LC-ESI-MS/MS method for the accurate quantification of eight nitrosamines in pharmaceutical products.

Abstract

In 2018, the Food and Drug Administration and the European Medicines Agency identified nitrosamine impurities in certain drugs, prompting detailed investigations by international regulatory authorities. According to ICH M7 (R1) guidelines, nitrosamines are classified as Class 1 substances, the most hazardous category, based on their carcinogenic and mutagenic properties. They are also recognized as probable human carcinogens by the International Agency for Research on Cancer. Since nitrosamine-induced DNA damage poses significant health risks, identifying potential nitrosamines in pharmaceutical products is crucial. In this study, a simple and efficient extraction method was designed to minimize matrix effects. These effects were evaluated using calibration curves prepared for each drug product in their respective matrices. The developed method was performed using an Agilent 1260 series HPLC system and an Agilent 6460 triple quadrupole tandem mass spectrometer. An Inertsil ODS-3 C18 (5 µm, 4.6 × 150 mm) column was employed for chromatographic separation. A triple quadrupole mass detector with electrospray ionization was used for detection, and multiple reaction monitoring was employed for quantification. The correlation coefficients (r²) were at least 0.999 for all eight nitrosamines. Limit of detection and limit of quantification values were determined as 0.05-0.8 ng/mL and 0.1-2.0 ng/mL, respectively. Validation results demonstrated satisfactory selectivity, accuracy, precision, and stability. The results demonstrated that the developed method is capable of reliably detecting potential nitrosamines present in pharmaceutical products at trace levels. This method contributes significantly to pharmaceutical safety and can serve as a valuable tool for future analyses.