Development and validation of LC–QTOF–MS/MS method for the identification and determination of low levels of a genotoxic impurity, 4,6‐dichloro‐5‐nitro‐2‐(propylthio)pyrimidine in ticagrelor API

Abstract

The foremost objective of the present study was to develop and validate a new LC-QTOF-MS/MS method for the identification and quantitative determination of 4,6-dichloro-5-nitro-2-(propylthio)pyrimidine (DPP) genotoxic impurity through the derivatization process in ticagrelor active pharmaceutical ingredient (API). Owing to the low response of DPP at the specification level, DPP was converted to 4,6-dibenzylamino-5-nitro-2-(propylthio)pyrimidine (DPP derivative) by addition of benzyl amine, then analyzed using mass spectrometry with a time-of-flight analyzer, and good separation was accomplished under the experimental conditions described.The effective separation of DPP derivative was achieved using an Acquity UPLC BEH C 18 reverse-phase column (100  4.6 mm  1.7 μm) with an isocratic program with mobile phase A as 0.1% formic acid in milli Q water and mobile phase B as acetonitrile in the ratio of 20:80 v/v. The flow rate was maintained as 0.4 ml/min, the injection volume was 2 μl, the autosampler temperature was 5 C, the column oven temperature was ambient and the run time was 6.0 min. The diluent used was 0.2% benzyl amine in water and acetonitrile in the ratio of 30:70 v/v. The retention time of the DPP derivative was 2.87 min. The limit of detection and limit of quantification were 0.03 and 0.08 ppm, respectively. The DPP derivative was linear from 1.68 to 12.78 ppm with R 2 of 0.9958. Thus, the developed method is valid for the identification and quantitative determination of DPP derivative in ticagrelor API. K E Y W O R D S 4,6-dibenzylamino-5-nitro-2-(propylthio)pyrimidine (DPP derivative), genotoxic impurity, LC-ESI-QTOF-MS/MS, ticagrelor, time-of-flight analyzer 1 | INTRODUCTION Ticagrelor is an antiplatelet agent used to thin the blood in patients with acute coronary syndromes (Danielak et al., 2018). It inhibits platelet aggregation in coronary blood vessels and reduces cardiovascular death and heart attack by preventing the formation of new blood clots (

What is the role of current mass spectrometry in pharmaceutical analysis?

“…The false positive results were almost three times above the actual NDMA amount. A similar strategy was demonstrated when determining 4,6-dichloro-5nitro-2-(propylthio)pyrimidine in ticagrelor API using an LC-Q-TOF instrument (Moorthy et al, 2022). To solve low analyte response at the specification level, the authors used a pre-column derivatization reaction with benzylamine.…”

Identification, characterisation and in silico ADMET prediction of ozenoxacin and its degradation products using high‐performance liquid chromatography‐photodiode array and liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectrometry

“…Precision refers to the degree of closeness between data points obtained from multiple measurements conducted under identical analytical conditions. It is considered at three levels: repeatability, intermediate precision and reproducibility 28 …”

A novel liquid chromatography with quadrupole time‐of‐flight‐tandem mass spectroscopy method for ultra‐trace level identification and quantification of the genotoxic impurity 2,6‐diamino‐5‐nitropyrimidin‐4(3H)‐one in valganciclovir hydrochloride

“…The regulatory agencies US Food and Drug Administration, European Medicines Agency and International Council for Harmonization [ICHM7 (R1)] have joined forces and proposed the use of a threshold of toxicological concern (TTC) (μg/day) and maximum daily dose (g/day), where the TTC is 1.5 μg/day and the maximum daily dose is 1.8 g/day. This threshold is considered an acceptable risk for potentially genotoxic impurities in most drugs (Lee et al, 2010; Manchuri et al, 2022; Moorthy et al, 2022; Robinson, 2010; Shaik et al, 2023). The ppm limit of DMNP in drug substances derived from TTC is calculated using the following equation based on the expected daily dosage: …”