Analysis of morphine in serum by high performance liquid chromatography with amperometric detection

In this report we describe a rapid and sensitive micromethod using high performance liquid chromatography (HPLC) with electrochemical detection (ED) to measure morphine concentration in serum or plasma. The separation of morphine and the internal standard 5-hydroxyquinoline, from interfering compounds present in plasma was achieved by paired-ion reverse phase chromatography using a 70 mM phosphate buffer at pH 5.80. The flow rate was 1 ml/min. Oxidation of morphine and the internal standard was obtained at a potential of 0.60 V. Only 100 microliter of serum or plasma was required. Analytical recoveries for morphine and 5-hydroxyquinoline were determined as 78% and 63% respectively. The between-day precision of serum samples containing 250, 100, and 25 microgram/L of morphine (n = 20) was 6.5%, 5.2%, and 9.5% respectively. The detection limit was determined as 1 microgram/L at a sensitivity of 5 nA/V. In our preliminary studies, 3 children between the ages of 0 and 5 years received a bolus of morphine of 11 microgram/kg, followed by an infusion of 2 microgram/kg/min during surgery. The time-concentration curves demonstrate an initial rapid fall in morphine concentration with subsequent attainment of a steady state concentration of approximately 90 microgram/L after 1 h. This concentration would be expected to produce optimal analgesia in conscious patients.