A note on HPLC as a Means of Following anin vitro Synthesis of 7-Hydroxymethotrexate

Excessive build-up of MTX in the patient leads to a syndrome of folate stress which, with the serum MTX level as a pointer, may be remedied by folinic acid therapy. The major metabolite of MTX is 7-OH-MTX which is much less water-soluble and may cause renal damage due to crystallization in the renal tubules [1]. Monitoring MTX and 7-OH-MTX is thus clinically relevant. We have recently developed an assay for MTX and its metabolites in serum (unpublished work) and detected what we believe to be 7-OH-MTZ behind the MTX peak. This position was atypical for an RP system; 3 mg of 7-OH were available to us and confirmed that the k′ for 7-OH-MTX was greater than MTX, but was insufficient to pursue the question. Anin vitrosynthesis was therefore attempted, with homogenates (stored on ice till needed) of freshly killed rabbit liver prepared with an ATO-MIX homogenizer in 10 mM pH 7.6 Tris-HCl containing 0.25 M sucrose and 10 mM MgCl2: from an 18,000 rpm supernatant (15 min, 4°) 20 ml was taken for incubation with 2–5 mg MTX. For monitoring and final isolation, see Fig. 1 legend.