Study On Impurity Profiling And Unknown Residue Solvent Of Folic Acid By Chromatography-mass Spectrometry

Impurity test is a propotion of drug safety tests.Physiological supply of folic acid prevents neural tube defects(NTDs).Folic acid is mostly used in pregnancy,so it is particularly critical to study its impurities.To improve the quality control,impurity profiling of folic acid drug substance and tablets were conducted,and unknown residual solvent of folic acid was also determined.At first,the methods for determination of the related substances of folic acid in the pharmacopoeias of various countries were compared,the formations of the six known impurities were analyzed.Then separation of known impurities in ChP 2015 and EP 8.0 were investigated.The chromatographic method of ChP 2015 was selected and validated.To provide a scientific basis for the quality control of folic acid,suggestions for improving the current ChP 2015 method were proposed: have control of the column temperature,determine amino-benzoyl glutamic acid as a special impurity and have control of the separation resolution between amino-benzoyl glutamic acid and pteridic acid,and improve the method of solution preparation of folic acid tablets.The optimized HPLC method was used for the qualitative and quantitative analysis of impurities of folic acid.To evaluate the quality from the results of related subsatances,comprehensive analysis was made to infer impurity profiling of folic acid in 7 batches of API from 2manufacturers and 10 batches of tablets from 4 manufacturers,and affect factors tests of folic acid and long-term tests of folic acid API and tablets were also conducted.The impurities of folic acid API are well controlled and the impurity profiling is stable.The impurity profiling of folic acid tablets is different and the impurities are more than APIs’.So the technology of pharmaceutics is closely correlated to their impurity profiling,which is also the key factor to have control of the impurities.The impurities were deduced: Impurity C、D、E、F、Ⅱ、Ⅳ are process impurities;Impurity Ⅲ is oxidative degradation impurity;Impurity A and Ⅰ are both process impurities and degradation impurity.An MS compatible HPLC method was optimized,and HPLC-Q TOF MS/MS was conducted to identify the impurities of folic acid.The possible structures of unknown impurities and its fragmentation rules were discussed.Impurity Ⅰ was deduced as pterin-6-carboxylicacid,impurity Ⅱ was L-Glutamic acid,N-[4-[[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-γ-glutamyl.In the fourth part,an HPLC method for determination of the optical isomer of folic acid was developed.Specificity,linearity,precision,limit of detection and quantitation,and stability of the solution were investigated,which provided the technical support for the quality control of folic acid.At last,a headspace gas chromatography method for determination of the residual solvents was developed.The unknown residual solvent that was not formed during the manufacturing process,was identified by GC-MS.The unknown peak in folic acid was determined as ethanol,which was used as equipment cleaning solvent.Equipment cleaning verification was recommended,ethanol can not be detected in folic acid substance.