Measurement of serotonin in platelet depleted plasma by liquid chromatography tandem mass spectrometry

5-Hydroxytryptamine (5-HT) in human platelet depleted plasma (PDP) is a biomarker in functional gastrointestinal disorders (FGID), with levels reflecting acute changes in circulating 5-HT concentration. PDP 5-HT is currently measured by reversed phase high performance liquid chromatography (HPLC) fluorimetric detection. We have developed a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method that is two times more rapid than the current HPLC methodology. Our method employs a simple protein precipitation requiring no further downstream sample preparation. 10 μL of extract was injected directly onto a SecurityGuard SCX cation exchange column followed by isocratic elution onto an Onyx Monolithic C18 analytical column and methanolic gradient elution. Eluant was connected directly to a Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring transitions m/z 160 > 114.9 and m/z 164.1 > 118.9 for 5-HT and d₄-5-HT, respectively. 5-HT and d₄-5-HT co-eluted at 2.79 min and cycle time between injections was 6 min. Mean recovery was 98%, limit of detection 1.5 nmol/L, lower limit of quantification 5 nmol/L, linearity to 1000 nmol/L (r² = 0.999), imprecision <10% and bias <13.4%. 5-HT eluted with no ion suppression. No interference was found with l-tryptophan or 5-hydroxyindoleacetic acid (5-HIAA). This assay was compared to a previously published HPLC method. Passing-Bablok regression analysis showed LC-MS/MS = 0.91 (HPLC)-0.83, r² = 0.97, n = 80. Bland Altman analysis showed general agreement, with a mean bias of 3.3 nmol/L. We have developed a simple and robust assay for PDP 5-HT that will increase throughput for clinical trials.