Reversed-phase high-performance liquid chromatography of polar lipids: II. Procedure for a phospholipid derivative of methotrexate

Phospholipid derivatives of methotrexate (MTX) having enhanced membrane penetration (DP-71 being the most important) are potential prodrugs for treatment of autoimmune and inflammatory diseases as well as diseases involving abnormal cell proliferation. The previously published reversed-phase HPLC methods for similar compounds, phospholipid derivatives of valproic acid and non-steroidal anti-inflammatory drugs (NSAIDs), could not be used for MTX derivatives due to highly basic character of the MTX core molecule. The new HPLC procedure using gradient elution was developed as a compromise between the pharmacopoeial method for MTX and the previous “generic” procedure for phospholipid derivatives of NSAIDs. The newly developed method is sensitive, selective, reproducible, and stability indicating. Identification of major related compounds was carried out. The bioanalytical applications of this method, as well as of the derived isocratic procedure, are discussed and illustrated by examples of pharmacokinetic studies.