With respect to serum 5HIAA analysis laboratory specifications, blood was collected in serum vacutainer tubes (BD Vacutainer; BD AB, Stockholm, Sweden) that were allowed to clot for at least 30 min, after which they were centrifuged for 7 min at 2000× g using a Hettich centrifuge (HETTICH Instruments LP, Beverly, MS, USA). Using a Tecan Evo 150 sampling robot (Tecan Group Ltd. Männedorf, Switzerland) 100 µL of serum supernatant was mixed with 400 µL methanol containing 100 ng/mL 5-HIAA-4,6,7-D3-3-acetic-D2 acid internal standard (Cerilliant, Round Rock, TX, USA) in a 96 well plate. The mixture underwent vortex mixing for 5 min. After 2 h of incubation at 8 °C the samples were centrifuged for 10 min at 4000× g. Samples were analyzed using a Waters Acquity UPLC system (Waters Chromatography Europe BV, Etten-Leur, The Netherlands) equipped with Waters HSS T3 column (1.8 µm, 2.1 × 100 mm) and a Waters Xevo TQ-S micro triple quadrupole MS-detector. Mobile phases A and B consisted of 0.1 % formic acid in water and 0.3 % formic acid in methanol, respectively. Quantification was performed after internal standard normalization against a standard curve of 5-Hydroxyindole-3-acetic acid (Sigma-Aldric, Saint Louis, MO, USA) at four levels (three plus blank). The method was linear at a range of 5–2000 nmol/L, with an extended linearity up to 10000 nmol/L. The mass spectrometric detection was performed using multiple reaction monitoring (MRM) with an electrospray ionization (ESI) source in positive mode. Results were calculated using the Waters MassLynx software. During method validation, a coefficient of variation (CV) for the total precision was measured at 9.0% at 45 nmol/L and 3.3% at 130 nmol/L.

Accordingly, with regards to urinary 5HIAA analysis, after 24-h urine collection, samples were acidified using 6 mol/L HCl and frozen at −80 Celsius Degrees. Before analysis, samples, controls and calibrators were thawed and centrifuged at 1500× g for 5 min using a Hettich Rotanta centrifuge (HETTICH Instruments LP, Beverly, Massachusetts, USA). Subsequently, internal standard, 5-hydroxy-2-indolecarboxylic acid (Sigma-Aldric, Saint Louis, MO, USA) was added to each sample. Samples were analyzed using a Waters HPLC Alliance 2695 (Waters Chromatography Europe BV, Etten-Leur, The Netherlands) with electrochemical detection. Results were calculated against a one-point standard curve with a 5-hydroxyindoleacetic acid calibrator from Bio-Rad (Bio-Rad, Hercules, CA, USA) using the Waters Empower software. The method was linear between 2–600 µmol/L. Method precision was measured as a coefficient of variation (CV) of 8.0% at 15 µmol/L and 6.0% at 130 µmol/L.

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