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 KH(2)PO(4) with 5 mM octane sulfonic acid: pH 3.7) and acetonitrile (77:33, v/v) through (flow rate=1.5 ml/min) a C(18) Symmetry column (150x4.6 I.D., 5 micrometer 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.
Aymard
G
G
Laboratoire de Pharmacocinétique, Service de Pharmacologie, Centre Hospitalier et Universitairé de la Pitié-Salpêtrière, Assistance-Publique Hôpitaux de Paris, 75634 Cedex 13, Paris, France. pharmaco.cinetique@psl.ap-hop-paris.fr
Warot
D
D
Demolis
P
P
Laville
I
I
Diquet
B
B
D000328:Adult; D019540:Area Under Curve; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018592:Cross-Over Studies; D004311:Double-Blind Method; D006801:Humans; D008297:Male; D009340:Nefopam / Q000097:blood* / Q000737:chemistry / Q000378:metabolism / Q000652:urine*
D009340:Nefopam