Development of a sensitive LC-MS/MS method for determination of N-nitrosopiperazine in levocetirizine

Abstract

Levocetirizine is one of the widely used antihistamines and has the potential to form N-nitrosopiperazine (NPZ) during drug synthesis, manufacturing, or storage. NPZ classified as a nitrosamine is a genotoxic impurity with carcinogenic properties. Controlling the presence of NPZ in the active pharmaceutical ingredient (API) and drug products is crucial with levels ideally maintained below 80 ppm. Herein, we developed a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique to analyze NPZ levels in levocetirizine API and various formulations. Chromatographic separation was carried out using an F5 column with mobile phases consisting of 2 mM ammonium formate in water and acetonitrile, employing gradient elution mode at a flow rate of 0.2 mL min⁻¹. The column oven temperature was set at 30 °C, and the injection volume was 2 μL. NPZ quantification was achieved using positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The developed method underwent rigorous validation according to regulatory guidelines. The limit of quantification (LOQ) was established at 1 ng mL⁻¹ within the range of 1–50 ng mL⁻¹, covering 10–500% of the specified NPZ limit in drugs. The effectiveness of the method was shown by utilizing it to analyze the NPZ impurity in both levocetirizine API and various drug products, including tablets, capsules, chewables, and syrups. The proposed method and the resulting data would be valuable for determining potentially present impurities in drug substances or products for quality assessment.