High-performance liquid chromatographic assay of methadone, phencyclidine, and metabolites by postcolumn ion-pair extraction and on-line fluorescent detection of the counterion with applications.

Methadone, phencyclidine, and their metabolites were extracted from plasma and separated on a high-performance liquid chromatographic (HPLC) column using the fluorescent 9,10-dimethoxyanthracene-2-sulfonic acid as a counterion. The chromatographed mobile phase was subsequently extracted on-line with chloroform. The separated organic phase, containing the fluorescent ion-pairs of the investigated amines, was analyzed in the flow cell of a fluorometer (excitation 380 nm, emission 445 nm). The phase separator volume was as small as possible to avoid dead volume. The method was also applied to the bioassay of cocaine with a sensitivity of 1-6 ng/ml of plasma. Application of these assays gave a red blood cell-plasma water partition coefficient for methadone of 3.39 +/- 0.26 (SD) in a concentration range up to 20 micrograms/ml, and demonstrated a time-dependent partition with a diffusion half-life of 1.44 min +/- 0.26 min (SD). The protein binding of methadone determined by ultracentrifugation was concentration dependent and varied between 75-62% at the highest concentration studied (9 micrograms/ml). The presence of the major metabolite did not have any influence on the protein binding. The results were confirmed by using the red blood cell-partitioning method to determine the protein binding.