Isolation and identification of process impurities in trimethoprim drug substance by high-performance liquid chromatography, atmospheric pressure chemical ionization liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy

Twenty-two lots of recently synthesized trimethoprim drug substance, from f ive different manufacturers, in three different countries of origin, China, Israel and the United States, were investigated for the presence of impuri ties. A liquid chromatographic system, using gradient elution, and a mobile phase consisting of 0.25% TEA/0.1% formic acid (pH 5.8)-acetonitrile, was used to separate and detect two significant, recurring impurities in trimet hoprim drug substance. The two impurities were isolated by preparative liqu id chromatography and identified, using a combination of liquid chromatogra phy/mass spectroscopy and nuclear magnetic resonance, as 2,4-diamino-5-(4-e thoxy-3,5-dimethoxybenzyl) pyrimidine and 2,4-diamino-5-(3-bromo-4,5-dimeth oxybenzyl) pyrimidine. These impurities were not detected by the compendial method and were present at significant levels in 17 of the lots tested. To tal impurity concentrations were in the range of 0.1-2.1%. (C) 1999 Elsevie r Science B.V. All rights reserved.