Quantification of Serotonin Concentration by LC-MS/MS

Serotonin hydrochloride, formic acid (FA), and heptafluorobutyric acid were obtained from Sigma Aldrich (St. Louis, MO). Serotonin-d4 hydrochloride was obtained from Santa Cruz Biotech (Dallas, TX). Optima LC/MS-grade water and acetonitrile (ACN) were obtained from Thermo-Fisher Scientific (Waltham, MA). Individual calibration standards were prepared at serotonin concentrations of 0.98, 3.9, 15.6, 62.5, 250, and 1000 ng/mL using an aqueous internal standard solution that contains 450 ng/mL of serotonin-d4 as the diluent.

To prepare samples for LC-MS analysis, frozen samples were thawed and immediately vortexed for 1 minute. The samples then were centrifuged at 10,000 × g for 5 minutes. Ninety microliters of an aqueous internal standard solution containing 500 ng/mL of serotonin-d4 was added to a 10-μL volume of each sample and vortex-mixed for 15 seconds; the internal standard (IS) concentration was consistent between calibration standards and samples. Samples then were loaded into 0.5-mL autosampler vials and submitted for LC-MS/MS analysis.

Chromatography was performed on a Shimadzu (Kyoto, Japan) Nexera-XR high-performance liquid chromatography system consisting of an SIL-20ACxr autosampler, a CTO-20AC column oven, and 2 LC-20ADxr binary pumps. Sample (5 μL) was loaded onto a Phenomenex Luna C18(2) (150 × 1 mm, 3 μm, 100 Å pore) (Torrance, CA) reverse-phase column equipped with a Phenomenex Security Guard C18 (4 × 2 mm) guard column. The aqueous mobile phase A (phase A) consisted of H₂O:ACN:FA:heptafluorobutyric acid (99.3:0.5:0.1:0.1 v/v/v/v) and the organic mobile phase B (phase B) consisted of methanol:FA (99.9:0.1 v/v). The mobile phase flow rate was 100 μL/min. The elution gradient was optimized as follows: started from 5% phase B and increased to 60% phase B over 9 minutes; decreased from 60% phase B back to 5% phase B over 0.1 minutes; and was maintained at 5% phase B for the duration of the 12-minute chromatographic run.

Selected reaction monitoring was performed on a Sciex (Framingham, MA) 6500 QTRAP MS system with a Turbo V source and TurboIonSpray probe installed. The MS was operated in the positive ion mode under the following conditions: curtain gas: 20 psi; collision gas: high; spray voltage: + 5 kV; ion source gas 1: 25 psi; ion source gas 2: 25 psi; interface heater temperature: 200°C; Q1 and Q3 resolution: unit/unit; dwell time: 220 ms; declustering potential (DP): +20 V; entrance potential (EP): +8 V; and collision exit potential (CXP): +10 V. The instrument was calibrated using Sciex PPG calibration standard and tuned to the manufacturer’s specifications. Selected-reaction monitoring (SRM) transitions monitored for serotonin were 177.1 → 160.1 (collision energy [CE]: +17 electron volts [eV]) and 177.1 → 115.1 (CE: +38 eV). For serotonin-d4, the SRM transitions 181.1 → 164.1 (CE: +17 eV) and 181.1 → 119.1 (CE: +38 eV) were monitored. Data were acquired with Analyst Software (SCIEX, Framingham, MA, version 1.6.2) and quantification was performed using Multiquant Software (SCIEX version 3.0.1).