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

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 1ml n-butylchloride from 200μl aliquots of plasma, after the addition of 20μl 4% (v/v) ammonium hydroxide and 100μl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC®BEH C181.7μm 2.1mm×100mm column eluted at a flow-rate of 0.250ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7min, with elution times of 1.3min for DX and 3HM, 2.8min for 3MM and 2.9min 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 (r2≥0.995) over the range of 0.500-100nM with the lower limit of quantitation validated at 0.500nM for all compounds, which is equivalent to 136, 129, 129 and 122pg/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.

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