Determination of N-nitroso folic acid in folic acid and multivitamin supplements by LC-MS/MS

Analysis of N-nitroso folic acid, a nitrosamine impurity found in folic acid, is challenging due to the complex sample matrices. Many of such supplements contain not only a variety of vitamins, including vitamin A, Bs, C, D and E, but also other ingredients such as minerals, docosahexaenoic acid (DHA), glucose syrup, sugar, and herbs. On the other hand, the strength of folic acid is typically low, ranging from 50 µg to 5 mg per unit. In this study, a highly selective and sensitive LC-MS/MS method was developed to accurately quantify N-nitroso folic acid in supplements containing folic acid. The sample was extracted by 0.1% ammonia solution: MeOH (9:1, v/v) containing 5 ng/mL of N-nitroso folic acid-d₄ (Isotope internal standard). The quantification was performed by MRM in negative ionization mode. Mobile phases A and B were 0.1% formic acid in deionized water and methanol, respectively. The method was validated and found to have sufficient linearity (R² > 0.995), accuracy (recovery 83-110%), precision (RSD 3%) and low LOD, LOQ (4 and 10 µg/g respectively, with respect to folic acid). The method was applied to the determination of N-nitroso folic acid in 40 supplements containing folic acid with different strengths and formulation. The content of N-nitroso folic acid was found to be up to 898 ng/unit (1794 µg/g with respect to folic acid). It enabled regulatory actions, such as product recall, to safeguard public health from unsafe products.