Analytes in plasma were quantified using liquid chromatography/mass spectrometry (LC/MS) following derivatization with benzoyl chloride (BZC)70 (link). 20 µL of blood was diluted with 60 µL acetonitrile:water (80:20), vortexed, and allowed to sit on ice for 10 min. The solution was then spun at 3.5 g for 5 min to pellet proteins. 5 µL of supernatant was then mixed with 10 µL each of 500 mM NaCO3 (aq) and 2% BZC in acetonitrile in an LC/MS vial. After 2 min, the reaction was stopped by the addition of 10 µL internal standard solution.
LC was performed on a 2.1 × 100 mm, 1.6 µm particle CORTECS Phenyl column (Waters Corporation, Milford, MA, USA) using a Waters Acquity UPLC. Mobile phase A was 0.1% aqueous formic acid and mobile phase B was acetonitrile with 0.1% formic acid. MS analysis was performed using a Waters Xevo TQ-XS triple quadrupole tandem mass spectrometer. The source temperature was 150 °C, and the desolvation temperature was 400 °C. The LC gradient and MS settings for 5-HT and 5-HIAA are shown in Supplemental TablesS1 and S2 .
LC was performed on a 2.1 × 100 mm, 1.6 µm particle CORTECS Phenyl column (Waters Corporation, Milford, MA, USA) using a Waters Acquity UPLC. Mobile phase A was 0.1% aqueous formic acid and mobile phase B was acetonitrile with 0.1% formic acid. MS analysis was performed using a Waters Xevo TQ-XS triple quadrupole tandem mass spectrometer. The source temperature was 150 °C, and the desolvation temperature was 400 °C. The LC gradient and MS settings for 5-HT and 5-HIAA are shown in Supplemental Tables
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