Metabolite and matrix interference in phenytoin immunoassays

Abstract

The major phenytoin metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin glucuronide (HPPG), was primarily responsible for the positive bias noted when uremic specimens were assayed with the Abbott TDx Free Phenytoin fluorescence polarization immunoassay. The amount of bias depended on both HPPG and phenytoin concentration, increasing with increases in either concentration. The new Abbott TDx II assays for phenytoin and free phenytoin exhibited no significant cross-reactivity with HPPG and no bias in clinical specimens from uremic patients. Both assays correlated well with Emit-based assays (r >0.98), had CVs of <3.5%, and had minimum detection limits of <0.1 mg/L. Calibration curves were stable for at least 6 weeks. All of the TDx assays cross-reacted with another metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), but expected HPPH concentrations are too low to cause a clinically significant bias. The Emit-based phenytoin assay exhibited a significant matrix effect when calibrators were prepared in defibrinated plasma processed to resemble serum.

Phenytoin immunoassay measurements in serum samples from patients with renal insufficiency: comparison with high‐performance liquid chromatography

“…In accordance with the findings of Rainey et al (13), our study suggests that both FPIA and EMIT provide accurate results for total serum phenytoin in specimens from patients with renal insufficiency. However, since Relationship of the relative differences among FPIA, EMIT, and HPLC phenytoin results with the GFR for serum samples with phenytoin levels lower () and higher (J) than 10 mg/mL.…”

“…Soldin and coworkers (17) suggested that HPPH presented a 19% cross-reactivity in the FPIA, which was the more affected of the most widely used immunoassays, and no doubt accounted for a significant HPPH interference in some phenytoin immunoassays (19). However, other authors found a lower HPPH cross-reactivity (5-15%) for FPIA (13)(14)(15)(16). In some studies the EMIT showed an even lower cross-reactivity with HPPH (12,13,17), and did not exhibit any significant interference in specimens from renally impaired patients (12,13).…”

“…The FPIA and EMIT phenytoin assays do not present a significant cross-reactivity with HPPG (13,18), although this metabolite appears to produce a slight negative interference in the EMIT (13). The phenytoin-N-glucuronide shows a significant cross-reactivity with FPIA and other phenytoin immunoassays (15); however, this is a minor phenytoin metabolite.…”

“…The dotted lines correspond to the limits of the acceptance criterion for accuracy. HPPH and HPPG are not strongly protein bound, and the ratios of the free metabolites to the free phenytoin concentrations will be increased in relation to the ratios of total concentrations (13,17), the possible interference of HPPH in free phenytoin measurements using FPIA would be more relevant.…”

“…High-performance liquid chromatography (HPLC) methods have the advantage of separating the parent drug from its metabolite HPPH. The cross-reactivity of different phenytoin immunoassays with the metabolites HPPH and HPPG has been explored in different studies, but conflicting results have been obtained regarding its clinical significance (9)(10)(11)(12)(13)(14)(15)(16)(17). Recent results obtained in a College of American Pathologists TDM Survey suggesting a significant HPPH interference in several phenytoin immunoassays, especially in the Abbott fluorescence polarization immunoassay (FPIA) (17), have reopened the debate regarding the possible interference of phenytoin metabolites in the phenytoin immunoassays in conditions of renal insufficiency (17)(18)(19).…”