Development and Validation of Estimation of Genotoxic Impurity (Benzimidamidecontent) in Leflunomide by Using RP-HPLC Technique

The measurement of Genotoxic contaminant, a simple, selective, linear, accurate, and specific reverse phase high-performance liquid chromatographic (RP-HPLC) process was proposed. A Benzimidamide impurity in the medication Leflunomide has been discovered. Separation and analysis were carried out on Zorbax SB phenyl (4.6 mm x 250 mm) with a particle size of 5.0 μm. with 0.1 % Triethylamine in purified water with a pH of 7.0 and a buffer of phosphoric acid (20% in water). The mobile phase is a 40:60 mixture of buffer and Acetonitrile with degassing. Isocratic program mode was used. The elution was carried out at a rate of 1.0 mL/min with UV detection at a wavelength of 289 nm. The temperature of the selected column oven is 25°C. The linearity and accuracy of Benzimidamide are covered in this approach, with a LOQ limit of 150 percent (i.e.0.03 to 0.45 ppm). The observed correlation coefficient is 0.99994, with a range of 100.01 to 104.8 for recovery. The measured percent RSD of six spiked test preparation is below 5.0 percent in procedure precision (i.e. repeatability) and intermediate precision (IP). When maintained at room temperature, the standard and sample remained stable for three days. System appropriateness characteristics such as tailing factor and percent RSD do not exhibit significant changes in robustness experiments. For the detection of Benzimidamide the present RP-HPLC method is act as selective, robust, linear and precise. Original Research Article Bhatale et al.; JPRI, 33(45B): 78-85, 2021; Article no.JPRI.74875 79


INTRODUCTION
Leflunomide (Fig.  1) is a Immune suppressive, anti-rheumatic (DMARD) drug [1] that is primarily used to treat active moderateto-severe rheumatoid arthritis [2,3] and psoriatic arthritis. 4-Isoxazolecarboxamide, 5-Methyl-N- [4-(trifluromethyl)-phenyl] is the chemical name for the drug Leflunomide. It functions as a pyrimidine synthesis inhibitor by inhibiting dihydroorotate dehydrogenase. Leflunomide has the chemical formula C12H9F3N2O2 and a molecular weight of 270.21 Leflunomide [4] is made from the starting materials 4-(trifluromethyl) aniline (TFMA) and 5-Methylisoxazole-4-carboxylic acid (5-MIA) in the production method. This Benzimidamide impurity is probable impurities and reveals the potential genotoxic agent in the essential starting material TFMA. As a result, the presence of this Benzimidamide impurity in Leflunomide must be identified and investigated. There is no specific method for determining the amount of Benzimidamide in Leflunomide in the literature. The goal of this research is to create a sensitive, and validated RP-HPLC method for determining the level of Benzimidamide impurity in Leflunomide.

MATERIALS AND METHODS
Emcure pharmaceuticals ltd, R & D, Hinjawadi, Pune provided a sample of Leflunomide and its impurity for development and validation. For mobile phase and diluent preparations, analytical grade ortho phosphoric acid, trimethylamine, acetonitrile, and purified water (HPLC grade) were employed. Gradient acetonitrile and triethylamine were used. Makemetler Toledo and waters HPLC with UV/ PDA detector and data acquisition, computation with Chromeleon software are used as analytical balances. Before they were utilised, all of the instruments were calibrated.

Preparation of Mobile Phase
Dilute Ortho phosphoric acid (OPA). Prepare 20 % of OPA in purified water. Mix well

Buffer Preparation
Mix 1 ml of triethylamine with 1000 ml of water, mix it and Degas. PH 7.0 (± 0.05) is adjusted by addition of dilute ortho phosphoric acid.

Mobile Phase Preparation
Prepare homogeneous mixture of buffer preparation and acetonitrile in ratio (40:60) mix and degas.

Diluent Preparation
Use acetonitrile as diluent.

Blank Preparation
Diluent is used as blank preparation.

Test Preparation
Prepared 20000 ppm of Benzimidamide sample solution in diuent.

Method Development
Leflunomide and its impurities are polar, a reversed phase chromatography method for detecting genotoxic Benzimidamide was established. In RP-HPLC, non-polar stationary phases such as phenyl, C4, C8, and C18 are used, while polar mobile phases such as water, acetonitrile, or buffer solution are used. Other parameters such as column compartment temperature, diluents, wavelength, and pH play a significant role during this evolution in terms of stationary and mobile phases. During stationary phase screening, Hypersil BDS C18 and YMC Triart, both C18 with (4.6 mm x 150 mm) and particle size3 were used. In addition, all display availability in 150 mm and 250 mm lengths. When the Zorbax SB Phenyl (4.6 x 250 mm) 5 was utilised, superior impurity separation, peak sharpness, and system suitability (tailing factor, column efficiency) were discovered.
Here the mobile phase is made up of triethylamine (TEA). Thus, a homogenous combination of 0.1 % triethylamine in pH 7.0 water with H 3 PO 4 added and degassed, and degassed acetonitrile for diluent. The total analysis time is 30 minutes. Different trial runs of standard preparation are used to select the optimal gradient programme, flow rate, and column oven temperature. The concentration limit in ppm of genotoxic impurity (Benzimidamide) in drug substance derived from the TTC (Threshold of Toxicological Concern) can be calculated based on the expected daily dose to the patient using equation: Concentration limit (ppm) = TTC [µg/day] /dose (g/day] = 1.5/0.100 = 15 ppm (Maximum daily dose of 100 mg/day and duration of treatment more than 10 years, the TTC limit for Benzimidamide impurity 1.5).
The chromatographic conditions are detailed in Table 1.
Furthermore, the HPLC analysis was performed using the isocratic programme, and the mobile phase was prepared using a 40:60 mixture of buffer and acetonitrile.

Specificity
By injecting Blank (diluent), standard (0.3 ppm Benzimidamide), and sample solution, the selectivity research parameter was done (20000 ppm). The chromatograms are analysed at the same wavelength as the method specifies. Table 2 contains the specificity data, as well as a chromatogram in Fig. 2. Blank (diluent) has no effect on the retention period of the Benzimidamide peak. All recognised and unknown peaks in the sample solution are well isolated from one another. Peak purity is more than 950, indicating that the peak is pure.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The signal-to-noise ratio approach was used to calculate the LOD and LOQ conc. of Benzimidamide impurity in Leflunomide. Injecting various concentration levels (between 10 and 100 percent) of standard solutions of hydroxylamine hydrochloride limit level concentrations to determine the projected LOD and LOQ concentrations. 1.50 ppm was the predicted LOQ concentration value for Benzimidamide impurity. The LOD concentration is calculated by multiplying the predicated LOQ concentration by a factor of 0.33. Table 3 shows the predicted LOD and LOQ values.

Linearity and Range
The capacity of a method to produce test findings that are proportionate to the concentration of analyte in a given test sample is known as linearity. Standard solutions of Benzimidamide impurity with LOQ Level to 150 percent specified limit (including 50, 80, 100, 120, and 150 percent) of concentration were used in the linearity investigation.
Table4 shows the correlation coefficient, slope, concentrations, and intercept of linearity data, and Figure 6 shows the linearity graph. Least squares linear regression analysis was used to examine the peak area versus concentration data. The Benzimidamide impurity has a correlation coefficient of 0.999940, which is higher than 0.999.

Precision
As stated in the technique of analysis, system precision was achieved by injecting five replicates of the standard preparation. For replicate injections, the observed percent RSD is 0.72, and the tailing factor is 1.0. For method precision, six distinct samples were prepared and analysed; for intermediate precision, six separate samples were created and analysed on various days, systems, and columns. The observed percent RSD in procedure precision and intermediate precision is 2.13 and 3.45, respectively. Overall percent RSD is 2.76, which is less than 5.0 percent, for twelve test preparations (six from procedure precision and six from intermediate precision). Table 5 provides the outcomes of method precision and intermediate precision.

Accuracy
Spiking test preparation with impurity at LOQ level, 50% level, 100 and 150 percent of specification limit concentrations was used to establish method accuracy. Table 6 shows the percent accuracy data for the Benzimidamide impurity. The percent accuracy observed at the LOQ level and 50% level, 100 and 150 percent, is between 100.02 and 104.8 percent, which is within acceptable limits. (An accuracy of 70 to 130 percent is recommended.).

Robustness
The method's resilience was tested by altering the flow rate by±10%. The flow rate is changed from 1.0 mL/min to 1.1 mL/min and 0.9 mL/min. In the actual procedure, the column oven temperature is varied by± 5 °C from 25 °C to 30 °C and 20 °C. Table 7 displays the observed area, standard deviation, and percent RSD. The retention times in all of the studies above differed by ±0.2 minutes from the original retention times. Tailing factor 1.01 to 1.15 was used to calculate the system appropriateness parameter. For robustness studies, the percent RSD ranges from 1.87 to 2.26. Changes in method parameters (flow rate and column oven temperature) had no significant impact on system suitability criteria tailing factor and percent RSD, according to Table 7. The values obtained are considerably within the acceptable range.

Solution Stability
The solution stability of the test preparation was tested at 25°C on a day-by-day basis for up to three days. Up to 3 days, the cumulative percent RSD values of the Benzimidamide impurity are substantially below acceptable limits. This implies that when stored at 25°C temperature, Analytical test preparations are stable for 3 days.

Mobile Phase Stability
The mobile phase was prepared according to the method of analysis, and the analysis was completed. After you've finished your analysis, keep a mobile phase at room temperature and show that it's stable. Initial system suitability parameter analysis and mobile stability research analysis were compared and checked.
In standard Benzimidamide impurity, the percent RSD and change in retention time are within criteria, and no haziness, precipitation, or appearance of mobile phase is seen up to 60 hours. As a result, mobile phase stability at room temperature is 60 hours.

CONCLUSION
The RP-HPLC method for Benzimidamide impurity content determination of Leflunomide is very exact, selective, accurate, and stable, and follows ICH criteria. Q2(R1) has been accurately developed and validated. The specificity demonstrates that the Benzimidamide impurity peak can be fully resolved from both known and unknown impurities. With LOQ at 150 percent level w.r.t. specification concentration, the method is linear, and the observed Correlation coefficient is 0.99994. Between 100.01 and 104.8 percent of Benzimidamide impurity was recovered. System appropriateness, such as tailing factor and percent RSD, has no substantial impact on robustness. The observed outcomes were deemed to be within acceptable bounds. For all of the technique parameters that have been examined, the validated method has shown satisfactory results. As a result, the current method is particular, linear, selective, precise, robust, and stable, and can be used well in analysis.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.