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Serotonin, Metabolites & Analogs
Simple method of analysis of neurotransmitters (NT) and their metabolites without Fluorescent Tags
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printable Application Sheet
Sample: Mix of 4 compounds:
- 5-hydroxy-3-indole acetic acid
(5-HIAA), metabolite of serotonin 192 m/z
- 3,4-dihydroxyphenylacetic acid (DOPAC) 169 m/z
- Serotonin 177 m/z
- Epinephrine 184 m/z
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Note: Neurotransmitters are important natural molecules that play significant roles in the mammalian central nervous
system. The method presented is sensitive enough to be used to quantitate the metabolite concentrations in blood
samples and may be applicable to relevant clinical studies.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 0.1% formic acid
B: acetonitrile + 0.1% formic acid |
| Mobile Phase |
Gradient t0 = 1.44 min
| Time |
%B |
Time |
%B |
| 0.0 |
95 |
5.00 |
70 |
| 2.00 |
95 |
7.00 |
45 |
| 4.00 |
70 |
8.00 |
45 |
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| Flow Rate |
0.4 mL/min. |
| Sample |
Mix of 4 compounds
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| Sample Prep |
153.5 µg /mL of each in water.
Dilution: 30 µL of the mix sample into 70 µL of 0.1% formic acid in acetonitrile.
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| Injection |
1 µL |
| Detection |
ESI – pos - Agilent 6210 MSD TOF mass spectrometer. |
Discussion
An Aqueous Normal Phase chromatographic method, coupled with ESI detection (LC-MS), was developed for the analysis of
neurotransmitters and their metabolites. Current HPLC-based methods which are used to analyze NT and metabolites in biological
fluids have several drawbacks since they often require a derivatization to convert NT into a fluorescent molecule or using ion
pairing reagents in the chromatographic process. The ion pair reagents typically are not MS compatible or hinder MS detection.
Mass spectrometry (MS) coupled to HPLC with Diamond Hydride™ column is a powerful technique which can be used within the
field of analytical toxicology or can be used for accurate determination of NT and metabolites in biological samples for routine
assessment of physiological or various pathological processes.
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