, Volume 75, Issue 21–22, pp 1257–1269 | Cite as

The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin, Dopamine and Melatonin

  • Merisa Moriarty
  • Aoife Lee
  • Brendan O’Connell
  • Mary Lehane
  • Helen Keeley
  • Ambrose Furey


Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L−1; R2 = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L−1 and the LOQs of 15.4–70.5 nmol L−1 for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.


LC–MS/MS HybridSPE-Precipitation Serotonin Dopamine Melatonin Ion suppression Serum 

Supplementary material

10337_2012_2330_MOESM1_ESM.ppt (87 kb)
Online resource 1: Example of a post-column infusion experiment for the evaluation of ion suppression in serum matrix for serotonin (a) and dopamine (b). Shaded areas correspond to elution times of analytes. Analyte standards (2.84, 3.26 and 2.15 μmol L−1 for serotonin, dopamine and melatonin) were infused at a rate of 10 μL min−1. This experiment was repeat with different serum samples (n = 5) (PPT 87 kb)
10337_2012_2330_MOESM2_ESM.ppt (70 kb)
Online resource 2: Dilution study for the assessment of matrix ion suppression on the target compound, serotonin, comparing various dilutions of serum. With no ion suppression effects from serum matrices at zero dilution, a 1 in 2 dilution of the samples should given exactly a 50 % response. This study determined that a 1 in 5 dilution is necessary to eliminate completely the influence of matrix ion suppression effects (PPT 69 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Merisa Moriarty
    • 1
  • Aoife Lee
    • 2
  • Brendan O’Connell
    • 2
  • Mary Lehane
    • 1
  • Helen Keeley
    • 3
  • Ambrose Furey
    • 4
  1. 1.Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research)Cork Institute of TechnologyCorkIreland
  2. 2.Department of Biological SciencesCork Institute of TechnologyBishopstownIreland
  3. 3.Child and Adolescent Mental Health Services, Health Service Executive, SouthNorth Cork Area and the National Suicide Research FoundationCorkIreland
  4. 4.Team Elucidate and PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research) research groupsCork Institute of TechnologyCorkIreland

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