DETERMINATION OF FEMTOMOLE MILLILITER CONCENTRATIONS OF ENPROSTIL ACID IN HUMAN PLASMA USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY LASER-INDUCED FLUORESCENCE DETECTION

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.