Efficient and Simultaneous LC-MS/MS Detection of N-Nitroso and Di-Nitroso Leucovorin in Leucovorin Drug Products: Method Development and Validation.

RATIONALE: The occurrence of complex nitrosamines, namely, N-nitroso drug substance related impurities (NDRSI) in pharmaceuticals is of great concern due to their potential carcinogenic and mutagenic properties. This creates the need for more rigid and more specific methods of analysis to determine NDSRI levels in pharmaceutical products. The main analysis problem is that it is difficult to separate these impurities and the active pharmaceutical ingredient by chromatography, which is due to their structural similarity. This study describes how a new method of measuring two specific impurities in the Leucovorin drug product, N-nitroso leucovorin and di-nitroso leucovorin, was developed and validated.

METHODS

Quantification was done by selective reaction monitoring by a positive electrospray ionization source. The required separation was achieved with the use of a reverse-phase C-8 column and gradient-elution in the methanol and water solutions, both adjusted with 0.1% formic acid.

RESULTS

Linearity of the method was established in a range of 0.1 to 1.5 ng/mL, which is effectually 10% to 150% of its necessary regulatory limit. The procedure had a detection limit of 0.05 ng/mL and a quantitation limit of 0.1 ng/mL. Additionally, it also demonstrated excellent specificity, precision, linearity, accuracy, and robustness when dealing with the two impurities.

CONCLUSIONS

This methodology will therefore offer a crucial solution to drug manufacturers to track and control N-nitroso leucovorin and Di-nitroso leucovorin concentrations, which will have a positive impact on regulatory compliance and patient safety.