High-performance liquid chromatographic assay for acetaminophen and phenacetin in the presence of their metabolites in biological fluids

Abstract

We propose a method in which tracer amounts of a radiolabeled compound are used as the internal standard for the same unlabeled compound in high-performance liquid chromatography. The approach is valuable when a response from the internal standard becomes undesirable due to the presence of interference by the metabolites. We tested our approach with phenacetin and its metabolites, acetaminophen, 2-hydroxyphenacetin, N-hydroxyphenacetin, phenetidine, acetaminophen sulfate conjugate and acetaminophen glucuronide conjugate in biological fluids with the use of [14C]phenacetin and [3H]acetaminophen as the internal standards, and were able to quantitate both phenacetin and acetaminophen simultaneously. We also tested the alternative approach in which the unlabeled drug was used as internal standard for tracer amounts of the same radiolabeled compound, with phenacetin and acetaminophen as the internal standards for tracer amounts of [14C]phenacetin and [3H]acetaminophen. Again, we were able to quantitate the two tracer radiolabeled compounds simultaneously.