Simple generic SPE assay for HPLC analysis of morphine and its glucuronides in serum samples

A very simple solid-phase extraction (SPE) generic assay has been developed for high performance liquid chromatography (HPLC)-DAD or HPLC-UV analysis of morphine and its two metabolites (morphine-3-glucuronide and morphine-6-glucuronide) in serum samples of cancer patients. Solid-phase extraction cartridges with OASIS polymeric hydrophilic-lipophilic sorbent have been applied for SPE off-line procedure. High extraction recoveries have been obtained for simultaneous isolation of all three analytes: 90% for morphine, and 96% and 99% for morphine-3-glucuronide M3G and morphine-6-glucuronide M6G, respectively. The main goal of this work was to optimize loading, washing, and elution steps of the SPE assay, especially pH of loaded sample, volumes of applied wash, and elution solvents with the aim to reduce time for sample preparation and to simplify the SPE procedure. Sorbents of a new generation has also been applied in the HPLC assay (Symmetry Shield RP-8) with mobile phases without any ion-pair agent, and without or with a very low content of organic modifier. The UV detection at 210 nm was used and the following detection limits were achieved: morphine 6.9 ng/mL, M3G 2.3 ng/mL, and M6G 20.0 ng/mL. The developed method has been recommended for routine monitoring of morphine and its glucuromides, in serum samples of cancer patients in clinical laboratories.