High-performance liquid chromatography of nalbuphine, butorphanol and morphine in blood and brain microdialysate samples: application to pharmacokinetic/pharmacodynamic studies in rats.

A rapid and sensitive assay for quantification of nalbuphine, butorphanol and morphine in blood (50 microL) and brain microdialysate ( approximately 40 microL) samples was developed. Blood samples were extracted with ethyl acetate. Analysis was performed with high-performance liquid chromatography (HPLC) coupled to an electrochemical detector. The mobile phase was a mixture of 0.1 M sodium phosphate buffer, methanol and octane-sulfonic acid with ratio and pH depending on compound and matrix. The limits of quantification in blood samples were 25, 50 and 25 ng/mL for nalbuphine, butorphanol and morphine, respectively and 0.5 ng/mL for morphine in microdialysate samples. Based on sample volume, sensitivity and reproducibility, these assays are particularly suitable for pharmacokinetic/pharmacodynamic studies in rodents.