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Analytical Method Validation for Estimation of Neurotransmitters (Biogenic Monoamines) from Cerebrospinal Fluid Using High Performance Liquid Chromatography

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Abstract

Biogenic amine neurotransmitters such as serotonin and dopamine are essential for signaling in both central and peripheral nervous system. Their metabolism is a multistep pathway and any defect in this results in alteration in metabolites of serotonin 5-Hydroxyindole acetic acid (5HIAA) and dopamine homovanillic acid (HVA) and 3-O-Methyl Dopa (3-OMD). Estimation of these metabolites in cerebrospinal fluid (CSF) assists in diagnosis of neurotransmitter defects. Their estimation is technically demanding and is currently available only in referral centers. We aimed to optimize a method for analysis of 5HIAA, HVA and 3-OMD. A high performance liquid chromatography (HPLC) method with electro chemical detector (ECD) was standardized for estimation. Analysis for method validation, reference range verification and clinical correlation was performed. Linearity obtained for 5-HIAA, HVA and 3-OMD was 65.35–2615.0 nmoles/l, 68.62–2745.0 nmoles/l and 236.5–4730.0 nmoles/l respectively. The coefficient of variation for internal quality controls ranged from 5 to 14% and the external proficiency testing samples (n = 16) were within peer group range. CSF metabolite levels of samples for reference range analysis overlapped with age matched ranges reported in literature. Among the 40 suspected patients analyzed for clinical testing four were found to have a neurotransmitter defect. These patients were then confirmed with molecular testing and clinical correlation. The method is validated and can be adapted in a clinical laboratory with analytical competence in HPLC.

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Acknowledgements

Resources: We acknowledge support extended by the Development committee, National Health and Education Society for funding the study, clinicians for referring patients and patients and their families for providing study samples.

Proficiency Testing: P. D. Hinduja Hospital and MRC acknowledge the use of data derived from ERNDIM EQA materials in this paper. The use of ERNDIM EQA materials does not imply that ERNDIM endorses the methods used or the scientific validity of the findings in this paper. ERNDIM (www.erndim.org) is an independent, not for profit foundation that provides EQA schemes in the field of inborn errors of metabolism with the aim of improving diagnosis, treatment and monitoring of inherited metabolic diseases.

Funding

Development committee, National Health and Education Society.

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Author notes

  1. Alpa J. Dherai

    Present address: Biochemistry Section, Department of Laboratory Medicine, P.D.Hinduja Hospital & MRC, Mumbai, India

Authors and Affiliations

  1. Biochemistry Section, Department of Laboratory Medicine, P.D.Hinduja Hospital & MRC, Mumbai, India

    Rohan V. Lokhande, Prasad R. Naik & Tester F. Ashavaid

  2. Department of Chemistry, Satish Pradhan Dnyanasadhana College, Thane, India

    Rohan V. Lokhande & Ganesh R. Bhagure

  3. Department of Paediatric Neurology, P.D.Hinduja Hospital & MRC, Mumbai, India

    Vrajesh P. Udani & Neelu A. Desai

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  1. Rohan V. Lokhande
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  2. Ganesh R. Bhagure
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Correspondence to Alpa J. Dherai.

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Lokhande, R.V., Bhagure, G.R., Dherai, A.J. et al. Analytical Method Validation for Estimation of Neurotransmitters (Biogenic Monoamines) from Cerebrospinal Fluid Using High Performance Liquid Chromatography. Ind J Clin Biochem 37, 85–92 (2022). https://doi.org/10.1007/s12291-020-00949-8

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  • DOI: https://doi.org/10.1007/s12291-020-00949-8

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