In Vivo Monitoring of Serotonin in the Striatum of Freely Moving Rats with One Minute Temporal Resolution by Online Microdialysis–Capillary High-Performance Liquid Chromatography at Elevated Temperature and Pressure

Online sampling/analysis is highly desirable for many applications, for example high throughput screening,1 analysis of synthetic reactions in microreactors,2 environmental3 and biological4 substance monitoring, and process analytical chemistry.5 Online sampling especially benefits the analysis of small volume samples since it avoids sample handling procedures. Another area in which it excels is in monitoring dynamic processes such as those occurring in the brain.6 Increasing the temporal resolution of online sampling/analysis will provide more details of dynamic processes. Serotonin (5-hydroxytryptamine, 5-HT) together with the 5-HT transporter and receptors modulate many brain functions7–10 and intestinal activities.11 Neurological disorders like depression and anxiety are associated with abnormalities of the serotonergic system. Two major tools to study 5-HT neurochemistry in the brain are fast scan cyclic voltammetry12–14 and microdialysis/HPLC. These techniques are complementary. The former has sub-second time resolution while microdialysis-HPLC can perform multi-analyte detection over long timescales with the potential for unambiguous analyte identification. Online measurements following microdialysis are continuous, e.g., using sensors,15 or discrete, e.g., using a separation.16–19 Here, we focus on the latter. Conventional microdialysis-HPLC is carried out with low temporal resolution. Temporal resolution can be improved by decreasing the scale of the separation method, e.g., capillary HPLC and capillary electrophoresis.20–23 Kennedy and Bowser measured changes in neurotransmitter concentrations (e.g., dopamine, glutamate, aspartate) in response to a stimulus using online microdialysis-capillary electrophoresis with online fluorescent derivatization.24 The temporal resolution was as high as 11 s for amino acids22 and 90 s for dopamine.21 While capillary electrophoresis is powerful, within the neuroscience community HPLC has become the preferred method for research on low concentration neurotransmitters like dopamine and 5-HT25,26 because of its reliability and reproducibility. Using microdialysis-HPLC, Newton and Justice20 studied dopamine dynamics with one-minute temporal resolution by using small, 1 µL samples in conjunction with a 0.5-mm-inner diameter (i.d.) microbore column. Richter et al.27 studied 5-HT and other neurotransmitters with a temporal resolution of 1.5 minutes using 3 µL samples in conjunction with a 2-mm-i.d. column. These researchers recognized the advantage of small diameter columns: they minimize sample dilution during the separation permitting reliable quantitation with small volume injections. However, they performed the chromatography offline. Using even smaller i.d. capillary columns offline Parrot et al.28 with 1 µL samples and Jung et al.29 with 0.5 µL samples achieved low detection limits for 5-HT, but the separations were performed offline and were approximately ten minutes long. The Andrews lab30 recently reported in vivo, online monitoring of 5-HT with a temporal resolution of three minutes. This is a significant advance. The work was done with commercially available equipment. They applied this fast microdialysis approach to awake animals for times long enough to perceive circadian changes. When considering the design of an online separation-based analysis system, it is important to understand the relationships that define and indeed connect the system’s figures of merit. Scheme 1 shows that the concentration of analyte 5-HT in the detector following a separation consists of two independent components: the animal/microdialysis part of the experiment defines the number of moles injected into the separation system and the separation system (here chromatography) dictates the volume of the solution in which the injected moles are found in the detector. We will call the latter volume the peak volume. The number of moles collected in each sample depends on the sample volume and the dialysate concentration, itself a function of the actual concentration in the brain and the microdialysis recovery. The peak volume is defined by the retention time and a number of other parameters related to column efficiency, (which may include extracolumn effects and overloading) as well as the inside diameter of the column. Ultimately, the moles acquired divided by the peak volume must exceed the concentration detection limit of the detector used. The integral relationship between sampling and measurement is even deeper. The microdialysis sample volume and flow rate define the sampling time and thus the temporal resolution. Ideally, the speed of the chromatographic system and the sampling time will “match” so that one chromatogram is achievable in the same time required to collect the sample. The constraints on an optimized system are therefore that under conditions where the sampling time is approximately equal to the separation time, the ratio of moles collected during the sampling time to the peak volume in the separation system must exceed the detector’s detection limit by some factor. While the foregoing is accurate, it should be borne in mind that the system’s time resolution may be limited by the dispersion within the microdialysis flow path (to be discussed below). Scheme 1 Yellow outlined boxes correspond to microdialysis; blue boxes correspond to the chromatography. Arrows imply “controls” or “affects”. Black triangles with two “inputs” and one “output” imply ... Recently, Liu et al.31 and Zhang et al.26 improved the HPLC analysis speed of 5-HT to the sub-minute level using elevated column pressure and temperature, a capillary column packed with sub-2-µm particles, and a sensitive, low dead-volume electrochemical detector. Using the smallest commercially available packing material at the time, Zhang et al. optimized a chromatographic system as described in Scheme 1 for maximum sensitivity given a required separation power (theoretical plate count), desired separation speed, and sample size. The column diameter is a key parameter and has an optimum value. This is the first time that both the microdialysis sampling time and the analysis time for 5-HT are one minute or less laying the groundwork for in vivo online microdialysis coupled to capillary UHPLC with electrochemical detection (UHPLC-EC) (here we use the rather ill-defined term UHPLC to represent our use of elevated temperature and pressure conditions). In this work, we carried out in vivo online microdialysis coupled to capillary UHPLC to monitor basal 5-HT concentrations and the subsequent changes in response to a stimulus with one-minute temporal resolution in the striatum of freely-moving rats. Optimization of both sampling and analysis were carried out, with emphasis on temporal resolution, long time continuous analysis sensitivity, and robustness. We observed basal concentrations and dynamic changes of 5-HT for up to sixteen hours and forty minutes.