Application Of Microdialvsis Coupled With High Performance Liquid Chromatography And Electrochemical Detection

Research of the brain has become a focus all over the world. In the brain, neurotransmitters are the material foundation of the information transfer. The information transfer between neurons in the brain mainly relies on the neurotransmitters release from the synapse. Due to the presence of the blood-brain barrier, it is difficult to obtain the neurotransmitters from the brain compared with other tissues (eg. blood, urine). In vivo microdialysis is a sampling technique which can get the neurotransmitters and other metabolites in the conscious animals. After implanted the microdialysis probes into the animal body and promote the perfusion fluid, neurotransmitter can be extracted from the extracellular fluid based on the concentration gradient. Microdialysis has become an important sampling technique in neuroscience and pharmacology. Compared with the conventional tissue homogenate and spectral analysis methods (eg. MRS, PET), microdialysis can obtain neurotransmitters from extracellular fluid which is more meaningful to the leaning of their functions.However, this technique still exists a lot of problems which limit its large-scale application. For example, determination of low concentration of neurotransmitters, calibration the recovery in different samples, etc. Because of these difficulties, the majority of microdialysis applications are limited in pharmacology. The goal of this research is to solve these problems and improve the method, to make it more suitable for basic research in neuroscience. The major contents of this dissertation are described as follows:1 Calibration of the microdialysis probes. Due to the differences between external and internal environment, the recovery measured in vitro cannot represent the in vivo probe recovery. Therefore, various in vivo probe calibration methods were developed for the calibration of the probes, such as reverse dialysis method, low flow rate method, extrapolation to zero velocity method, zero net flux method, etc. However, all of these methods have their advantages and disadvantages. How to choose a suitable method for calibration of the probe in the study is difficult. In this part, the common in vivo and in vitro probe calibration methods are compared and used for the determination of neurotransmitter y-aminobutyric acid in the extracellular fluid of the thalamic with various methods.2 A simple method was developed for simultaneous determination of five amino acid neurotransmitters (taurine, glutamate, aspartate, glycine, and γ-aminobutyric acid) in brain microdialysates by high performance liquid chromatography with coulometric detection. The microdialysates were derivatized with o-phthalaldehyde in 5 mM sodium sulfite and separated within 17 min using isocratic elution. The separation was performed in an Agilent Eclipse Plus C18 (2.0×150 mm, particle size 3.5μm), and the mobile phase consisted of 100 mM citrate (pH 3.75) and 10% acetonitrile (V/V). Detection parameters for these transmitters were linearity (R)> 0.999 over the concentration range from 0.10 to 15.00μM (0.01 to 1.00 μM for y-aminobutyric acid); reproducibility 2.88-11.67%(relative standard deviation); the limits of detection 4.4-17.2 fmol and recovery 86.66%-103.42%. This method was successfully applied to analyze these neurotransmitters in the brain. The changes of neurotransmitters were also investigated via local administration of potassium chloride and bicuculline, which was a y-aminobutyric acid receptor antagonist. It was found that the developed method is more efficient as compared to previously reported methods in simultaneously determining five major neurotransmitters because it shows quick separation, sensitive detection, and is easy to operate. This method can be a practical choice for continuous monitoring of the amino acid neurotransmitters in vivo.3 A fast and sensitive method was developed for in vivo determination of histamine in the brain microdialysate by reverse ion pair chromatography with electrochemical detection. The microdialysates were derivatized with o-phthalaldehyde and sodium sulfite and separation was achieved using isocratic elution within 10 min. The separation was performed in an Agilent Eclipse Plus C18 (3.0×150 mm, particle size 3.5m), and the mobile phase consisted of 100 mM monosodium phosphate (pH 6.0),500mg/L OSA, and 20% methanol (V/V). The linearity (R2) was found to be greater than 0.999, with a range from 2 to 50 nM; with excellent repeatability (relative standard deviation,2.29% to 6.04%), and the limit of detection was 4.0 fmol. This method was successfully applied to analyze the extracellular concentration of histamine in the hypothalamus of rats, with probe recovery calculated in vitro and in vivo.4 A highly sensitive, rapid detection method for the determination of thiols (glutathione and cysteine) and guanosine metabolites (guanosine, guanine, xanthine and uric acid) in the rat brain using high performance liquid chromatography with electrochemical detector was established. Using 100 mM phosphate buffered saline (pH 2.0, containing 350mg/L octane sulfonic acid sodium salt,95%)-methanol (5%) as the mobile phase, the detection potential was set to 800 mV. All of the six compounds with good linearity in three orders of magnitude, the detection limit was 22.6-56.9 nM. The recovery of different spiked levels were ranged from 94.4% to 106.3% and the coefficient of variations were between 0.21%-3.85%. The constructed method can be employed for the determination of the six compounds in the rat brain.5 A simple method was developed for the simultaneous determination of five hydrophilic whitening agents (ascorbyl glucoside, magnesium ascorbyl phosphate, arbutin, hydroquinone and kojic acid) in bleaching cosmetics by high performance liquid chromatography with electrochemical detection. The samples were prepared using the microdialysis approach and separated within 10 min using isocratic elution. Linearity was achieved with correlation coefficients larger than 0.999 over a concentration range of 0.10 to 10.00μM. The detection limit ranged from 0.6 to 2.0 nM, the average recovery was between 91.43% and 108.04%, and the relative standard deviation was less than 6.88%.This method was successfully applied to analyze whitening compounds in several commercial bleaching cosmetics. The on-line microdialysis coupled with zero net flux method was also developed and applied. The entire separation procedure is fast, simple, and selective, and it is suitable for routine analysis of hydrophilic whitening agents in commercial cosmetics.