Simultaneous and Trace Level Quantification of Five Potential Genotoxic Impurities in Ranolazine Active Pharmaceutical Ingredient Using LC-MS/MS

Highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of five potential genotoxic impurities in ranolazine active pharmaceutical ingredient. Chromatographic separation achieved using Poroshell C18 PFP 150 × 3.0 mm 2.7 μ column and 0.1% formic acid in water as Mobile phase A and 0.1% formic acid in methanol as mobile phase B using gradient elution and a flow rate of 0.4 ml/min with a run time of 18 minutes. Mass spectrometric conditions were optimized using electrospray ionization in positive mode. Method shows excellent linearity from 0.05 5.0 ppm of the ranolazine test concentration for all the five impurities. The correlation coefficient was observed greater than 0.99. Satisfactory recoveries were observed for all the five impurities within the range of 102.9% 112.3%. Method has been validated as per ICH recommended guidelines with a LOQ of 0.15 ppm achieved. The developed method was able to quantify all the five impurities at a concentration level of 1 ng/ml (0.5 ppm with respect to 2 mg/ml ranolazine).


Introduction
Pharmaceutical impurities are the unwanted chemicals that remain with active pharmaceutical ingredients which may arise during synthesis or may be derived from sources such as starting materials, intermediates, reagents, solvents, catalysts, reaction by-products and can also form due to degradation of API. Potential genotoxic impurities (PGI) are the impurities that have potential to interact with genetic material and can cause DNA alteration or damages. During toxicological assessment, these PGIs are proven to be genotoxic with established data for carcinogenicity. Several regulatory agencies like United States Food and Drug Administration (USFDA) [1] [2] and European Medicines Agency (EMEA) [3] and very recently International council of Harmonization of technical requirements for pharmaceuticals use (ICH) [4] have released guidelines regarding the regulatory issues related to the presence of genotoxic impurities in new drug substances and products. Threshold of toxicological concern refers to a threshold exposure level of 1.5 µg/day was also established. It is of utmost importance to quantify these genotoxic impurities at trace levels which would require highly sensitive analytical techniques like LC-MS/MS to establish the safety of the drug product for human use.
Ranolazine, a piperazine derivative sold under the trade name Ranexa, is a well-tolerated medication that selectively inhibits the late sodium current. Ranolazine is currently approved in the United States and Europe as a second-line agent in the management of chronic stable angina pectoris (CSAP) [5] One study also demonstrated that ranolazine was an effective anti-anginal in patients with stable coronary artery disease despite maximal doses of amlodipine [6]. In addition, ranolazine decreases angina episodes and increases exercise tolerance in individuals taking concomitant atenolol, amlodipine or diltiazem. Ranolazine indirectly prevents the calcium overload that causes cardiac ischemia [7].
The aim of the current research work is to quantify five potential genotoxic impurities observed in ranolazine drug substance at trace levels. During extensive literature survey for analytical techniques for the determination of impurities in ranolazine, there were several chromatographic methods reported which are stability indicating for ranolazine drug substance and formulation [8] [9] [10] [11]. Several methods were reported for determination of ranolazine in bulk and tablet dosage form [12] [13] and some LC-MS/MS methods reported for determination of ranolazine in biological matrices [14] [15] [16]. One LC-MS/MS method was reported for the determination of single genotoxic impurity 2-[(2methoxy phenoxy) methyl] oxirane content in ranolazine drug substance [17].
There were no data in literature available for LC-MS/MS method for simultaneous determination of five genotoxic impurities in ranolazine. However, based on the results obtained from computational structural analysis for mutagenicity alerts, the possible genotoxic impurities were procured. Considering the maximum allowable dosage, the GTI concentration must be controlled in ranolazine at concentration lower than 0.5 ppm. In this paper, we present the highly sensi-  Dichloro impurity (f).

Reagents and Chemicals
All the solvents and reagents used are of LC-MS grade with the highest purity of >99.8%. Water, Acetonitrile and Methanol were purchased from Honeywell (Charlotte, NC, USA). Formic acid was purchased from Fluka. Ranolazine and its five potential genotoxic impurities were procured from PS3 labs LLP, Hyderabad, India.

Mobile Phase Preparation
Mobile phase A was prepared by addition of 1 ml of formic acid in 1000 ml of water and mobile phase B was prepared by addition of 1 ml of formic acid in 1000 ml of Methanol. Both the mobile phases were degassed and stored at ambient temperature for further use. Fresh mobile phases prepared before each set of analysis.

Method Validation
We

Chromatographic Method Development
This study was conducted to develop highly sensitive and selective analytical method that could separate and quantify all the five potential genotoxic impurities in Ranolazine API.
To achieve good peak shapes and critical separation between ranolazine and impurities, several mobile phase pH and gradient conditions were evaluated but 0.1% formic acid provided the better peak shapes and sensitivities.

Method Validation
The optimized method was successfully validated as per ICH recommended guidelines and established all the critical parameters required to show the efficiency of the method.

Specificity
A single ranolazine with five impurity mix solution was prepared at specification level in the diluent. The spiked ranolazine solution was then subjected to LC-MS/MS analysis. The obtained results have shown that there is no interference of ranolazine API with all the five impurities Dimethyl aniline, Related compound A, ((2,6-Dimethyl)amino carbonyl methyl) chloride, Chlorohydrin impurity.
The chromatogram acquired was captured in Figure 2.

Linearity
The linearity of the method was established from 0.1 ng/ml to 10 ng/ml (0.05 -5 ppm) for all five genotoxic impurities. The slope, intercept and regression coefficient values were derived using least squares linear regression analysis of average peak areas versus concentration of impurities. Good correlation between peak areas and concentrations of impurities observed as can be seen in Table 2.

LOD and LOQ
The LOQ and LOQ values for all the five impurities were determined based on S/N ratios of 10.0 and 3.0 respectively, by injecting known standard concentrations and the results are captured in Table 2. Peak to peak algorithm used to derive the S/N ratio values for all the five impurities. Reproducibility and recovery were also evaluated at LOQ level using triplicate injections.

Accuracy and Recovery
Accuracy referred as deviation from linearity was evaluated by injecting impurity mixture from LOQ which is 30% of specification limit and on 10 times the specification limit. The acceptance criteria for accuracy is between 70% -130% for such a low concentration range. Accuracy values were observed at all levels for all the impurities within 10% which are well within the required acceptance criteria. The accuracy (as recovery) was further evaluated by standard addition method in triplicate at two concentrations at 0.15 ppm and 0.5 ppm levels in ranolazine API. The acceptance criteria for recovery is 80% -120%. The percentage recoveries for all the impurities are presented in Table 3.

Robustness
To evaluate the method robustness, different conditions of the method including the flow rate of the mobile phase and column oven temperatures were intentionally changed. The optimized flow rate of the mobile phase was 0.4 mL/min and the same was altered from 0.36 to 0.44 ml/min. The effect of column oven temperature on resolution was studied at 36˚C and 44˚C (altered by 4.0˚C). The results showed that there was no impact on chromatographic performance of all the impurities due to the mentioned changes proving the method robustness.

Repeatability and Solution Stability
The developed method was evaluated for repeatability by injecting six replicate injections at 1 ng/ml (0.5 ppm) mixture of five impurities and observed the %RSD after including one bracketing standard. The acceptance criteria for %RSD is less than 15%. The RSD values achieved for all the five impurities are less than 7% which are well within the acceptance criteria which are captured in Table 4. Repeatability chromatogram for all the five impurities captured in Figure 9. The solution stability study of ranolazine and five impurities was evaluated by placing spiked and unspiked sample solutions at 25˚C for 24 h and measured against freshly prepared standard solutions and there were no significant changes observed for any of the impurities. Therefore, we confirmed the stability of impurities in sample solution for at least 24 hours. Figure 9. Repeatability overlay of seven injections including bracketing standard for all the five potential genotoxic impurities in Ranolazine.

Conclusion
In summary, the work presented here is novel in terms of simultaneous determination of multiple genotoxic impurities in ranolazine active pharmaceutical ingredient by single method using LC-MS/MS and there is literature available only for single impurity determination currently. We could also establish all the critical parameters to prove the method performance and done method validation as per ICH recommendations. The LOD and LOQ values determined for all five impurities are very low showing the high sensitivity performance of the method. The method is fully validated and presents good reproducibility, linearity, recovery and robustness. The method presented here could be very useful for the determination of five impurities in ranolazine during routine manufacturing process which will increase the throughput and could help in establishing the safety of the active pharmaceutical ingredient.

Conflicts of Interest
To the best of our knowledge this is the first method published for simultaneous determination of five potential genotoxic impurities in Ranolazine and the authors hold no conflicts to declare.