3.2. Preparation of Calibrators, Quality Controls, and Patient Samples

Stock solutions (1 mg/mL) were prepared by dissolving HVA and VMA in 70% (v/v) methanol; these were frozen and stored at −80 °C until use. The stock solutions were diluted with blank urine (Mass Spect Gold Human Urine) to prepare the calibrators at six concentrations (0.5, 1.0, 5.0, 10, 50, and 100 mg/L HVA or VMA). Stock solutions of internal standards (IS) (2 mg/mL) were prepared by dissolving HVA-d₅ and VMA-d₃ in methanol. Quality control (QC) samples were prepared at two concentrations (1.7 mg/L and 15.7 mg/L for HVA; 2.7 mg/L and 15.0 mg/L for VMA) with 0.05% hydrochloric acid as per the manufacturer’s instructions.

For the LC–MS/MS assay, 50 μL of patient urine samples or blank/calibrators/controls and 100 μL of protein precipitation solution at −20 °C (50% acetonitrile in methanol) including IS (5 μg/mL) were added to Eppendorf tubes. The tubes were sealed and vortexed for 30 s and then centrifuged at 20,600× g for 10 min. A volume of 20 μL of the supernatant was transferred to the wells of a 2.0 mL polypropylene 96-deep-well plate. Next, 100 μL of distilled water was added to each well of the plate, mixed for 30 s, and then transferred into an autosampler at an injection volume of 10 μL.