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

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.

Cerebrospinal Fluid Biogenic Monoamine Analysis for Diagnosis of Primary Neurotransmitter Disorders.

Biogenic amine neurotransmitters metabolism is a multistep pathway with pterin and pyridoxal phosphate (vitamin B6) as cofactors. A defect in biogenic amine and cofactor metabolism and vesicular transporters result in a primary neurotransmitter disorders. These are a well-recognized groups of inherited disorders and often present with features overlapping with other neurological conditions. Their diagnosis is made by analysis of biogenic amine metabolites in cerebrospinal fluid (CSF) and other body fluids and respective enzyme assays. Many of these disorders are treatable and deficits can be reverted by timely intervention. CSF biogenic amine or cofactor metabolite analysis is one of the primary indicators of a neurotransmitter disorder. In this paper, 3 cases are reported-2 of cofactor deficiency and 1 with enzyme deficiency wherein biogenic amine estimation has assisted in diagnosis.