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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

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Abstract

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; R 2 = 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.

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Acknowledgments

We gratefully acknowledge funding from the Irish Research Council for Science, Engineering and Technology (IRCSET) funding M. Moriarty. The Council of Directors, Technological Sector Research-Strand III 2006 Grant Scheme, awarded to Dr. A. Furey is also acknowledged for funding the formation of the ‘Team Elucidate’ research group. The Higher Education Authority (Programme for Research in Third-Level Institutions, Cycle 4 (PRTLI IV) National Collaboration Programme on Environment and Climate Changes: Impacts and Responses is also acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research), Cork Institute of Technology, Cork, Ireland

    Merisa Moriarty & Mary Lehane

  2. Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland

    Aoife Lee & Brendan O’Connell

  3. Child and Adolescent Mental Health Services, Health Service Executive, South, North Cork Area and the National Suicide Research Foundation, Cork, Ireland

    Helen Keeley

  4. Team Elucidate and PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research) research groups, Cork Institute of Technology, Cork, Ireland

    Ambrose Furey

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  1. Merisa Moriarty
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  2. Aoife Lee
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Correspondence to Ambrose Furey.

Electronic supplementary material

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10337_2012_2330_MOESM1_ESM.ppt

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

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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Moriarty, M., Lee, A., O’Connell, B. et al. 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. Chromatographia 75, 1257–1269 (2012). https://doi.org/10.1007/s10337-012-2330-5

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  • DOI: https://doi.org/10.1007/s10337-012-2330-5

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