Impurity Profiling, Separation of Impurities, Characterisation by LCMS and 2DNMR Spectroscopy and Ultrafast Liquid Chromatography Method Development
Keywords:
Sulfametapyrazine (SMP), Low Pressure Gradient Mode, UFLC, Impurity Profiling, Electro Spray Ionisation (ESI).Abstract
Sulfametapyrazine is an opportunistic candidate for Impurity profiling. The
development of method and validation in presence of related substances by RP-UFLC and
separation of identified impurities by preparative HPLC in Sulfametapyrazine (SMP) is a
challenging candidate. The identification, separation and quantification was performed
using RP-UFLC, LCMS and 2DNMR spectroscopy. A new RP-UFLC and LCMS method was
developed for identification of impurity in SMP, using mobile phase composed of
ammonium formate buffer pH 4.0 (B) and acetonitrile (A) (40:60 v/v), with 1.0 ml/min
flow rate, Phenomenex Hyperclone BDS C18 employed as column. The eluent was
examined using PDA detector at 254 nm. The impurity and SMP was efficiently separated
with the retention time of 3.04 and 4.12 min respectively. The DSC analysis showed the
actual melting point of SMP between 174- 177 °C, SMP peak was seen at 175 °C and the
impurity peak at 158 °C. The LCMS studies were carried out by using ESI through positive
ion mode. The m+1 prominent peak appeared for SMP and impurity at 281 g/mol and 449
g/mol. The detected impurity was separated by using preparative HPLC. The impurity
structure was characterised by HNMR, 13CNMR, COSY, HSQC and Mass spectroscopy. The
method was validated following guidelines.