Sensitive determination of nefopam and its metabolite desmethyl-nefopam in human biological fluids by HPLC.

Nefopam (NEF) and desmethyl-nefopam (DMN) were assayed simultaneously in plasma, globule and urine samples using imipramine as internal standard. A liquid–liquid extraction procedure was coupled with a reverse phase high-performance liquid chromatography system. This system requires a mobile phase containing buffer (15 mM KH2PO4 with 5 mM octane sulfonic acid: pH 3.7) and acetonitrile (77:33, v/v) through (flow rate=1.5 ml/min) a C18 Symmetry column (150×4.6 I.D., 5 μm particle size: Waters) and a UV detector set at 210 nm. Internal standard was added to 1 ml of plasma or globule sample or 0.5 ml of urine sample, prior to the extraction under alkaline ambiance with n-hexane. The limits of quantification were 1 and 2 ng/ml for both molecules in plasma and globule, respectively; 5 and 10 ng/ml for NEF and DMN in urine, respectively. The method proved to be accurate and precise: the relative error at three concentrations ranged from −13.0 to +12.3% of the nominal concentration for all molecule and biological fluid; the within-day and between-day precision (relative standard deviation %) ranged from 1.0 to 10.1% for all the molecules and biological fluids. The method was linear between 1 and 60 ng/ml for both molecules in the plasma; 2 and 25 ng/ml for both molecules in the globule; 25 and 250 ng/ml for NEF and 50 and 500 ng/ml for DMN in the urine: correlation coefficients of calibration curves (determined by least-squares regression) of each molecule were higher than 0.992 whatever the biological fluid and during the pre-study and in-study validations. This method was successfully applied to a bio-availability study of NEF in healthy subjects.