An automatic 96-well solid phase extraction and liquid chromatography-tandem mass spectrometry method for the analysis of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma

Abstract

A bioanalytical method using automated sample transferring, automated solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for morphine (MOR), and its metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma. Samples of 0.25 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated SPE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C(18) sorbent. The extract was injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 3.5 min per injection, with retention times of 1.5, 2.0 and 2.6 min for MOR, M6G, and M3G, respectively. The detection was by monitoring MOR at m/z 286-->152, M6G and M3G at m/z 462-->286. The deuterated internal standards were monitored at m/z 289-->152 for MOR-d(3), and m/z 465-->289 for M6G-d(3) and M3G-d(3). The standard curve range was 0.5-50 ng ml(-1) for MOR, 1.0-100 ng ml(-1) for M6G, and 10-1000 ng ml(-1) for M3G. The inter-day precision and accuracy of the quality control samples were <8% relative standard deviation (RSD) and <7% relative error (RE) for MOR, <5% RSD and <2% RE for M6G, and <2% RSD and <4% RE for M3G.