Effective phospholipids removing microelution-solid phase extraction LC-MS/MS method for simultaneous plasma...

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 μl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well μElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-μm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five μl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18-110 ng/ml and 0.35-100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies.