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|Peaks||tR (min)||Conc. (pg/mL)||Precursor Ion||Product Ion|
|Column||Raptor HILIC-Si (cat.# 9310A52)|
|Dimensions:||50 mm x 2.1 mm ID|
|Particle Size:||2.7 µm|
|Pore Size:||90 Å|
|Diluent:||Mobile phase A:mobile phase B (10:90)|
|Inj. Vol.:||10 µL|
|A:||Water, 100 mM ammonium formate, pH 3.0|
|Sample Preparation||Charcoal stripped human plasma was fortified with analytes at 50 pg/mL (0.25, 0.27, and 0.30 nmol/L for metanephrine, normetanephrine, and 3-methoxytyramine, respectively). Internal standard (IS) was prepared at 4 ng/mL for metanephrine-d3 and at 8 ng/mL for normetanephrine-d3 and 3-methoxytyramine-d4 in methanol. The plasma sample (200 μL) was mixed with 10 μL of IS solution and 600 μL of 50 mM ammonium acetate solution. The mixture was loaded in an EVOLUTE EXPRESS WCX 96-well plate (30 mg) and washed with 1 mL water and 1 mL methanol:acetonitrile (50:50). The elution was performed twice with 0.9 mL of 5% formic acid in methanol:acetonitrile (50:50) and evaporated to dryness at 55 °C under a gentle stream of nitrogen. Dried extract was reconstituted with 100 μL of diluent and injected (10 μL) for analysis.|