Identification of methotrexate and folic acid analogs by mass spectrometry

Methotrexate and folic acid analogs are polar molecules and attempts to obtain electron impact and chemical ionization mass spectra of these compounds by several laboratories have failed. We have found that methylation of this important class of compounds with diazomethane produces derivatives which are sufficiently volatile to allow their mass spectra to be recorded. The mass spectra of the methyl derivatives of five compounds, 4‐amino‐4‐deoxy‐N10‐methylpteroylglutamic acid, 4‐amino‐4‐deoxypteroylglutamic acid, pteroylglutamic acid, 4‐amino‐4‐deoxypteroic acid and N10‐methylpteroylglutamic acid are presented, and the fragmentation pathways of these compounds discussed.