Identification and Characterization of Unknown Impurity of Benzocaine in Lozenges

A workflow was developed to identify and characterize the trace level of pharmaceutical unknown impurity of Benzocaine in lozenges observed at 0.58 Relative retention Time (RRT) above the threshold limit (0.2 % w/w) in 18 Month stability samples at 30oC/65 % Relative humidity. Identification of unknown impurity mass number were carried out with MS-Agilent technologies-6130 Quadrupole LC/MS interfaced with an MM-ES+APCI ion source operating in both the positive and negative ion mode to identify the mass of unknown impurity. The mass spectrum of the unknown in positive ionization mode peak at m/z 328.2. Atomic mass unit which corresponds to the exact molar mass of known impurity ethyl 4-[[3,4,5-Trihydroxy-6-(Hydroxymethyl)Tetrahydropyran-2-yl]Amino]Benzoate or BenzocaineN-Beta-D-glucose.LC-MS showed molecular mass of two compounds are same, and are eluting at different RRT in HPLC. Enrichment of unknown impurity was done under thermal stressed conditions 60 °C for 24 hour and got 26.28 % of unknown impurity; Isolation was done by preparative HPLC and characterized by LC-MS, 1H-NMR, 13C-NMR, NOE in-depth analysis of the full characterization of an unidentified impurity in a drug product to confirm unknown impurity at 0.58 RRT as benzocaine N-alpha-D-glucose.