MODIFIED METHOD OF NALBUPHINE DETERMINATION IN PLASMA - VALIDATION AND APPLICATION TO PHARMACOKINETICS OF THE RECTAL ROUTE

A solid-phase extraction (SPE) procedure was developed for the quantif ication of nalbuphine in a small volume (500 mu l) of human plasma wit h subsequent assay by high-performance liquid chromatography (HPLC) an d electrochemical detection using 6-monoacetylmorphine as internal sta ndard. Plasma was extracted using Bond flute certified extraction colu mns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tr is buffer (pH 8). Elution was performed with a CH2Cl2-isopropanol-NH4O H (79:20:1, v/v). The organic phase was evaporated to dryness and resu spended 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 plasm a samples resulted in recoveries of 95+/-5.4% and 98+/-6.2% for nalbup hine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variati on (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and th e inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The det ection limit for 500 mu l plasma sample was 0.02 ng/ml and the limit o f quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed meth od 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 p resented.