High-performance liquid chromatographic assay for morphine in small plasma samples: Application to pharmacokinetic studies in rats

In order to perform a reliable pharmacokinetic study of morphine during subchronic treatment in rats, an easy, rapid, sensitive and selective analytical method was proposed and validated. The analyte and internal standard (naloxone) were extracted from plasma samples (100 μL) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography (HPLC) along with electrochemical detection (ED). Standard calibration graphs were linear within a range of 3.5–1000 ng/mL (r = 0.999). The intra-day coefficients of variation (CV) were in the range of 5.8–8.5% at eight concentration levels (7–1000 ng/mL) and the inter-day coefficient of variation ranged from 7.4 to 8.8%. The intra-day assay accuracy was in the range of −5–10% and the inter-day assay accuracy ranged from 3.0 to 9.3% of relative error (RE). The limit of quantification was 3.5 ng/mL using a plasma sample of 100 μL (15.8% of CV and 12.8% of RE). Plasma samples were stable for 2 months at −20 °C. This method was found to be suitable for pharmacokinetic studies in rats, after subcutaneous administration of morphine (5.6 mg/kg per day) in subchronic treatment for 6 and 12 days.