Protocol | Published:

Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC

Nature Protocols volume 12, pages 23592375 (2017) | Download Citation

Abstract

The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

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Acknowledgements

This work was supported by grants from the Instituto de Salud Carlos III (ISCIII; FIS PI15/01082 and PI14/00032) and the FEDER Funding Program from the European Union. R.A. was supported by 'programa de intensificación de la actividad investigadora', from the ISCIII. The CIBERER is an initiative of the ISCIII.

Author information

Author notes

    • Marta Batllori
    •  & Marta Molero-Luis

    These authors contributed equally to this work.

Affiliations

  1. Department of Clinical Biochemistry, Institut de Recerca Sant Joan de Déu (IRSJD), Barcelona, Spain.

    • Marta Batllori
    • , Marta Molero-Luis
    • , Aida Ormazabal
    • , Mercedes Casado
    • , Cristina Sierra
    •  & Rafael Artuch
  2. Centre for Research in Rare Diseases (CIBERER-ISCIII), Barcelona, Spain.

    • Marta Molero-Luis
    • , Aida Ormazabal
    • , Angels García-Cazorla
    •  & Rafael Artuch
  3. Department of Pediatric Neurology, IRSJD, Barcelona, Spain.

    • Angels García-Cazorla
  4. Molecular Neurosciences, UCL-Institute of Child Health, London, UK.

    • Manju Kurian
  5. Developmental Neurosciences, UCL-Institute of Child Health, London, UK.

    • Manju Kurian
  6. Department of Neurology, Great Ormond Street Hospital, London, UK.

    • Manju Kurian
    •  & Simon J Heales
  7. Department of Chemical Pathology, Great Ormond Street Hospital, London, UK.

    • Manju Kurian
    •  & Simon J Heales
  8. Neurometabolic Unit, National Hospital, Queen Square and UCL-Institute of Child Health, London, UK.

    • Simon Pope
    •  & Simon J Heales

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Contributions

M.B., M.M.-L., A.O., M.C. and C.S. performed the experiments and collected all technical data regarding the monoamine and PLP determinations. S.P. managed the technical aspects of the pterin analysis and prepared the quality-control scheme. A.G.-C. and M.K. directed the sample collection and the management and establishment of the preanalytical protocols and clinical discussions. S.J.H. and R.A. designed the methods and planned the strategies for further methodological developments. M.B., M.M.-L., A.O. and R.A. wrote the manuscript. All authors critically reviewed the content of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Rafael Artuch.

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DOI

https://doi.org/10.1038/nprot.2017.103

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