Applications:
HPLC Separation of Vanilmandelic, Hydroxyindoleacetic, and Homovanillic Acids
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Condition 1
Column |
Newcrom B , 3.2x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 20/80% |
Buffer |
H3PO4 - 0.1% |
Flow Rate |
0.5 ml/min |
Detection |
UV, 275 nm |
Vanilmandelic, hydroxyindoleacetic and homovanillic acids are metabolites of various neurotransmitters that are used in urine tests to measure the levels of those neurotransmitters in the body. They can be baseline separated on a reverse-phase Primesep D HPLC column by varying the buffers and pH of the mobile phase consisting of water and acetonitrile (MeCN, ACN). UV detection at 230nm.
Condition 2
Column |
Primesep D, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
TFA, AmFm |
Flow Rate |
1.0 ml/min |
Detection |
UV, 230 nm |
Description
Class of Compounds
|
Acid, Monocarboxylic acid, Hydrophilic, Ionizable |
Analyzing Compounds |
Vanilmandelic, Hydroxyindoleacetic, Homovanillic Acid |
5-Hydroxyindole-3-Acetic AcidHomovanillic AcidVanilmandelic Acid
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
HPLC Separation of Homovanillic Acid, Dopamine, and DOPAC using Hydrogen Bonding
Condition
Column |
Sharc 1, 3.2x50 mm, 5 µm, 100A |
Mobile Phase |
Gradient MeCN/MeOH - 100/0 - 30/70% |
Buffer |
Formic Acid - 0.5% AmFm 0.05% 7 min |
Flow Rate |
1.0 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Acid, Monocarboxylic acid, Hydrophilic, Ionizable, Hormone |
Analyzing Compounds |
DOPAC, Homovanillic Acid, Dopamine |
DOPAC (Dihydroxyphenylacetic Acid)DopamineHomovanillic Acid
HPLC Separation of DOPAC, Homovanillic Acid and Dopamine on the Primesep 100 Column
Application Notes: Neurotransmitters DOPAC, homovanillic acid and dopamine were separated by mixed-mode chromatography on Primesep 100 and Primesep 200 HPLC columns. The method can be used for quantification of neurotransmitters with LC/MS compatible conditions. The compounds are retained by combination of reversed-phase, ion-exchange or ion-exclusion mechanisms. The retention time and selectivity of separation can be adjusted by variation of amount of acetonitrile, buffer pH and buffer concentration.
Application Columns: Primesep 100
Application compounds: Dopac, Homovanillic Acid, Dopamine
Detection technique: UV, LC/MS, ELSD/CAD
Condition
Column |
Primesep 100, 4.6x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
AmFm pH 3.0- 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 |
DOPAC, Homovanillic Acid, Dopamine |
DOPAC (Dihydroxyphenylacetic Acid)DopamineHomovanillic Acid
HPLC Separation of DOPAC, Homovanillic Acid in Dopamine on the Primesep 200 Column
Application Notes: Neurotransmitters DOPAC, homovanillic acid and dopamine were separated by mixed-mode chromatography on Primesep 100 and Primesep 200 HPLC columns. The method can be used for quantification of neurotransmitters with LC/MS compatible conditions. The compounds are retained by combination of reversed-phase, ion-exchange or ion-exclusion mechanisms. The retention time and selectivity of separation can be adjusted by variation of amount of acetonitrile, buffer pH and buffer concentration.
Application Columns: Primesep 100, Primesep 200
Application compounds: Dopac, Homovanillic Acid, Dopamine
Detection technique: UV, LC/MS, ELSD/CAD
Condition
Column |
Primesep 200, 4.6x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN/H2O - 5/95% |
Buffer |
AmFm pH 3.0- 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 |
DOPAC, Homovanillic Acid, Dopamine |
DOPAC (Dihydroxyphenylacetic Acid)DopamineHomovanillic Acid
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