Simultaneous quantification of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

Abstract

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the simultaneous quantitative determination of dextromethorphan (DM) and its metabolites dextrorphan (DX), 3-methoxymorphinan (3MM) and 3-hydroxymorphinan (3HM), in human lithium heparinized plasma. The extraction involved a simple liquid–liquid extraction with 1 ml n-butylchloride from 200 μl aliquots of plasma, after the addition of 20 μl 4% (v/v) ammonium hydroxide and 100 μl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC ® BEH C 18 1.7 μm 2.1 mm × 100 mm column eluted at a flow-rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7 min, with elution times of 1.3 min for DX and 3HM, 2.8 min for 3MM and 2.9 min for DM. The multiple reaction monitoring transitions were set at 272 > 215 ( m/ z), at 258 > 133 ( m/ z), at 258 > 213 ( m/ z) and at 244 > 157 ( m/ z) for DM, DX, 3MM and 3HM, respectively. The calibration curves were linear ( r 2 ≥ 0.995) over the range of 0.500–100 nM with the lower limit of quantitation validated at 0.500 nM for all compounds, which is equivalent to 136, 129, 129 and 122 pg/ml for DM, DX, 3MM and 3HM, respectively. Extraction recoveries were constant, but ranged from 39% for DM to 83% for DX. The within-run and between-run precisions were within 11.6%, while the accuracy ranged from 92.7 to 110.6%. The applicability of the bioanalytical method was demonstrated and is currently implemented in a clinical trial to study DM as probe-drug for individualized tamoxifen treatment in breast cancer patients.