Abstract
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‐3β‐glucuronide, morphine‐6β‐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 μElution 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 2.1×100 mm, 1.8 μm 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.