When microdialysates are manually collected and analyzed offline, the minimal sample volume required for the CE analysis must be determined. Sample injections are currently made at one side of the capillary by applying pressure.
The volume of the sample must be sufficient 1 to allow the capillary to plunge into it, preventing the injection of air microbubbles with the sample, and 2 to avoid any significant loss by evaporation when a series of dialysates i. The authors have found that nl is the minimal volume required for the analysis of a large series of sample with a good reproducibility. With a shorter series of samples, determination of amino acid neurotransmitter content can be performed using nl of derivatized microdialysates. The reduction of microdialysis sample volumes needed for neurotransmitter analysis using CE-LIF allows slower perfusion rates, thereby increasing temporal resolution.
With slow perfusion rates, the concentration gradients developed by the probe are less marked, a characteristic that improves the spatial resolution and decreases disturbance of neural tissue. Moreover, the increased concentration of sample that is removed at slow perfusion rates improves the detection limit for concentration-limited measurements. As a consequence, in vivo calibration methods such as quantitative no-net-flux or extrapolation to zero flow rate, which are time consuming, could be avoided. Since a derivatization reaction with a fluorescent tag is needed to detect the molecules of interest and since most of the fluorescent reagents used react with primary amines, a large number of the neurochemical constituents of brain microdialysis samples can potentially be detected.
In this respect, the use of CE-LIFD makes possible the high-sensitivity determination of various neurotransmitters in a single microdialysis sample with improved temporal resolution. This can be performed via several analyses.
The low volume requirement of CE-LIFD allows several subsequent offline analyses to be performed on the same sample of microdialysate. The advantage of such multiple assays is the ability to study functional or drug-induced interactions between neurotransmitters.
Some of these are due to characteristics of the dialysate in general and some to characteristics of brain dopamine systems. These issues are as follows. To maximize diffusion into the dialysis probe, the perfusate is pumped through the probe at slow rates 0. The entire sample is usually needed for analysis, which allows only one opportunity to assay each sample.
HPLC - High Performance Liquid Chromatography
If larger volumes are collected, the sample can be split and analyzed for a variety of transmitters e. Dialysates are usually analyzed immediately after collection. Therefore, run times must be kept short to keep up with sample collection. A sensitive assay is required because the basal extracellular concentration of neurotransmitter is low and dialysate levels are even lower because of fractional recovery.
At a detector sensitivity that is optimal for dopamine, metabolites may exceed the linear range of the detector, and can interfere with the resolution of dopamine. Conversely, a detector sensitivity that is optimal for metabolites is not sufficient for the quantification of dopamine.
Dialysates recovered from the primate brain show several uncharacterized peaks that occur close to that of dopamine. During routine analysis, it is a constant struggle to prepare a mobile phase that provides good separation of dopamine from its metabolites. In the authors' experience, minimizing organic solvent, manipulating pH, and prolonging the run time improve chromatographic separation of the dopamine peak from other electrochemical signals.
Mobile phase and electrochemical detector conditions need to be modified to suit the characteristics of the transmitter of interest.
How Chromatography Packing Materials Used in HPLC are Affected by the Uniformity of Particle Shape
Similarly, protocols for amino acid analysis in rat samples unit 7. It is critical that mobile phase and detector conditions be modified for optimum detection of the transmitter of interest. Retention times are affected by alterations in room temperature as well as by pH, the concentration of ion-pairing reagent, and the inorganic solvent employed.
These can be varied to modify analyte resolution. Increasing pH decreases the retention time of acidic metabolites e. Increasing the concentration of ion-pairing reagents e. Increasing solvent concentration results in a decreased retention time of amines and their metabolites. Each of these parameters can be altered to increase analyte resolution. Additional troubleshooting guidelines can be found in Table 7. The microbore column is easily clogged, making an inline precolumn filter or guard column useful.
Guard columns should be replaced on a regular basis, especially when increased pressure or a loss of performance is observed. Retention times of acetylcholine and choline primarily depend on the mobile phase ionic strength. A higher ionic strength will result in shorter retention times. Platinum is a soft material and can scratch easily. If a loss of sensitivity can be traced to the performance of the electrode, avoid unnecessary polishing and try first to activate the electrode electrochemically. Until recently, detection of dialysate levels of acetylcholine could only be achieved from samples perfused with an aCSF solution containing the acetylcholinesterase inhibitor neostigmine.
There is, however, increasing evidence indicating that the micromolar concentrations of neostigmine employed can markedly modify the neurochemical effects of various drugs. Therefore, attempts should be made to use as low a concentration of neostigmine as possible. Several laboratories are now able to measure basal levels of acetylcholine in various brain areas in the absence of neostigmine. Therefore, pilot studies should be conducted to determine the minimum concentration of neostigmine needed for the region examined. Bacterial contamination of the pump results in the production of enzymes that scavenge hydrogen peroxide.
Methods and Concepts in the Life Sciences/Chromatography
Therefore, the system should be flushed periodically. The addition of a preservative Proclin to the mobile phase is also recommended. When setting up the method for the first time, it is critical to passivate the system to ensure a clean system. Acetylcholine and choline are hygroscopic.
Therefore, the solids should be allowed to equilibrate to room temperature before weighing.
The solids should be stored in a freezer. Columns should not be in contact with organic solvents such as methanol or acetonitrile. A decrease in sensitivity may be caused by bacterial contamination, loss of enzymatic activity in the IMER, a dirty electrode, or insufficient oxygen in the mobile phase. Deterioration of the OPA reagent can lead to decreased sensitivity. The pH must be basic and 2-mercaptoethanol must be present as the nucleophile for the reaction to yield fluorogenic amino acid isoindole derivatives. Therefore, stock OPA reagent should be prepared weekly.
The OPA reaction is also dependent on an aqueous medium.
It is important to dilute the stock OPA reagent with an aqueous solution other than the mobile phase, preferably with the perfusate buffer used in the collection of microdialysates. Un-reacted OPA will elute immediately after aspartate. The presence of this peak can indicate improper mixing of the sample with the OPA reagent, resulting in an underestimation of microdialysate amino acid concentrations. Note that the amino acids asparagine and histidine may appear as peaks directly preceding and following glutamate elution. Detection of amino acids e.
The main problems encountered with this method are exogenous contaminants, increases in column pressure, especially when using microbore columns, and high background due to air in the system. Contrary to other neurotransmitters like catecholamines, indoleamines and acetylcholine, amino acids are very stable at room temperature.
They are also ubiquitous in dust, sweat, and organic matter. For this reason, exogenous contamination of samples and solutions is common. It is good practice to wear gloves when handling solutions and to thoroughly clean glassware. It is also necessary to run blanks of the aCSF or Ringer solutions used for preparing standards and running the dialysis experiments. Increases in HPLC system pressure are also a common problem. The microbore columns are especially sensitive to clogging by particulate matter due to the small inner diameter. Adequate filtering of all the solutions is required.
It is advisable to install a precolumn filter with minimal dead volume to trap any particulate matter coming from the injector even if samples are clean, a faulty valve seal or other moving components in the injector can be the source of particles. Sudden increases in pressure are likely caused by particulate matter trapped in the filter. Sonicating or replacing the filter normally takes care of the problem.
Eventually, pressure builds up over time. If replacing the filter does not help, this means that pressure has built up in the column. The repetitive acetonitrile flushes pose a strain on the column packing and eventually it will become loose and pack at the end of the column, increasing the pressure. Finally, slow buildup in pressure may also be due to accumulation of proteins.
Since dialysates do not contain proteins, accumulation of proteins in the column likely reflects bacterial contamination in the system upstream from the column. The precolumn filter will not trap proteins in solution. In this case, a guard column may be installed instead of the precolumn filter. This may be fixed by either preparing a fresh degassed mobile phase or sometimes just degassing the old mobile phase.
This problem is related to the high concentration of acetonitrile in the mobile phase since it is normally observed with the GABA mobile phase and it seems related to oxygen in the mobile phase quenching the fluorescent signal. Since the CE-LIF technique was developed for the analysis of brain extracellular fluid obtained by microdialysis, the derivatization procedure has to be performed on small volumes and with standards dissolved in the perfusion fluid. The derivatization rate is typically not affected by the higher ionic strength of biological samples when compared to standards in aqueous solution, as evidenced by lack of variation in peak areas when samples are prepared either in water or aCSF A.
Zapata, V. Chefer, T. Shippenberg, and L. Denoroy, unpub. The rate of derivatization, stability of the labeled amino acids, and amino acid quantification varies for each amino acid. In contrast, derivatives of GABA seems less stable that those of Glu and Asp; this may be due to the greater hydrophilic properties of Glu and Asp derivatives. Thus, the time spent by the solutes in crossing the membrane is not a limiting factor for monitoring short-lasting events.
However, high-sampling-rate microdialysis can be performed only if dead volumes are minimized both at the outlet and at the inlet if local application of drugs by reverse dialysis is to be carried out of the dialysis system. The local application of drugs through the microdialysis probe is performed by switching perfusion fluid from aCSF to an aCSF containing known concentrations of drugs.