Simultaneous determination of plasma epinephrine and norepinephrine using an integrated strategy of a fully automated protein precipitation technique, reductive ethylation labeling and UPLC–MS/MS

A novel, automated, simple, sensitive, specific, accurate, precise and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS)-based method has been developed for simultaneous determination of epinephrine (E) and norepinephrine (NE) in plasma by using the combination of a fully automated protein precipitation technique for plasma sample preparation, reductive ethylation labeling with UPLC–MS/MS. A simple protein precipitation procedure was used to clean up 50 μL calibration samples prepared in stripped human plasma and 50 μL quality control plasma samples containing 25 μL plasma and 25 μL stabilizing additives. The supernatants were subsequently dried down and then reconstituted with commercially available and cost-effective reductive ethylation labeling reagents, followed by UPLC–MS/MS analysis. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and quality control samples, shaking of 96-well plates, adding and transferring liquids. Processing time, which included the preparation of standards and quality control samples, protein precipitation and reductive ethylation labeling, is less than 2 h per 96-well plate. The chromatographic run time is 3.5 min per sample. The limits of detection of UPLC–MS/MS-based methods for E and NE, with/without reductive ethylation labeling, are 0.025/0.40 and 0.025/2.00 ng mL−1, respectively. Reductive ethylation labeling of amino groups of E and NE affords 16 and 80 times increased detection sensitivity of corresponding native counterparts during the UPLC–MS/MS analysis. The linearity of this method was established from 0.05 to 25 ng mL−1 for E and NE with accuracy and precision within 15% at all concentrations. The intra-run and inter-run assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QC samples prepared in commercially purchased plasma samples.