Determination of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmospheric pressure chemical ionization mass spectrometry

A selective assay of morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), morphine, codeine, codeine-6-glucuronide (C6G) and 6-monoacetylmorphine (6-MAM) based on liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) is described. The drugs were extracted from serum, autopsy blood, urine, cerebrospinal fluid or vitreous humor using C18 solid-phase extraction cartridges and subjected to LC-APCI-MS analysis. The separation was performed on an ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (5:95), using a flow-rate gradient from 0.6 to 1.1 ml/min (total analysis time was 17 min). The quantitative analysis was done using deuterated analogues of each compound. Selected-ion monitoring detection was applied: m/z 286 (for morphine, M3G-aglycone and M6G-aglycone), 289 (for morphine-d3, M3G-d3-aglycone and M6G-d3-aglycone), 300 (for codeine and C6G-aglycone), 303 (for C6G-d3-aglycone), 306 (for codeine-d6), 328 (for 6-MAM), 334 (for 6-MAM-d6), 462 (for M3G and M6G), 465 (for M3G-d3 and M6G-d3), 476 (for C6G) and 479 (for C6G-d3). The limits of quantitation were: 1 microg/l for morphine, 2 microg/l for 6-MAM, 5 microg/l for M3G, M6G and codeine and 200 microg/I for C6G. The recovery ranged from 85 to 98% for each analyte. The method appeared very selective and may be used for the routine determination of opiates in body fluids of heroin abusers and patients treated with opiates.