UPLC Determination of Process Impurity Hydrazine in Neisseria meningitidis A/C/Y/ W-135-DT Conjugate Vaccine Formulated in Isotonic Aqueous 1x PBS

Purpose : To develop and validate a rapid and sensitive UPLC method as a limit test to determine traces of hydrazine process impurity in a Neisseria meningitidis A/C/Y/W-135-DT conjugate vaccine formulated in isotonic aqueous 1x PBS. Methods : Hydrazine was derivatized in a single step with salicylaldehyde at 60°C for 60 minutes and the adduct was resolved from the un-reacted salicylaldehyde on a Waters UPLC BEH C18, (2.1 mm ID x 50 mm length) column with mobile phase- water: acetonitrile (40:60 v/v) at a flow rate of 0.2 mL/ minute and detected by TUV detector at 209 nm. Sample matrix was prepared from final PBS formulated vaccine to study the % spike recovery. Results : UPLC C-18 column and optimized conditions achieved better sensitivity and resolution than previously reported HPLC method. Clear linearity (r 2 >0.99) was exhibited with hydrazine standard (in PBS) at concentrations ranging from 0.25 to 12.5 ppm. Spike recovery of the method was >99%. Detection limit (DL) and quantitation limit (QL) were 0.08 ppm 0.25 ppm respectively. Hydrazine impurity in the formulated single dose vialed final product is lower than detection limit by this method. Conclusions : Results indicated that this method is sensitive, rapid, reproducible, accurate and compatible with PBS formulation.