Simultaneous analysis of morphine-related compounds in plasma using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-mass spectrometry

A bioanalytical method using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the analysis of morphine, morphine-3 beta-glucuronide, morphine-6 beta-glucuronide, 6-acetylmorphine, morphine N-oxide, and 10-hydroxymorphine in porcine plasma. All six compounds, along with four deuterated internal standards, were simultaneously extracted using mixed-mode strong cation exchange SPE in a 96-well mu Elution plate format. Due to analyte instability, a neutralizing solvent was used during the elution step to minimize degradation of 6-acetylmorphine. Separation was subsequently performed in 8 minutes on a 2.1 x 100 mm, 1.8 mu m C(18) column designed for retention of extremely polar compounds using a formic acid and methanol gradient. Analytes were detected by positive electrospray ionization in multiple reaction monitoring mode using, a fast-scanning triple quadrupole mass spectrometer. Recovery was 73-123% depending on the analyte, and inter-day variability was less than 6%. Linearity was determined in porcine plasma by spiking the analytes prior to SPE. Correlation coefficients were >= 0.998, and% deviation from the actual concentrations was less than 15%. The lower limit of quantitation (LLOQ) for all compounds was between 0.1 and 0.25 ng/mL.