Sensitive and Selective Analytical Method for the Quantification of Two Potential Genotoxic Impurities in Azilsartan Drug Substance by using LC-MS/MS with Multiple Reaction Monitoring (MRM mode)

The main aim of the present study to Synthesize,method development and method validation for quantification of two potential genotoxic impurities i.e., Methyl(Z)-2-ethoxy-1-((2'-(N'hydroxycarbamimidoyl)-[1,1'-biphenyl]-4-yl) methyl)-1H-benzo[d]imidazole-7-carboxylate(Impurity-A) and 2-ethoxy-1-((2'-(N'-hydroxycarbamimidoyl[)-[1,1'-biphenyl]-4-yl)methyl)-1H-benzo[d]imidazole-7carboxylicacid(Impurity-B) by using LC-MS/MS MRM mode at trace level determination inAzilsartan drug substance. The new LC-MS/MS MRM mode method was developed by using Inertsil ODS-3V 150x4.6mm,5μm column as stationary phase. The mobile phase used is the composition of 10Mm Ammonium formate pH3.00buffer:Acetonitrile(9:1)%v/v as mobilephase-A and 10Mm Ammonium formate pH3.00buffer:Acetonitrile(2:8)%v/v as mobile phase-B, isocratic elution of mobile phase-A and Mobile phase-B (60:40)v/v at flow rate of 0.8mL/min.The concentration limits of the both genotoxic impurities were calculated a limit of 37.5ppm based on the concept of TTC (threshold of toxicological concern) and MDD (maximum daily dosage which is 40mg/day for Azilsartan drug substance.The limit of detection(LOD) was found to be 1.4ppm for both Impurity-A and ImpurityB.The limit of quantification(LOQ) for Impurity-A was 4.7ppm and Impurity-B was 4.5ppm Original Research Article Alluri et al.; JPRI, 33(63A): 467-478, 2021; Article no.JPRI.85510 468 respectively The method was found to be linear from 4.7ppm to 78.4ppm for Impurity-A (correlation coefficient:1.000) and 4.5ppm to 75.7ppm for Impurity-B (correlation coefficient:0.999). The method was precise and found percentage of relative standard deviation for six replicate sample preparations of Impurity-A and Impurity-B was below 5.0%. The method accuracy was confirmed based on the recovery studies. Based on the method validation study method was sensitive and selective for quantification both genotoxic impurities.

Literature survey show some work related to Azilsartan and Azilsartanmedoxomil [8][9][10][11][12][13][14][15]related substances by using high performance liquid chromatographic methods.So the accurate quantification of Impurity-A and Impurity-B at ppm levels the above Literature methods are inadequate. Literature survey reveals that there was no sensitive and selective method available for the quantification of Impurity-A and Impurity-B by using LC-MS/MS Multiple reaction monitoring (MRM)mode. . So, the objective of this work is to develop and validate a highly Sensitive, accurate and selective LC-MS/MS MRM mode method developed and validated for the determination of trace level quantification of Impurity-A and Impurity-B in Azilsartan drug substance.
Quantitative structure-activity relationship (QSAR) analysis carried out for all the raw materials, reagents, intermediate, impurities and reagents used in the process of Azilsartan drugsubstance to identify the Mutagenic impurities. We have found the Impurity A and Impurity B are mutagenic due to certain electrophilic moieties within a chemical structure. Both compounds are mutagenic as well as DNAreactive. i.e. Impurity-A is intermediate of Azilsartan and Impurity-B is process impurity due to hydrolysis of intermediate. Following illustration below shows.

MATERIALS AND METHODS
Azilsartan samples were obtained as a gift samples from reputed pharma company upon request. All chemicals were purchased from Sigma Aldrich and used directly. Reaction progress was monitored by thin-layer chromatography (TLC) using silica gelaluminum sheets (60F-254) and UV light. 1H nuclear magnetic resonance (NMR) spectra were recorded on BrukerAvance 400 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Splitting patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), or doublet of doublet (dd) and multiplet (m). The broad (br) signals were also indicated. The value of chemical shifts (d) is given in ppm and coupling constants (J) in Hertz (Hz). Mass spectra were obtained using waters XEVO TQ LCMS instrument was used with an electrospray(ESI) positive and negative ionization modes.

Method Development and Optimization
The objective of LC-MS/MS in this study to develop a sensitive, selective and Accurate method for quantification of Impurity-A and Impurity-B in Azilsartan drug substance. Different acidic mobile phases such as formic acid, trifluoroacetic acid, diflouoroacetic acid mix with organic modifiers such as Acetonitrile and methanol isocratic mode elution have been tested. Different stationary phases like C18,C8 and Phenyl HPLC columns has been tested and found Inertsil ODS-3V(150x4.6mm),5µm has been(Make:GLSciences,Japan) separation of Impurity-A,Impurity-B and Azilsartan drug substance.Gaussian curve peak shapes observed in Ammonium formate mobile phase pH=3.00, pre-mix with Acetonitrile in the isocratic mode elution with flow rate 0.8mL/min.Finalised mobile phase conditions pre mix of 10Mm

Methodology
LC-MS grade of Ammonium formate and formicacid from sigma-Aldrich.LC-MS grade Acetonitrile from Fisher chemicals. Purified water collected from Mill-Q plus water purification system. The method development and method validation was performed in Water's Acquity UPLC H-Class connected to Xevo TQ MS/MS detector. The data were collected and processed using Mass lynx software.

Preparation of standard solution
Weighed about each 20mg of Impurity-A and Impurity-B and transferred in 100mLvolumetric flask and dissolved with diluents (Stock-1). Pipette out 1.0mL from Stock-1 into 100mLvolumetric flask and make up to the mark with diluents (Stock-2). Further transferred 1.0mL of stock-2 solution into 100mL volumetric flask and makeup to the mark with diluent (Standard solution).
Preparation of sample solution: Weighed about 50mg of Azilsartan drug substance sample transferred into 100mL volumetric flask, dissolved and make up to the mark with diluent.

LC-MS/MS Operating Conditions
Experimentation performed using Water's Acquity UPLC H-Class connected to Xevo-TQ MS/MS detector with ESI Source (Electron spray ionization). Inertsil ODS-3V (150x4.6mm),5µm column used to separate the Impurity-A, Impurity-B and Azilsartan. Chromatographic method developed using isocratic mode of elution with Mobile phase-A and Mobile phase-B(60:40)%v/v with a flow rate of 0.8mL/min and a runtime of 10minutes for standards solution and 25mins runtime for samples solution. Column oven temperature maintained at 30°C and auto sampler temperature maintained at 10°C with an with a injection volume of 5µL.
A triple quardrupole MS equipped with a positive electron spray ionization (ESI) source was used in the MRM mode. The equipment was set with a Capillary voltage3.2kV, Cone voltage20V, Source temperature 150°C, Desolvation temperature 600°C, Desolvation Gas flow 850L/hr.

Specificity
The specificity of the method was verified by injecting the individual impurity standards Impurity-A and Impurity-B each at about 37.5ppm level with respect to 0.5mg/mL analyte concentration, azilsartan drug substance at 0.5mg/mL, Spiked sample solution of Azilsartan drug substance containing Impurity-A and Impurity-B.

Sensitivity
The Limit of detection(LOD) and Limit of quantification (LOQ) was determined from Signal to noise ratio(S/N) method. Prepared and injected a series of diluted solutions from individual standard solutions. Based on S/N ratios of diluted solutions reported LOD and LOQ concentrations of Impurity-A and Impurity-B reported. Injected LOQ solution of Impurity-A and Impurity-B standards six replicates to conform the precision at LOQ.

Linearity
Linearity studies were performed for Impurity-A and Impurity-B at different concentrations from QL to 200% ( QL, 25, 50,100,150 and 200%)of the specification level with respect to analyte concentration. Plotted a linear graph by taking the MRM peak areas on Y-axis and corresponding concentration on X-axis. Reported the values of correlation co-efficient, slope, yintercept and residual sum of squares from linearity study.

Precision
Prepared the spiked sample solution in six times containing each Impurity-A and Impurity-B at specification level at each preparation and injected each once. Calculated the content of each Impurity-A and Impurity-B and reported % RSD for Impurity-A, Impurity-B content from six spiked sample preparations.

Accuracy
The accuracy of the test method was demonstrated by prepared the un spiked sample solutions and spiked sample solution with known concentration of Impurity-A, Impurity-B at LOQ level,50%,100% and 150% of the specification limit. Calculated the %recovery of Impurity-A and Impurity-B at each level.

Validation results of the method
The developed method for the quantification of trace level determination of Impurity-A and Impurity-B in Azilsartan drug substance was validated as per ICH guidelines. The method was evaluated for its specificity, LOD (limit of detection),LOQ(Limit of quantification),Linearity, Accuracy and Precision .

Specificity
From specificity results of Table:2, it was observed that Impurity-A, Impurity-B and Azilsartan peaks were well resolved from each other. No blank interference was not observed at retention times of impurity-A and Impurity-B. So the method was specific for quantification of Impurity-A and Impurity-B in Azilsartan drug substance.

Sensitivity
Limit of detection (LOD) concentration for Impurity-A observed as 1.4ppm(signal to noise ratio:3.2) and Impurity-B observed as 1.4ppm (signal to noise ratio:3.4). Limit of quantification(LOQ) concentration forImpurity-A observed as 4.7ppm (signal to noise ratio:10.4) and Impurity-B observed as 4.5ppm ((signal to noise ratio:10.2). %Relative standard deviation was observed from LOQ precision experiment for Impurity-A and Impurity-B was 2.9 and 4.7 respectively.From these results, current method was sensitive for quantification of Impurity-A and Impurity-B in Azilsartan drug substance.

Linearity
From Linearity study, it was found that analyte response directly proportional to the concentration of analyte, it clearly indicates that linear relationship between analyte response and analyte concentration. Correlation co-efficient for Impurity-A and Impurity-B observed as 1.0000 and 0.9997 respectively (refer table no-6&table no-7).Method was found linear from LOQ(4.7ppm) to 200%(78.4ppm) for Impurity-A and LOQ(4.5ppm) to 150%(56.8ppm) for Impurity-B.

Precision
Repeatability expresses the closeness of agreement between the series of measurments. From method precision study of 100% Spiked sample solution 6 preparations found that the %RSD(relative standard deviation) for Impurity-A and Impurity-B was 1.39 and 1.14 respectively(refer table no-8).LC-MS/MS MRM mode method was precise for trace level quantification of Impurity-A and Impurity-B in Azilsartan drug substance.

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.