Advantages of HILIC Mobile Phases for LC–ESI–MS–MS Analysis of Neurotransmitters

Mar 01, 2013
Volume 26, Issue 3, pg 128–140

This article investigates the influence of liquid mobile phase composition on the response measured in electrospray ionization-mass spectrometry (ESI–MS) for 12 selected neurotransmitters. The aim was to find the optimal conditions to achieve sufficient limits of detection (LOD) that would permit their detection by liquid chromatography-tandem mass spectrometry (LC–MS–MS) in biological samples (brain extracts). The advantages of solvent-rich mobile phases typically used in hydrophilic interaction liquid chromatography (HILIC) are clear. The HILIC–ESI–MS–MS system optimized for the 12 selected compounds analysis presented significant advantages over other existing methods.

Neurotransmitters are important polar biological compounds. For example, catecholamines and indolamines are very important because anomalies in their concentrations lead to a number of diseases, including Parkinson's, Alzheimer's or Down's disease, depression, schizophrenia and epilepsy (1). Their physiological concentrations are very low and they are usually analysed in the presence of complex matrices. Mass spectrometry (MS) has become the preferred tool for their detection and quantification to reach very low limits of detection (LOD) (2–6). While some derivatization is required to analyse neurotransmitters with gas chromatography (GC) (7), their direct separation (without the need for a prior derivatization step) can be conducted with capillary electrophoresis (CE) (8) or liquid chromatography (LC). The latter can be achieved with different elution modes: ion-exchange (IEC), reversed-phase liquid chromatography (RPLC), ion-pairing liquid chromatography (IPLC) or hydrophilic interaction liquid chromatography (HILIC).

IEC (9) is of limited interest because it can only separate compounds under a given charge state (positive or negative), while neurotransmitters, their precursors and metabolites may be either neutral, cationic, anionic or zwitterionic in the same sample. IPLC, an alternative approach to RPLC can effectively increase the retention of ionizable polar compounds allowing satisfactory separation of both anionic, cationic and neutral neurotransmitters in the same run (10). However, the anionic ion-pairing agent added to the mobile phase prevents the MS detection under a negative ion (NI) mode and thus renders MS detection impossible for anionic analytes. RPLC (2,3,6,10-12) and HILIC (13–15) do not have any of these disadvantages. Both have proven to be capable to retain and separate neurotransmitters and compatible with a MS detection. RPLC separations can be made easier by using stationary phases possessing some polar character (such as mixed-mode phases like pentafluorophenyl phases or porous graphitic carbon) to get sufficient retention of the polar neurotransmitters.

Our group previously developed satisfying RPLC (10) and HILIC separations of catecholamine and indolamine neurotransmitters in varied mobile phase compositions (13), and we were keen to select those conditions that would provide the best MS response to allow for both robustness and low detection limits. For the biological analysis of neurotransmitters sample size is usually very limited, even if adequate sample preparation to concentrate samples prior to the analysis is performed. Thus the loest possible LOD with ESI–MS would be advantageous. However, it is well known that electrospray ionization (ESI) strongly depends on the eluent nature, thus detection issues cannot be considered independently of the chromatographic system.

Although it is generally accepted that the buffered solvent-rich HILIC mobile phases are most favourable for ESI–MS detection (16), only a few studies have investigated the effect of the chromatographic operating parameters on the MS response. Some research has simply shown how HILIC conditions compared favourably to RPLC conditions when using MS detection (17,18) but those examples were only provided for a couple of compounds. Fountain et al. (19) investigated the influence of HILIC mobile phase pH on MS response for 21 small test compounds, comprising acids, bases and organophosphonic acids.

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