Liquid chromatography neurotransmitter analysis

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

With the introduction of modern electronics, inexpensive computers, and new materials there is a resurgence of voltammetric techniques in various branches of science as evident in hundreds of new publications. Now, voltammetry can be performed with a nano-electrode for the detection of single molecular events [1], similar electrodes can be used to monitor the activity of neurotransmitter in a single living cell in subnanoliter volume electrochemical cell [2], measurement of fast electron transfer kinetics, trace metal analysis, etc. Voltammetric sensors are now commonplace in gas sensors (home CO sensor), biomedical sensors (blood glucose meter), and detectors for liquid chromatography. Voltammetric sensors appear to be an ideal candidate for miniaturization and mass production. This is evident in the development of lab-on-chip... 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

S.6.4.2 Reversed-phase ion-pair liquid chromatography. Reversed-phase ion-pair chromatography is an alternative approach for controlling the retention of ionic compounds. This approach is particularly useful for the separation of amines on silica-based columns and it has had a profound effect on the analysis of this class of compounds. In particular, the combination of reversed-phase ion-pair liquid chromatography and electrochemical detection revolutionized the analysis of neurotransmitters in the brain (Tomlinson et al., 1978 and refs, therein). 

Analysis of Neurotransmitters and Their Metabolites by Liquid Chromatography... 

DNLM 1. Neurotransmitters—physiology. 2. Histochemistry— methods. 3. In Situ Hybridization— methods. 4. Chromatography, Liquid— methods, 5. Spectrum Analysis, Mass— methods. W1 ME 9616J V. 72 1997/QV 126 N4939 1997]... 

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