A Rapid and Sensitive LC-MS/MS Method for Quantification of NDSRI in Melatonin Drug Product via Controlled In Situ...

Area of Science:

• Pharmaceutical Analysis
• Analytical Chemistry
• Drug Safety

Background:

• Nitrosamine drug substance-related impurities (NDSRIs) present mutagenic and carcinogenic risks, necessitating stringent regulatory control (ICH M7).
• Reactive amines in active pharmaceutical ingredients can lead to in situ NDSRI formation during analysis, compromising data accuracy.
• Melatonin, with its reactive amine group, serves as a model to address these analytical challenges.

Purpose of the Study:

• To develop and validate a precise LC-MS/MS method for quantifying N-Nitroso melatonin.
• To mitigate in situ NDSRI formation during sample preparation using a scavenger.
• To ensure the reliability and regulatory compliance of analytical methods for NDSRI monitoring.

Main Methods:

• Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
• Incorporation of a scavenger during sample preparation to inhibit in situ nitrosamine formation.
• Validation of the method according to regulatory standards, including specificity, linearity, accuracy, precision, sensitivity, and robustness.

Main Results:

• The LC-MS/MS method demonstrated excellent linearity (R² = 0.9983) and sensitivity (LOD: 0.9 ng/mL, LOQ: 1.8 ng/mL).
• Accuracy (85.3%–95.9% recovery) and precision (%RSD < 2%) were within acceptable limits.
• The scavenger effectively prevented false positives by suppressing in situ nitrosamine formation, confirming method specificity and data integrity.

Conclusions:

• A validated LC-MS/MS method with a scavenger effectively quantifies N-Nitroso melatonin, preventing analytical artifacts.
• This approach ensures data integrity and supports regulatory compliance for NDSRI monitoring in pharmaceuticals.
• The validated methodology offers a transferable framework for analyzing other drug products containing reactive amines.