Novel RP-HPLC Method Development And Validation Of Deferasirox And Deferiprone In Bulk And Dosage Forms

Deferasirox:        

DEFERASIROX is chemically known as 4-[bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl] benzoic acid. Deferasirox is an oral iron chelator. Its main use is to reduce chronic iron overload in patients who are receiving long term blood transfusions for conditions such as beta-thalassemia and other chronic anemias Two molecules of deferasirox have the ability of binding to 1 iron atom. Deferasirox function is to manage iron toxicity by binding trivalent (ferric) iron (for which it has a strong affinity), forming a stable complex which is eliminated via the kidneys. The symptomatic adverse reactions caused by Deferasirox are more or less tolerable and if they become severe, they can be treated symptomatically, these include Nausea, Vomiting, Diarrhea, Skin rash, Stomach pain

Deferiprone:

Deferiprone is chemically known as 3-hydroxy-1,2-dimethyl-1,4-dihydropyridin-4-one. Deferiprone is an oral iron chelator used as a second line agent in thalassemia syndromes when iron overload from heme transfusions occurs. Thalassemias are a type of congenital anemia due a defect in the production of hemoglobin. As a result, erythropoiesis, the production of new red blood cells is impaired. Deferiprone is an iron chelator that binds to ferric ions (iron III) and forms a 3:1 (deferiprone: iron) stable complex and eliminated through urine. Deferiprone is more selective for iron in which other metals such as zinc, copper, and aluminum has a lower affinity for deferiprone.

MATERIALS AND METHODS:

Chemicals and reagents:

All reagents used were analytical grade, and solvents used were HPLC grade; Methanol and Water was procured from MOLYCHEM Pharmaceuticals. Potassium dihydrogen orthophosphate

was purchased from MOLYCHEM Pharmaceuticals. Acetonitrile for HPLC was purchased from MERCK Pharmaceuticals. Deferasirox was (100% pure) procured from MYLAN. Deferiprone (100% pure) was procured from CIPLA.

Instrumentation and software

Waters HPLC system Alliance 2695 separation module with an auto-injector, temperature controller for sample storage, and Empower Software Build 3471 SPs were used to monitor the signal output. Feature Release DB ID: 2639633283 has been installed. UV/VIS spectrophotometer -Model LABINDIA UV 3000. The column Xterra C18 (4.6 x 250mm).  Digital Weighing balance -Model BSA224SCW, Ultra sonicator (model: SE60US), pH Meter (model: AD102U), Suction pump (model:VE115N) Thermal oven (make: NEWTRONIC) were employed in this work.

Chromatographic conditions

The chromatographic separation was achieved by using the Xterra C18 column (4.6 x 250mm) with phosphate buffer 0.05M (pH 4.6) and ACN 55:45 (%v/v) was used as a mobile phase. The mobile phase was filtered through a 0.45µm filter, and the flow rate was 1.0 mL.min-1 with an isocratic elution method. Detection and quantitation of the main active pharmaceutical ingredients were achieved using a PDA detector at 255 nm.

Standard preparation

10mg of Deferasirox and Deferiprone working standard was accurately weighed and transferred into a 10ml clean dry volumetric flask and about 2 ml of DMF is added. Then it is sonicated to dissolve it completely and make volume up to the mark with the diluent. (Stock solution). Further 10.0 ml from the above stock solution is pipette into a 100 ml volumetric flask and was diluted up to the mark with diluent.

Sample preparation

Accurately 10 tablets are weighed and crushed in mortar and pestle and weight equivalent to 10 mg of Deferasirox and Deferiprone (marketed formulation) sample into a 10mL clean dry volumetric flask and about 7mL of Diluents is added and sonicated to dissolve it thoroughly and made volume up to the mark with the same solvent.  (Stock solution) Further 3 ml of above stock solution was pipetted into a 10 ml volumetric flask and diluted upto the mark with diluent.

RESULTS AND DISCUSSION

Method development strategy and optimization:

To provide a suitable method for the routine quality control analysis of this multicomponent drug mixture. The developed process was carefully planned and optimized to separate the cited compounds. The most critical aspect in RP-HPLC method development is the achievement of sufficient resolution of the analytes with good peak symmetry in a reasonable analysis time. Many experiments were conducted to optimize both the stationary and mobile phases for better results. In these trials, estimation was based on efficient resolution between the two analytes peaks. For optimization of the mobile phase, different types of mobile phase were tested entirely, such as Methanol: water (60:40 v/v),water: Methanol (40:60 v/v),Phosphate buffer (0.05m) pH 5.0: Methanol (50:50%v/v),Phosphate buffer (0.05M) pH 4.6: MeOH, Phosphate buffer (0.05M) pH 4.6: ACN (30:70%v/v).The most desirable clear separation between the two primary compounds within a relatively short run time was obtained. Method Development Trails are done using the Inertsil C18 (4.6 x 250mm) column; consequently, it became the Mobile phase of choice for this mixture. Other Mobile phase exhibited poor separation between the peaks of the target compounds.  The excessive tailing for the peaks was another disadvantage of using the Methanol: water (60:40 v/v),water: Methanol (40:60 v/v),Phosphate buffer (0.05m) pH 5.0: Methanol (50:50%v/v),Phosphate buffer (0.05M) pH 4.6: MeOH, Phosphate buffer (0.05M) pH 4.6: ACN (30:70%v/v).The multi-wavelength ranges were evaluated to measure each analyte at its maximum wavelength to verify the sensitivity. DFX and DFP show stronger UV absorption with prominent peaks at 255 nm. Further optimization was carried eluting peaks with optimal separation by the flow rate 1.0 mL/min and column temperature (range from 25°C to 40°C). Estimation of Deferasirox and Deferiprone in different mobile phases, solvent-buffer ratios were tried to propose final chromatographic conditions. The shape of the peaks, the symmetry, and resolution of Deferasirox and Deferiprone were good with mobile phase containing phosphate buffer (0.05M) pH 4.6 and ACN in a ratio of (30:70v/v). Isocratic elution at a 1mL/min flow rate, sample, and column temperature was Ambient. The developed method was successfully helpful to estimate the amount of Deferiprone and Deferasirox in bulk and tablet dosage form. The Optimised Chromatographic Conditions are shown in table 2, Chromatogram was illustrated in (Figure 3)

Method validation

Analytical method validation is necessary to ensure that the analytical method employed for a specific test is appropriate for its intended purpose. After method development, analytical techniques were validated before the duration of routine use. The parameters evaluated during contemporary method development include specificity, linearity, range, accuracy, robustness, & precision. The proposed method was to conform based on the International Conference on Harmonization (ICH) Q2 (R1) guidelines.

Specificity:

An essential obligatory ICH guideline for method validation is specificity or selectivity. In other words, specificity is the ability to evaluate the purity of the analyte in the presence of the co-eluting or co-migrating impurity. The method specificity was demonstrated by demonstrating that no excipients mediate with the retention time of both drugs in the assay sample chromatogram.

Method precision:

In method precision, a homogenous test of a single batch was analyzed six times. The results assure whether a method produces consistent results for a single batch. Calculate the percent relative standard deviation (%RSD). The suggested method was found to be precise since the RSD values method precision was below 1.0. The summary results were shown in table 3.

Intermediate precision:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day by using different make column of the same dimensions. The % RSD for the area of five standard injections results should not be more than 2%. The summary results were shown in table 3.

LOD (Limits of Detection) and LOQ (Limits of Quantification):

The LOQ and LOD are calculated using signal-to-noise ratios at analytical responses of 3×10 times the background noise. The method validation results were shown in table 3.

LOD (mg/L) =3 × Noise/signal × Lowest concentration of the linearity samples

LOD (mg/L) =10 × Noise/signal × Lowest concentration of the linearity samples

Linearity:

An assay can obtain test results directly proportional to the concentration of an analyte in the sample. The determination of this parameter will describe the range of the analytical assay. The linearity of the method was intended by drawing the calibration curves. Standard solutions of Deferasirox and Deferiprone of different concentration levels (10%-150%) prepared by serial dilution of standard stock solution) were used for this purpose. The summary results were presented in table 3, and the linearity curve was shown in (Figure 4,5).

Accuracy:

The accuracy of an analytical method is the closeness of the test results obtained by the process to the actual value. Accuracy may often be expressed as a percent of recovery by testing known added amounts of analyte. Accuracy was the measurement of the exactness of the analytical method. In this HPLC method, the recovery of the samples was verified with three concentration levels (50%, 100% & 150%). The recovery was performed by API + placebo and injected into the HPLC (triplicate).

The summary results were presented in table 3.

Robustness:

To demonstrate the robustness of the method, changes were made to the chromatographic conditions and system suitability parameters, such as tailing factor (<2>3000), and resolutions were between the nearest peaks (>2.0). Based on the results, the maximized method was proved robust, even under changed conditions. The outline results were presented in table 4.

Filter validation and solution stability

Two different types of 0.45?m filters (Nylon and PVDF) were used to determine the filter’s effect on the sample. Concentrations of both types of filtered samples were calculated and compared against the centrifuged sample and showed no difference in results. The sample solution was stable for up to 24 h on the bench.

FIGURES

Deferasirox:

Deferiprone:

       
           
       
Figure 1: Chemical Structures of Levamisole and Albendazole.

       
           
       
Figure 2: Dilute

Standard Chromatogram of Deferasirox and Deferiprone

       
           
       
Chromatogram for Deferasirox and Deferiprone Standard Preparation

Retention time of Deferiprone - 4.342 min.

       
           
       
Figure 5: Calibration curve of Deferiprone at 255 nm

TABLES

       
           
       
Table 1: System suitability evaluation.

* All the parameters R², S/N Ratio, %RSD should be within limits.

       
           
       
Table 4: Robustness evaluation.

* Results for actual Mobile phase composition (55:45 Buffer: ACN) have been considered

from the Accuracy standard.

CONCLUSION

A sensitive & selective stability indicating RP-HPLC method has been developed & validated for the analysis of Deferasirox and Deferiprone in bulk and pharmaceutical dosage form. Based on peak integrity results, obtained from the analysis of samples using demonstrated method, it can be certified that the absence of co-eluting peak along with the main peak of Deferasirox and Deferiprone suggested that the developed method is precise for the analysis of Deferasirox and Deferiprone in the bulk and pharmaceutical dosage forms. Further the proposed RP-HPLC method has acceptable sensitivity, precision and reproducibility.

ACKNOWLEDGEMENT: The authors are thankful to the Administration of Mother Teresa Pharmacy College, Kothuru, Sathupally-507303, and Telangana, India, for providing the essential

 research facilities.