Enantiospecific analysis of ibuprofen by high performance liquid chromatography: Determination of free and total drug enantiomer concentrations in serum and urine

A reversed-phase high-performance liquid chromatographic (HPLC) assay, based on the indirect approach to enantiomeric analysis, for the determination of ibuprofen in human serum and urine has been developed. Following the addition of (R,S)-flurbiprofen, as internal standard, the enantiomers of ibuprofen were isolated from plasma and urine samples by liquid-liquid extraction at acidic pH. The enantiomers of flurbiprofen and ibuprofen were derivatized with (R)-1-(naphthen-1-yl)ethylamine, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole as coupling reagents, to yield the corresponding diastereoisomeric amides. Chromatographic resolution of the derivatives was achieved using a C-18 column (Waters Resolve C-18; 5 mu m, 150 x 3.9 mm) using a mobile phase of phosphate buffer (pH 3.5, 0.01 M): acetonitrile (50:50 v/v) at a flow rate of 1.5 mt min(-1) at ambient temperature. Quantification was carried out using a spectro-fluorometer with excitation and emission wavelengths of 290 and 330 nm respectively. The use of a semi-microbore column (150 x 2.1 mm) containing the same stationary phase facilitated the analysis of the free drug enantiomer concentrations following equilibrium dialysis. The derivatization procedure was carried out as described above but with a reduction in the quantities of the reagents used in order to reduce the background noise in the chromatographic analysis. The HPLC methodology for the determination of free drug enantiomer concentrations was validated against a previously reported method employing the radiolabelled drug.