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

Phillip J. Monaghan, Heather A. Brown, Lesley A. Houghton, Brian G. Keevil

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2163-2167
Number of pages5
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume877
Issue number22
DOIs
StatePublished - Jul 15 2009

Keywords

  • Functional gastrointestinal disorders
  • LC-MS/MS
  • Protein precipitation
  • Serotonin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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