Mixture of polar acidic and basic compounds is separated on a Primesep AB mixed-mode HPLC column. Dopamine and tyrosine are retained by combination of reversed-phase and cation-exchange mechanisms. Maleic acid is retained by anion-exchange mechanism, and benzoic acid is retained by reversed-phase mechanism. Primesep AB is a trimodal column with a C12 hydrophobic chain and cation-exchange and anion exchange groups on the surface. Method utilizes UV detection but can be used with other detection techniques (ELSD, LC/MS, Corona).

The catecholamine neurotransmitters are amino-acid derivatives of tyrosine. DOPA, tyrosine, phenylalanine, norepinephrine, epinephrine, and dopamine and baseline are resolved on a Primesep 100 column with UV-transparent phosphate buffer. This method can be used for analysis of catecholamines and related impurities in various matrices. Peak order and retention time can be changed by changing the amount of ACN, buffer concentration and buffer pH. Various buffers can be used to accommodate desired detection technique. Primesep 100 is a reversed-phase cation-exchange mixed-mode column that can be used for analysis of polar neutral, polar ionizable, polar zwitter-ionic, hydrophobic neutral, and hydrophobic ionic compounds in the same run. Column can be operated in reverse-phase, cation-exchange, anion-exclusion, HILIC and mixed-modes depending on the mobile phase selection and nature of analytes. Column is compatible with LC/MS and does not require use of ion-pairing reagents.

Dopamine and epinephrine were separated on a Primesep S2 HILIC mixed-mode column. Dopamine and epinephrine are retained by HILIC and cation-exchange mechanisms. Retention time is controlled by the amount of ACN, buffer and buffer pH. Method is compatible with LC/MS and can be used for analysis of polar metabolites in biofluids.

Introduction

Hydrogen-bonding interaction offers unique selectivity based on number of “interaction points” available for hydrogen bonding. One of the useful characteristics to determine retention patterns in hydrogen-bonding mode is the molecular polar surface area (PSA). This calculated parameter is usually used for prediction of drug transport properties, but we successfully applied it to hydrogen-bonding interactions. Polar surface area is defined as a sum of surfaces of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.  Since those polar  atoms can participate in hydrogen-bonding interaction, estimation of elution order can often be made based on PSA. While PSA is a good indicator of elution time, it must be noted that polar surface area does not account for the accessibility of hydrogen-interaction sites. Not every polar surface participates in intermolecular hydrogen interactions with the stationary phase.

Proximity of “interaction points” to each other within one molecule also needs to be considered since molecules can form an intramolecular hydrogen-bonding, which competes with intermolecular interaction between analyte and stationary phase. This reduces retention time in hydrogen-bonding mode.  Such structural factors provides unique selectivity among similarly structural (isomers, homologs, degradation products, precursors) molecules.

Since SHARC 1 column is a mixed-mode column, pKa is another useful parameter in method development for these columns. SHARC columns operate in non-aqueous mobile phase, but some effect of charge interaction of stationary phase and ionizable molecules still exists and contributes to the retention profile.

Mixture of basic and acidic compounds containing hydroxy and amino groups can be separated in one run utilizing hydrogen-bonding interaction between stationary phase and hydrogen-bonding groups of analytes. Method was used for separation of three neurotransmitters (homovanillic acid, DOPAC and dopamine). 

Application Notes: Dopamine is a naturally occurring neurotransmitter found in the brain. Dopamine is a well studied compound because dopamine is an important neurotransmitter known to regulate many functions from pain response to behavior disorders. According to USP methods, dopamine hydrochloride contains not less than 98% and not more than 102% dopamine hydrochloride calculated on the dried basis. The USP HPLC method for the separation of hydrocortisone was developed on Legacy L1 column according to the US Pharmacopeia methodology. L1 classification is assigned to reversed-phase HPLC column containing C18 ligand. Support for the material is spherical silica gel with particles size 3-10 um and pore size of 100-120A. Resolution between critical pairs corresponds to rules and specifications of USP.


Application Columns: Legacy L1 C18 HPLC column

Application compounds: Dopamine hydrochloride

Mobile phase: Water with 1% acetic and 5mM octanesulfonic acid/MeCN

Detection technique: UV

Reference: USP35: NF30

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

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