Individual solutions (50-[micro]L aliquots) of phenytoin, HPPH, HPPH-glucuronide, phenytoin-N-glucuronide, and the purified fosphenytoin metabolite were incubated with 200 [micro]L of deuterium oxide for 1 h at ambient temperature.
As expected, phenytoin and HPPH did not interact strongly with the column.
Assuming that the metabolite possessed the phenytoin or HPPH core as part of its overall structure, we performed reversed-phase chromatography with a shallow acetonitrile gradient, monitoring m/z 251 and 268 in the negative-ion mode.
The data for HPPH cross-reactivity in the two TDx assays also agree with those of Rainey et al.
In summary, the ACS:180 Phenytoin assay does not cross-react with HPPH or oxaprozin.
Cross-reactivity of oxaprozin and HPPH in TDx Phenytoin, TDx Phenytoin-II, and ACS:180 Phenytoin assays in serum pools with and without phenytoin.