UHPLC with electrochemical detection for in vivo neurochemical research

Introduction and aim: In vivo microdialysis is a widely used sampling tool to investigate changes in neurotransmitter levels. To determine the monoamines dopamine, noradrenaline and serotonin in these samples, ion-pair liquid chromatography (LC) with electrochemical detection (ECD) is typically applied. Because of the low concentrations in the extracellular space (pM range), further dilution in the microdialysis samples, the complexity of the samples and the required temporal resolution, the analysis is a challenging issue. Furthermore, there is an increasing demand in the development of high-throughput analysis. In recent years, ultra-high performance LC (UHPLC) has been introduced as a way to increase the efficiency and speed of analysis by use of columns packed with sub-2 µm particles.
The aim of this study is to transfer the existing LC-ECD method [1] to a UHPLC-ECD method. Both methods will be compared in terms of throughput and sensitivity.
Methods: The ALEXYS neurotransmitter analyzer consists of an AS 110 autosampler, an LC 110S pump (up to 1000 bar), an OR 110 degasser unit and the DECDADE II electrochemical detector, all provided by Antec (Zoeterwoude, The Netherlands). Instrument control and data acquisition are carried out by Clarity chromatography software of Data Apex (Prague, The Czech Republic). The mobile phase consists of a buffer at pH 5.5, containing 0.1 M sodium acetate.3H2O, 20 mM citric acid.1H2O, 2 mM sodium decanesulfonate, 0.5 mM Na2EDTA, and 13% acetonitrile. Separations are performed on an Acquity UPLC BEH C8 column (100 x 1.0 mm, 1.7 µm) from Waters (Milford, Massachusetts, United States). The injection volume is 10 µL and the flow rate 60 µL/min, the column and detection temperature are set at 35 °C and the detection potential is + 450 mV vs. Ag/AgCl. Basal microdialysis samples are obtained from the hippocampus of the rat, using modified Ringer's solution as perfusion fluid.
Results and discussion: The use of a column with smaller particles (1.7 µm instead of 5 µm) leads to much faster analysis and higher peak efficiencies. This results in improved signal/noise ratios and detection limits below 100 pM for the three monoamines. However, some issues need to be addressed. First, the application of a low pass noise filter is not able to lower the detection limits without loss in peak shape, peak efficiency and resolution. Fast Fourier transformation on the unfiltered chromatogram is suitable for this purpose. Secondly, some loss of sensitivity occurs when the flow rate is increased to achieve smaller analysis times. Furthermore, with flow rates above 90 µL/min two peaks are visible for serotonin. This is not observed without ion-pair reagent. Further experiments will be performed to elucidate this problem. Thirdly, the separation of noradrenaline towards the front peak remains challenging because of its hydrophilic nature. Optimization of different parameters (mobile phase composition, column temperature,...) is necessary to improve its selective and sensitive determination in microdialysates.
Conclusion: UHPLC coupled to ECD is promising for the fast and sensitive analysis of the three monoamines in microdialysates. However, further investigations are necessary to optimize the late eluting serotonin and the early eluting noradrenaline within an acceptable time frame.

References: [1] Sarre S et al. J Chromatogr. 1992; 575(2):207-12.