Identification of metabolism pathways of anticancer drugs by high-pressure liquid chromatography in combination with field desorption mass spectrometry.

The separation and structure elucidation of the metabolites of many anticancer drugs by combined gas chromatography-mass spectrometry using conventional ionization techniques is not possible because of their high polarity and involatility. For the direct analysis of anticancer drug metabolites without their derivatization and extraction from biological fluids, high-pressure liquid chromatography (HPLC) in combination with field desorption (FD) mass spectrometry have developed as powerful methods. Specific techniques and preconditions for the HPLC separation of metabolites suitable for their subsequent analysis by FD mass spectrometry have been worked out. Most important for the isolation by reversed-phase HPLC is the selection of suitable volatile aqueous eluents and the purification of metabolites by removal of excess alkali metal ions from the biological sample. Essential advances for FD mass spectrometric analyses with high sensitivity are the development of adjustable insertion probes permitting optimal alignment of the field ion emitter, precise control of the emitter temperature and spectra accumulation techniques which provide enhanced reproducibility of FD spectra. In metabolism studies of the clinical anticancer drugs, methotrexate (MTX), cyclophosphamide (CP) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), the combined application of HPLC and FD mass spectrometry enabled the identification and quantitation of involatile and labile metabolites that were previously resistant to structural analysis. The toxicologically important metabolites of MTX, 7-hydroxy-MTX and MTX-oligoglutamyl conjugates were identified by their FD mass spectra in serum, urine and erythrocytes of patients receiving high-dose MTX. 4-Hydroxy-CP, the unstable key metabolite for the antitumor selectivity of CP, has been directly characterized by FD mass spectrometry, and its presence as a urinary product in man was first demonstrated by FD mass spectral analysis of its 4-benzylthio derivative. The particular advantage of the FD technique for the analysis of highly polar, polyfunctional metabolite conjugates was demonstrated by the structural identification of phosphoramide mustard-nucleoside adducts, and of several metabolites in the glutathione conjugation pathway of m-AMSA, from rat bile. In addition to HPLC, FD mass spectrometry has been successfully applied to the quantitation of MTX, CP and their metabolites in serum and urine and, because of its outstanding specificity, may prove useful in selected pharmacokinetic studies of anticancer drugs.