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Quantitative Method For The Determination Of Phenyl Hydrazine, A Potential Genotoxic Impurity Of Ondansetron Hydrochloride Using Tandem Mass Spectrometry

To estimate the level of Phenylhydrazine, a potential genotoxic impurity in Ondansetron Hydrochloride API, a new simple, sensitive and accurate method was developed using Liquid Chromatographic Mass Spectrometry (LC-MS/MS). The chromatographic separation was achieved on Inert Sustain Swift C18, 5μ (150 x 4.6) mm column with gradient programme and elution was monitored by mass spectrometer in Multiple Reaction Monitoring mode using electrospray ionization. The LOD and LOQ values found to be 5 ppm and 15 ppm for the impurity with respect to the test concentration 2 mg/ml. The method was linear (r>0.99), precise (RSD<2%), accurate and well within acceptable ICH limits.


INTRODUCTION
Synthesis of drug substances involves many reactive chemicals, solvents and reagents. Impurities are generated as by-products during the synthesis or can be formed due to subsequent degradation of the drug substance or drug products. For majority of these impurities, guidance for qualification and their control is provided in ICH Q3A and ICH Q3B [1][2] . There are certain impurities that are reactive to DNA and even when present at low levels have the ability to modify the DNA and as a consequence can cause cancer. These are called as potential genotoxic impurities (PGIs). ICH M7 guideline 3 published by International Conference on Harmonization highlights the requirement for assessment and control of PGTIs. The allowable levels of PGI's are determined by a staged toxicological threshold of concern (TTC) based on both the dose and duration of the intended clinical study. This allowable amount can be in low ppm range, which is much lower than the permissible levels of non-PGI impurities controlled under ICH Q3A guideline. This TTC value was estimated to be 1.5 μg/person/day. For example a drug dosed at 1g/day the genotoxic impurity level would be 1.5 ppm. [4][5][6][7][8][9][10][11][12][13][14][15] .
Phenyl hydrazine ( Figure 2) is used during the synthesis of one of the intermediates of Ondansetron Hydrochloride drug substance. Hydrazine is a known genotoxic impurity and there have been many reported methods used to detect and quantify hydrazine using different analytical techniques [18][19][20] . Thus Phenyl hydrazine has been considered as a potential genotoxic impurities as per structural alert and must be controlled in Ondansetron Hydrochloride drug substance at low ppm level. The maximum daily dose of Ondansetron HCl is 24 mg and the TTC value for Ondansetron is 62.5 ppm (1.5/24*1000) justified.
Literature survey revealed that currently there is no method for the low level quantification of this potential genotoxic impurity Phenylhydrazine in Ondansetron Hydrochloride. The proposed method is a direct, sensitive and robust which involves MRM mode with electrospray ionization to achieve very low detection and quantification of potential genotoxic impurity Phenylhydrazine in Ondansetron Hydrochloride. Centre, and Hyderabad, India.

Instrumentation:
The LC-MS/MS method development and validation was done using Waters Xevo TQ-S micro system including Acquity UPLC H-Class system connected to Mass detector equipped with electrospray ionization in positive mode.

LC-MS/MS chromatographic conditions:
The analysis was carried out using Inert Sustain Swift C18, 5μ (150

Limit of Detection (LOD) and Limit of Quantification (LOQ):
The LOD and LOQ were calculated with signal to noise ratios of 3:1 & 10:1 respectively and by injecting a dilute solution having known concentrations of Phenylhydrazine and established the minimum level at which the Phenylhydrazine can be reliably detected. The LOD was 5 ppm and LOQ was 15 ppm obtained for the impurity.

System precision and system suitability:
The precision and system suitability was performed by injecting six replicates of the working standard solution of 62.5 ppm Phenylhydrazine with respect to the test sample concentration. The %RSD for the peak areas obtained was calculated. The data presented in Table 1 establishes system precision. Reference solution chromatogram for Phenylhydrazine is represented in Figure   3.   Tables 2 and Table 3. The mean responses recorded for Phenylhydrazine were plotted against concentration. The correlation coefficient of linear regression was found to be greater than 0.99, indicating good linearity. Corresponding linearity graph is shown in Figure 6a and overlaid chromatogram is represented in the Figure 6b.    Table 4. A representative spiked chromatogram is shown in Figure 7.