Development of an Analytical Method for Quantifying Nitrosamine Impurities in Metformin Using Liquid Chromatography-Mass Spectrometry

Nitrosamine-containing active pharmaceutical ingredients (APIs) have raised safety concerns due to their potential carcinogenic nature. Regulatory agencies such as the United States Food and Drug Administration (FDA) have established guidelines limiting nitrosamine impurities, including N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA), among others. To ensure compliance, this study presents an analytical method for detecting and quantifying nitrosamine impurities in metformin API, utilizing liquid chromatography coupled with mass spectrometry (LC-MS/MS).A Waters XSelect High Strength Silica (HSS) pentafluoro phenyl (PFP) column was employed along with a mobile phase gradient of 0.1% methanoic acid in water and methanol for impurity separation. The retention times for Metformin, NDMA, NDEA, NEIPA, NDIPA, NMPA, and NDBA were recorded at 8.47, 15.83, 19.35, 20.54, 21.74, 22.11, and 24.85 minutes, respectively. The detector exhibited linearity across concentrations ranging from 8.8 to 111.8 ng/mL. The correlation coefficients for NDEA, NEIPA, NDIPA, NMPA, and NDBA ranged between 0.9973 and 0.9954, with a linear response function established between 20% and 150%.The determined limits of detection (LOD) and quantification (LOQ) were as follows: NDMA at 50.9 ng/mL and 25.5 ng/mL, NDEA at 7.5 ng/mL and 1.9 ng/mL, NEIPA at 7.1 ng/mL and 1.8 ng/mL, NDIPA at 8.8 ng/mL and 4.4 ng/mL, NMPA at 7.0 ng/mL and 3.7 ng/mL, and NDBA at 7.4 ng/mL and 1.9 ng/mL. The method’s precision, expressed as relative standard deviation (%RSD), was determined to be 18.67 for NDMA, 9.81 for NDEA, 7.58 for NEIPA, 14.0 for NDIPA, 18.50 for NMPA, and 10.41 for NDBA. The developed method aligns with the FDA guidelines for LOD and LOQ, making it suitable for routine analysis of nitrosamine impurities in pharmaceutical products. Additionally, it optimizes solvent consumption while maintaining the necessary sensitivity and specificity for detecting nitrosamine impurities in metformin. As per the International Council for Harmonization (ICH) guidelines, the method is validated, demonstrating efficiency, sensitivity, and reliability.