Quantitative determination of phenobarbital and phenytoin by dry-phase apoenzyme reactivation immunoassay system (ARIS).

Abstract

We assessed the performance of the apoenzyme reactivation immunoassay system (ARIS) reagent strip tests for determination of phenobarbital (PB) and phenytoin (PHT) with the Seralyzer reflectance photometer. In the assay, the drug of the sample competes with a flavine adenine dinucleotide (FAD)-drug conjugate for binding to a specific antibody; the unbound conjugate then activates apoglucose oxidase to reconstitute glucose oxidase, whose activity is kinetically monitored by a coupled chromogenic reaction. Within-run coefficients of variation (CVs) were less than or equal to 5.0% for PB and less than or equal to 5.6% for PHT; between-run CVs were less than or equal to 6.1% for PB and less than or equal to 6.5% for PHT. Mean analytical recoveries were 100.3% for PB and 100.2% for PHT. Test results were not significantly affected by bilirubin (5 mg/dL), hemoglobin (25 mg/dL), triglycerides (500 mg/dL), uric acid (15 mg/dL), or elevated levels of other antiepileptic drugs. Reagent strip tests correlated very well with substrate-labeled fluorescent immunoassay (r = 0.9923 and 0.9944 for PB and PHT, respectively), enzyme multiplied immunoassay technique (r = 0.9941 and 0.9919), and gas-liquid chromatography (r = 0.9980 and 0.9960). These homogeneous competitive colorimetric immunoassays are particularly suitable for emergency use, for testing small batches of samples, wherever prompt results are needed.