Routine determination of morphine, morphine 3-beta-D-glucuronide and morphine 6-beta-D-glucuronide in human serum by liquid chromatography coupled to electrospray mass spectrometry.

A robust liquid chromatographic mass spectrometric method capable of quantifying morphine, morphine 3-beta-D-glucuronide and morphine 6-beta-D-glucuronide down to 1.0 ng/ml, 5.0 ng/ml and 2.0 ng/ml respectively in human serum is presented. The method was validated over linear ranges of 1.0 to 20.0 ng/ml for morphine, 5.0 to 500.0 ng/ml for morphine 3-beta-D-glucuronide and 2.0 to 100.0 ng/ml for morphine 6-beta-D-glucuronide using deuterated morphine as internal standard. In tandem mass spectrometry conditions, the product ions of morphine-3-glucuronide and morphine-6-glucuronide were the ion m/z corresponding to the morphine moiety. By contrast morphine which presented numerous product ions after collision did not allowed a tandem methodology. Compounds were extracted on 100 mg C18 columns and analysed on the PE Sciex API 300 system equipped with a C18 column and electrospray ionisation interface. The interrun precision of quality controls (1.0, 2.0, 10.0, 20.0 ng/ml for morphine, 5.0, 10.0, 250.0, 500.0 ng/ml for morphine 3-beta-D-glucuronide and 2.0, 4.0, 50.0, 100.0 ng/ml for morphine 6-beta-D-glucuronide) was < or =9.3% and accuracy was between 97.9 and 109.8% for each analyte. Sample stabilities in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of morphine, morphine 3-beta-D-glucuronide and morphine 6-beta-D-glucuronide in human serum.