase system containing methanol: 0.1% formic acid (85:15, v/v) was employed for the determination of deferasirox because this system gave symmetric peak shape, and minimum of tailing (retention time - 4.1 minutes).

Linearity of deferasirox was found over the range of 100-1000 ng/mL, and correlation coefficient value was found to be 0.9994 showing good correlation between concentration and peak area response. The LOD and LOQ were found to be 0.8 and 50 ng/mL, respectively. Precision of the developed method was studied under inter-day, intra-day and repeatability studies. A low relative standard deviation value shows that the developed method is precise. Recovery study was carried out at 80%, 100% and 120% levels. Good recovery values show that the method is free from interferences. System suitability parameters like number of theoretical plates (N), asymmetry factor (As), resolution (Rs) etc. were studied. The validated RP-HPLC method was applied to the determination of deferasirox in tablet dosage form.

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Ravi kiran kaja., et al., reported a stability indicating LC method for the determination of deferasirox in bulk drugs and pharmaceutical dosage forms. The developed RP-HPLC method has the following advantages when compared to the reported method.

It is more sensitive

This method uses more simple and cheaper mobile phase system

The run time was less

Among the developed methods RP-HPLC method was more sensitive. RP-HPLC and HPTLC methods were found to be more precise and accurate. All the above developed methods were successfully validated according to ICH guidelines and these methods can be used for the analysis of deferasirox from formulation.

DETERMINATION OF DEFERASIROX FROM HUMAN PLASMA BY HPTLC

In order to extract the deferasirox from human plasma, Liquid-liquid extraction method was selected. Paracetamol was selected as the internal standard. After extraction, various concentrations of drug solutions were applied on the plate and development of the plate was done by using mobile phase system consisting of methanol: n-butyl acetate (2.5: 7.5, v/v). Rf values of deferasirox and internal standard were found to be 0.23 and 0.61, respectively. The developed method was validated according to the US-FDA guidelines. Linear regression data revealed an excellent linear relationship between concentration and peak area ratio over a concentration range of 100 to 700 ng/spot. The slope, intercept and correlation co-efficient values were found to be 0.0056, -0.1981 and 0.997. The concentration of 100 ng/spot was found as a 'Lower Limit of Quantification'. Good recovery values demonstrated the extraction efficiency. Accuracy and precision of the developed method was found to be within the permitted limit. The stability of deferasirox in prepared sample was found to be 12 hours and 22 hours, under room temperature and refrigeration, respectively. Hence the developed method can be used for the analysis of deferasirox from human plasma.

IN VITRO INTERACTION STUDY

Effect of aluminium hydroxide on in vitro protein binding of deferasirox

In the absence of aluminium hydroxide, percentage protein binding of deferasirox was found to be 98.16% and the percentage free fraction of deferasirox was found to be 1.84%. But after interaction with aluminium hydroxide, the free fraction of deferasirox increased from 1.84% to 7.27%, fig. 1.

Fig. 1: Effect of aluminium hydroxide on in vitro

protein binding of deferasirox

Therefore, from the study it may concluded that deferasirox should not be co-administered with aluminium containing antacid. Otherwise, the concomitant therapy may increase the free fraction of deferasirox and it may lead to the unwanted side effects like abdominal pain. In order to prevent this, the patients who are taking deferasirox should be advised, to avoid therapy with aluminium containing antacids.