Applications:
Separation of Serotonin, Dopamine, and Related Compounds

Catecholamines are chemical compounds derived from the amino acid tyrosine containing catechol and amine groups. Some of them are biogenic amines. Retention of compounds of the catecholamine pathway is achieved on Obelisc N column. All polar compounds are well retained by combination of HILIC and ion-exchange mechanisms. Obelisc N columns produce very good peak shapes for all analytes. The method is very sensitive to amount of ACN, buffer and buffer pH. The retention time changes with variation of the main parameters. This method can be used for quantitation of biogenic amines and related compounds (homovanillic acid, dihydroxyphenyl acetic acid, serotonin, dopamine, epinephrine, hydroxytryptophan, epinephrine and DOPA) in urine, blood and other biological fluids. Further optimization of this HPLC method can be used during screening and validation. Amines and acids can be analyzed in the same run and retained by a combination of polar organic mode, cation-exchange and anion-exchange modes. Various buffers within specified pH can be employed (ammonium formate, ammonium acetate, sodium phosphate, etc.).
Condition
Column |
Obelisc N, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
AmAc pH 4.0- 30 mM |
Flow Rate |
1.0 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Acid, Monocarboxylic acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Homovanillic acid, Dihydroxyphenyl acetic acid, Serotonin, Dopamine, Epinephrine, Hydroxytryptophan, DOPA |
DOPA (3,4-dihydroxy-L-phenylalanine)DOPAC (Dihydroxyphenylacetic Acid)DopamineEpinephrineHomovanillic AcidHydroxytryptophanNorepinephrineSerotonin
Generic Screening Method for Complex Mixtures
Condition
Column |
Primesep 200, 4.6*150 mm 5 µm, 100A |
Mobile Phase |
MeCN/H2O |
Buffer |
H2SO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV, 215 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
Uracil, Epinephrine, DOPA, 2,6-Lutidine, Benzylamine, Hydroxytrypthophan, Homovanillic acid, Phenol, Tryptophan , 2,3-DHBA, Benzoic acid, Methylparaben, Ethylparaben, Toluene, Amitriptyline |
2,3-Dihydroxybenzoic Acid2,6-LutidineAmitriptylineBenzoic AcidBenzylamineDOPA (3,4-dihydroxy-L-phenylalanine)EpinephrineEthylparabenHomovanillic AcidHydroxytryptophanMethylparabenPhenolTolueneTryptophanUracil
HPLC Separation of Neurotransmitters

The neurotransmitters dl-DOPA, creatinine, hydroxytryptophan (5-HTP), and serotonin are separated in less than 4 minutes on a short 50 mm Primesep 200 column. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoroacetic acid (TFA) and UV detection at 250 nm. Ion-pair reagents were not needed for retention of these polar, hydrophilic compounds, instead, a combination of ion-exchange and reversed-phase interactions were used.
Condition
Column |
Primesep 200, 4.6x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 80/20% |
Buffer |
TFA - 0.05% |
Flow Rate |
1.0 ml/min |
Detection |
UV, 250 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
dl-DOPA, Creatinine, Hydroxytryptophan (5-HTP), Serotonin |
CreatinineDOPA (3,4-dihydroxy-L-phenylalanine)HydroxytryptophanSerotonin