Determination of naloxone in blood by high-performance liquid chromatography

A rapid, sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of naloxone in small blood samples of premature infants. Naloxone and the internal standard, naltrexone, are extracted from alkalinized blood into diethyl ether and subsequently back extracted into 0.05% (v/v) phosphoric acid before chromatographing on a reversed-phase system. The mobile phase comprises 85 parts of acetonitrile and 15 parts of 0.06% (v/v) triethylamine in an aqueous phosphoric acid solution at pH 5 and is pumped at 1.5 ml/min. The retention times of naloxone and naltrexone were observed to be 5.4 and 7.5 min respectively. Ultraviolet detection at 214 nm enabled a limit of detection of 1 ng to be achieved. The reproducibility of the method was good at both 100 ng (C.V. = 3.4%; n = 9) and 10 ng (C.V. = 5.1%; n = 6). The high sensitivity and speed with which this assay can be performed makes it especially useful for the estimation of naloxone in small volumes (0.3-0.6 ml) of blood. It is thus particularly valuable for the determination of naloxone blood concentration-time profiles in premature infants where the minimization of the volume of blood collected is of paramount importance.