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Pharmaceutical Article

Simple, Optimized HPLC Analysis of Catecholamines:

Increase Retention by Using an Allure™ PFP Propyl Column

By Rick Lake, Pharmaceutical Innovations Chemist, and Bruce Albright, HPLC Innovations Chemist
  • No derivatization or ion-pairing—save time, ensure reproducible results.
  • Excellent retention and resolution of low molecular weight amine compounds.
  • Excellent peak shapes for reliable quantification of basic compounds.

Biogenic amines are low molecular weight intercellular messengers that relay much of the body’s chemical signaling. Many synthesized drug compounds are chemically similar to these very biologically active compounds, including stimulants, hallucinogens, antidepressants, and bronchodilators.

Members of one group of biogenic amines, the catecholamines, act as hormones or neurotransmitters, exciting and inhibiting reactions in the peripheral and central nervous systems. These actions are associated with stress, and include stimulation of the circulatory and respiratory systems, psychomotor activity, and metabolism. Chemically, catecholamines are substituted phenethylamines (Figure 1) and, specifically, they are phenethylamines with hydroxyl groups in positions 3 and 4 of the phenyl ring. In our analysis, we included the most commonly assayed catecholamines among our target analytes: epinephrine (adrenaline), norepinephrine (noradrenaline), levodopa (L-dopa), and dopamine (Figure 2). We also included the aromatic amino acid tyrosine, the metabolic precursor of the catecholamines (Figure 2).

Traditionally, these analytes have been assayed by either GC or HPLC, but either approach must be modified. When assayed by GC, catecholamines’ low volatility and poor stability in non-acidic solutions makes derivatization necessary, and stability issues pose a problem as well. Limited retention on hydrophobic alkyl (e.g., C18) or polar embedded (cyano) HPLC phases makes derivatization or ion-pairing techniques necessary. These modified HPLC techniques are laborious and disrupt reproducibility, and many derivatizing reagents are not LC/MS compatible.

Relative to cyano phases, pentafluorophenyl (PFP) HPLC phases show greater retention for compounds that have electrophilic properties, like protonated amine groups in basic compounds, and a propyl spacer between the functional group and the silica surface — a pentafluorophenyl propyl phase — further increases retention. Our intent with this analysis was to determine if an Allure™ PFP Propyl column, with its pentafluorophenyl propyl stationary phase, sufficiently increases retention for biogenic amines to allow an analysis without ion-paring or derivatization. If so, we would be able to develop a simple reversed phase HPLC analysis.

We used an acidic mobile phase to induce protonation of the analytes’ amine groups. Using this mobile phase and an Allure™ PFP Propyl column retention was suitable, as expected, and the analytes were separated (Figure 3). Norepinephrine, the first eluting analyte, is the key sample component to developing a method for effectively retaining catecholamines. A nearly 100% aqueous mobile phase was needed to adequately retain norepinephrine but, retention was achieved. Because the Allure™ PFP Propyl provides optimum retention, a small percentage of organic modifier could also be included in the mobile phase. By changing the organic modifier, selectivity of the catecholamines can be altered (Figure 4). This gives the analyst more flexibility in optimizing specific separations.

This same principle holds true for other, structurally similar amines — retention and selectivity are superior on an Allure™ PFP Propyl column, relative to retention and selectivity on a C18, cyano, or PFP column, without modification of the analytes or the mobile phase. By using an Allure™ PFP Propyl column, an analyst can achieve simple, reproducible analyses of low molecular weight amine-containing pharmaceutical compounds.


Figure 1  General structure of substituted phenethylamines.



Figure 2  Chemical structures of catecholamines and their parent compound, tyrosine (aromatic amino acid).
Epinephrine (Adrenaline) Norepinephrine (Noradrenaline) Levodopa (L-dopa)
Dopamine Tyrosine  
 


Figure 3  Superior retention of catecholamines on an Allure™ PFP Propyl column — better separations, without ion pairing.

  1. norepinephrine
  2. levodopa
  3. epinephrine
  4. tyrosine
  5. dopamine

LC_BA0349

Sample:

Inj.:

10µL

Conc.:

40µg/mL each component

Sample diluent:

mobile phase

Sample temp.:

ambient

Column:

Allure™ PFP Propyl (cat.# 9169565)

Dimensions:

150 x 4.6mm

Particle size:

5µm

Pore size:

60Å

Conditions:

Mobile phase:

50mM ammonium phosphate (pH 3.0, adjusted with formic acid)

Flow:

1mL/min.

Temp.:

35°C

Det.:

UV @ 266nm


Figure 4  Change organic modifiers to alter selectivity of an Allure™ PFP Propyl column, for more flexibility in optimizing catecholamine separations.

  1. levodopa
  2. epinephrine
  3. dopamine

LC_BP0360, LC_BP0359

Sample:

Inj.:

10µL

Conc.:

100µg/mL each component

Sample diluent:

mobile phase

Column:

Allure™ PFP Propyl (cat.# 9169565)

Dimensions:

150 x 4.6 mm

Particle size:

5µm

Pore size:

60Å

Conditions:

Mobile phase:

20mM ammonium formate (pH 3):methanol, 90:10 or 20mM ammonium formate (pH 3):acetonitrile, 90:10

Flow:

1mL/min.

Temp.:

ambient

Det.:

UV @ 280 nm