INTRODUCTION:

The Antidote class includes Deferasirox (DFS) it’s chemically known as 4-[3, 5-bis (2-hydroxyphenyl)-1H-1, 2, 4-triazol-1-yl]-benzoic acid. DFS is an iron chelator that can be swallowed by mouth. The main application of DFS reduce severe iron overload in patients who need long-term blood exchange for disorders like beta-thalassemia and other severe anemias.

DFS is an iron (as Fe3+) selective orally active chelator. It's a tridentate ligand that complex with iron exceptional affinity in a 2:1 ratio. Despite the fact that DFS has a low affinity for zinc and copper, there are varied declines in serum concentrations of these trace metals following DFS administration. These declines aren't known to have any therapeutic impact. DFS is fairly soluble in Dimethylformamide, Dimethyl sulfoxide, and slightly soluble in methanol, practically insoluble in water. DFS is a powder that is white to slightly yellow in colour. DFS has molecular formula C₂₁H₁₅N₃O₄, and its mass is 373.4g/mole. DFS finished products have different types of inactive ingredients. The structures of DFS are shown in (figure 1).

Few LC approaches have been published in human plasma determination by LC¹, cardiac iron overload in -thalassemia, blood², calcium and argon were involved in an iron evaluation by ICP-MS³, biological fluids/drug product determination by sensitized fluorescence^4,5, Pharmacokinetics, distribution of drug, metabolic pathway, and eliminationiron chelation in animal models⁶, few were published in LC chromatography^{7-12, 17-20}, iron overload and chelation¹³, Electrocatalytic oxidation¹⁴, and capillary zone electrophoresis¹⁵. few were published in other LC chromatography²¹^-40.

Figure 1. Chemical structure of Deferasirox

MATERIAL AND METHODS:

Chemicals and Reagents:

DFS (potency-100.0%) was procured from Glen mark Life sciences, Gujarat, India.VWR International, and LLC provided the AR grade Orthophosphoric acid. JT Baker provided an HPLC grade of Acetonitrile with a purity certificate of 99.9%. For the experimental analysis high quality double distilled water (Milli-Q) was used (Total Organic Carbon - TOC 500ppb, pH 5.0-7.0, Conductivity 1.2 s/cm)

Instrumentation and Software:

It isa HPLC system with an Alliance e2695 elution module, water software, auto-injector, sample storage heat controller column was used in this work. Build 3471 SPs of Empower 3 Software The following signal output was recorded after installation: DB ID: 2639633283, Feature Release 3. The Inertsil ODS-3V C18 (150 x 4.6 mm), 5m LC column was made by GL Sciences Inc. An analytical balance model CP225D (made by Sartorius), a top load balance model GP5202 (produced by Sartorius), a sonicator (manufactured by LIFECARE), and a thermal oven were also used in this experiment (made by NEWTRONIC).

Chromatographic conditions:

A mobile phase of 60:40 v/v Acetonitrile and buffer (0.05% Orthophosphoric acid in water) was used to produce chromatographic separation at a flow rate of 1.5 mL min^-1. The LC column utilized was an Inertsil ODS-3V C₁₈ 150mm length, 4.6mm ID, and 5µm particle size at a 10µL injection volume. The temperature of the column and sample is kept at 25°C. The PDA and UV detectors were used to identify and quantify the DFS at 250nm.

Standard Preparation:

To acquire a concentration of 108µg/mL, the DFS standard was prepared in the diluent (the diluent was prepared by mixing water and acetonitrile in a 50:50 v/v ratio). Representative chromatograms of dilute standard preparation were presented in (figure 2). (Table 1) shows the system suitability results and evaluation.

Figure 2. Chromatogram of Standard

Table 1. System suitability evaluation

Parameters	Deferasirox	Acceptance criteria
Retention Time(min)	3.653	±10%
USP Tailing factor	1.1	NMT 2.0
USP Plate count	3686	NLT 3000
% RSD	0.2	NMT 2.0

Preparations of Sample:

Accurately Weigh and transfer 360mg of crushed tablet powder (equal 180mg DFS in DFS tablets) to a 50mL volumetric flask, and add diluent about 35mL, afterwards sonicate it for 30 minutes with intermediate shaking, allow to stand at room temperature before diluting with diluent to volume, and thoroughly mix. Centrifuge a portion of the solution for 10 minutes at 3000rpm.

Pipette 3mL test stock supernatant solution in 100 mL volumetric flask, diluted to volume with diluent, and thoroughly mixed.

Method development and optimization:

Analytical method development is a procedure of proving that the developed chromatography method is appropriate for its intended use in the improvement and manufacturing of the pharmaceutical drug substance and drug product.

The chemical structure and functional groups of DFS, as well as component solubility, were used to develop analytical methodologies. The acidic component pka of DFS is the most powerful (pka 4.55). The structural polarity of the component was the starting point for HPLC technique development. The completion of any analytical process is impossible without the use of a detector. The majority of organic/drug compounds have UV–vis absorption and are aromatic or unsaturated in nature. When it comes to quantifying and characterizing molecules and their impurities, this is advantageous. The compound's absorbance maxima will be measured using a either UV–visible spectrophotometer or diode array detector (DAD) in HPLC. The detection was done at the same wavelength as the DFS's UV absorbance maxima, which are at 250nm.

For stationary phase optimization, many types of columns were thoroughly examined, including Inertsil ODS-3V C18, (150*4.6mm), 5µm, Phenomenex kinetex C18 (4.6*150mm), Spherisorb 80A^o C18, (4.6*250mm), and Symmetry C18, 250mm x 3.0mm, 5.0µm. Different individual and combinations of solvents such as water, acetonitrile, methanol, and buffer solutions were investigated for analyte separation using the above-mentioned columns. The buffer and its efficiency have a big role in peak symmetry and separation. A range of organic and inorganic buffers are used to produce the desired separation. To optimize the chromatographic peak shape of the chemical, 0.05 percent ortho phosphoric acid was used. DFS is soluble in CH₃OH and a combination of CH₃CN and H₂O. With 60:40 v/v Acetonitrile and buffer ratios, the peak forms and symmetry of DFS were good (0.05 percent ortho phosphoric acid into water). A series of method development tests were carried out by changing conditions of chromatography those including method flow rate (1.0-2.0 mL min^-1) and the temperature of column (between 30-40°C) to determine the peak shape and symmetry of the compound As a result, the Inertsil ODS-3V C18 (150 x 4.6 mm), 5µm column became the column of choice for this mixture since it gave the most optimal unambiguous separation of DFS in a relatively short run time. The target compound's peak shape was poor in other columns. Other tests found that employing the Phenomenex kinetex C18 (4.6*150 mm), Spherisorb 80A^oC18 (4.6*250 mm), and Symmetry C18, 250 mm x 3.0 mm, 5.0µm, resulted in significant peak tailing. Using an isocratic method with a constant flow rate (1.5 mL/min) column, the method was found to have appropriate peak symmetry and reasonable retention durations based on the overall optimizations. The temperature of the sample is kept constant at 25°C.

METHOD VALIDATION:

The approach was validated using ICH Q2 (R1) guidelines from the International Conference on Harmonization¹⁶.

Specificity:

The research was undertaken to determine the involvement of blank and placebo. According to the test technique, the assay was done twice on placebo, with each dose matching to the mass of the placebo in a fraction of the test preparation. There were no peaks at the RT of DFS peak on chromatograms of Blank and Placebo solutions. This means that the excipients employed in the formulation have no effect on the DFS content of DFS tablets. Representative chromatograms of blank were presented in (figure 3).

Figure 3. Chromatogram of Blank

Limit of Quantification and Limit of Detection:

To establish the LOD and LOQ of DFS was verified by the slop method. The LOD &LOQ were calculated by using the below following formula. The method validation results are shown in (Table 2).

3.3 X SD of Y-intercept

LOD = ––––––––––––––––––––––––

Slope of a calibration curve

10 X SD of Y-intercept

LOQ = ––––––––––––––––––––––––

Slope of a calibration curve

Linearity:

As per current ICH guidelines, the linearity was tested. The linearity of detector response was verified by preparing the individual seven concentrations ranging from 10% to 150% of the standard concentration. In the diluent, the linearity solutions were created. The linearity curve was calculated by injecting each concentration level of the sample into the HPLC and measuring the value. The calibration curve was drawn between peak areas versus the concentration of samples. The linear regression equations were found to be satisfactory. The linearity of the approach is indicated by the regression coefficient value R2 = 0.999, as seen in (figure 4). The method validation results are shown in (Table 2).

Figure 4. Linearity Curve for Deferasirox

Precision:

A homogeneous test of a single batch was evaluated six times for method precision. The results determine whether a method produces consistent results for a single batch. Six samples were prepared in the same way using DFS samples, and six samples were prepared the next day to test the method's inter-day precision. The accuracy of the procedure was determined by calculating the % Relative Standard Deviation (RSD). The summary findings are shown in (Table 2).

Table 2. Method validation results

Parameters	Deferasirox
Linearity (10-150%)
Range (µg mL^-1)	10.8-162
Slope	49434.212
Intercept	-2440.971
Correlation Coefficient	1.000
STYX SD	6312.013
LOD (µgmL^-1)	0.421
LOQ (µgmL^-1)	1.277
Accuracy^(a) ( percent of Recovery)
50% Mean ± SD	100.5±0.54
100% Mean ± SD	99.1±0.98
150% Mean ± SD	100.2±1.22
Precision ^(b)(%RSD)
Repeatability	1.2
Intermediate precision	1.5

^(a)Each concentration level is average of three determinations

^(b)Percent RSD is six determinations of each component.

Accuracy:

Accuracy was established to determine drug extraction capability diluent at different concentrations, and the results were within the acceptable range. The present improved approach's recovery illustrates how close the method is to actual theoretical values. The recovery of the samples was exhibited with three concentration levels in this HPLC procedure (50 percent, 100 percent, and 150 percent). API+ placebo was used to perform the recovery, which was then injected into the HPLC (triplicate). For each level, the percent recovery was computed. The findings are summarized in (Table 2).

Robustness:

To demonstrate the robustness of the method, the chromatographic conditions were purposefully modified, and Tailing factor (2.0) and theoretical plate counts (>3000) were validated as system suitability requirements. Based on the results, the optimal process approach proved to be robust, even when the parameters were changed.

Filter validation and solution stability of the sample:

To evaluate the filter's effect on the sample, two different types of 0.45m filters were used (Nylon and PVDF). When compared to the centrifuged sample, there were no significant differences in the concentrations of both types of filtered samples. Sample and standard solution was stable for up to 72 h on the bench top.

CONCLUSION:

To determine deferasirox in bulk and tablet dosage form, an accurate, sensitive, and precise RP-HPLC method with PDA detection was developed and validated in accordance with ICH guidelines. For Deferasirox, the suggested method is extremely fast, with a total analytical run time of less than 5 minutes. Routine quality control analysis can also be easily changed using this procedure.

CONFLICT OF INTEREST:

The authors have declared no conflicts of interest.

ACKNOWLEDGEMENT:

There was no funding from any of the organizations. Thank you to the Aurex Laboratories LLC. Management for agreeing to publication.

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Received on 01.12.2021 Modified on 05.02.2022

Asian J. Pharm. Ana. 2022; 12(2):73-77.

DOI: 10.52711/2231-5675.2022.00013