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 journalArticle

25 Citations (Scopus)

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 d 4-5-HT, respectively. 5-HT and d 4-5-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 (r 2 = 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, r 2 = 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
Externally publishedYes

Fingerprint

Liquid chromatography
Platelets
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
Serotonin
Blood Platelets
Plasmas
High performance liquid chromatography
High Pressure Liquid Chromatography
Assays
Hydroxyindoleacetic Acid
Gastrointestinal Diseases
Biomarkers
Mass spectrometers
Reverse-Phase Chromatography
Regression analysis
Tryptophan
Limit of Detection
Cations

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology
  • Clinical Biochemistry

Cite this

Measurement of serotonin in platelet depleted plasma by liquid chromatography tandem mass spectrometry. / Monaghan, Phillip J.; Brown, Heather A.; Houghton, Lesley A.; Keevil, Brian G.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 877, No. 22, 15.07.2009, p. 2163-2167.

Research output: Contribution to journalArticle

Monaghan, Phillip J. ; Brown, Heather A. ; Houghton, Lesley A. ; Keevil, Brian G. / Measurement of serotonin in platelet depleted plasma by liquid chromatography tandem mass spectrometry. In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2009 ; Vol. 877, No. 22. pp. 2163-2167.
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AB - 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 d 4-5-HT, respectively. 5-HT and d 4-5-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 (r 2 = 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, r 2 = 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.

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