Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal...

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 microliters) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH2Cl2-isopropanol-NH4OH (79:20:1, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5-100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95 +/- 5.4% and 98 +/- 6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n = 8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n = 10), respectively. The detection limit for 500 microliters plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V. = 12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.