Content study of N-nitroso impurities in 5 types of β-blockers

Abstract

OBJECTIVE To determine the contents of N-nitroso impurities in raw materials/formulations of propranolol，metoprolol，atenolol，esmolol and bisoprolol，and clarify the attention threshold. METHODS Ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q/Orbitrap HRMS）was adopted. An ACE Excel 3 C18-AR column was used for the separation and a mixture of 0.2% formic acid solution with 0.01 mol/L ammonium acetate and methanol was employed as the mobile phase by gradient elution，at a flow rate of 0.60 mL/min. The column temperature was set at 40 ℃ ，and the sample size was 5 μL. The heated electrospray ionization source was employed in the positive full mass spectra-selected ion monitoring mode. The contents of N-nitroso impurities in raw materials/formulations of 15 batches of β-blockers from 10 manufacturers were determined by this method. Discovery Studio software was applied to predict the toxicity of the impurities and estimate the attention threshold. RESULTS Among 5 kinds of β -blockers，the linear ranges of N-nitroso propranolol，N-nitroso metoprolol，N-nitroso atenolol，N-nitroso esmolol and N-nitroso bisoprolol were 1.01-503.38，1.02-508.38，0.97-483.63，1.11-554.27 and 1.05-523.92 ng/mL，respectively（r＞0.999）. The limits of quantitation were 1.04，0.25，0.05，0.55 and 1.05 ng/mL，and the limits of detection were 0.52，0.08，0.02，0.17 and 0.52 ng/mL，respectively. RSDs of precision，reproducibility，recovery，stability and durability tests were all lower than 7.5% （n＝6 or n＝5）. Among the 15 batches of samples，except for 1 batch，N-nitroso propranolol（1.07-8.91 ng/mg），N-nitroso metoprolol（1.43-3.37 ng/mg），N-nitroso atenolol （1.33 ng/mg），N-nitroso esmolol（0.19 ng/mg）and N-nitroso bisoprolol（1.27 ng/mg）were detected in all other batches. According to predictions，the above 5 impurities had varying degrees of reproductive toxicity，mutagenicity and carcinogenicity，with attention thresholds of 1.0，0.4，4.3，0.2 and 46.7 ng/mg，respectively. CONCLUSIONS The established method is simple，rapid，sensitive and specific，the estimated attention thresholds are clear，which can be used for the control of N-nitroso impurities in various β-blockers.