Microdialysis Instrumentation

The microdialysis probe is the heart of the method, as a chromatographic column is the heart of the HPLC instrument. Rigid CMA probes, Models 10, 11, and 12, are used for stereotaxic implantations into the brain, where the probe can be fixed (cemented) to the skull. A flexible probe design (CMA 20) allows the placement of such a catheter into the moving tissues (muscle) or peripheral organs for studies in freely moving animals. The technical difficulties of microdialysis experiments impose requirements for precise liquid delivery, minimized dead volumes, and the capability of handling small sample volumes. 

When not in use, microdialysis probes should be stored wet and protected from mechanical damage. Also, testing the probes by in vitro recovery is easily accomplished if the probes are fixed in an appropriate stand. Fractions can be collected manually, but it is more convenient to use fraction collectors capable of collecting fractions as low as 1 fiL. 

Several commercial constructions are available for triple (CMA 140, Fig. 6.3B) or dual (CMA 142) probe collecting and with refrigeration (CMA 170) for collecting easily degradable compounds. An alternative is direct coupling of the microdialysis probe s outlet to the high pressure injection value (CMA 160) of an HPLC system (Johnson and Justice, 1983). This enables on-line 

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Besides the basic apparatus for microdialysis perfusions, fraction collection, and HPLC analysis, several additional instruments and devices are needed, depending on where the microdialysis probe is to be implanted. The most complicated instrumental setup is probably that required for brain dialysis. A stereotaxtic instrument and a stereomicroscope are necessary for precise positioning of microdialysis cannulae into various brain structures. Inhalation anesthesia is preferable and more convenient than injections. However, this type of anesthesia calls for additional equipment, such as air lines, valves, and mixing chamber for halothane or other anesthetic gases, as well as good ventilation of the operation theater. 

The selectivity characteristic of biosensors allows one to avoid separation procedures after the microdialysis collection, thus greatly improving the overall analysis, in terms of both instrumentation and the time required. Moreover, since microdialysis and chromatography entail dilutions, the avoidance of chromatographic separation also allows one to obtain a more concentrated sample which can be malysed more easily and accurately. 

By exploiting the microdialysis technique, the miniaturization of the whole instrumentation was achieved, also through the use of a peristaltic pump which turned out to be the only type of pump suitable for coupling to an electrochemical cell. The high sensitiviiy of the latter, in fact, revealed that all the syringe pumps tested were not really pulse-free . 

The actual glucose sensor (a platinum electrode covered by three membranes ceUulose acetate, nylon net with covalently Unked GOD, and a polycarbonate protective membrane) is located in a miniaturized waU-jet cell. The sensor exhibits excellent performance, with a linear range extending up to 27 mM, thanks to the microdialysis dilution effect which was estimated to be 1 10 for the probe length used and for the flow rate set by the instrumentation. Long-term stability tests revealed that the biosensor stiU maintains its initial activity after incubations of 4 weeks at 45°C, 11 weeks at 37°C, and 32 weeks at room temperature (see Table 12.2). From these results, a shelf life of more than 2 years at 2-8°C can be extrapolated [119]. 

All the current efforts directed at miniaturization of the analytical techniques are addressing the problem of interfacing the miniaturized probes used in microdialysis with suitable instrumentation, so that smaller sample volumes can be subjected to appropriate analyses, thus increasing the time resolution of the overall systems. 

A relatively new and powerful tool in metabolite identification is the (J-LIT instrument. In this triple-quadrapole instmment, the third quadrupole can be apphed as a scanning quadmpole, but also as a linear ion trap (Ch. 2.4.2). Potential and additional features of a Q-LIT in metabolite identification have been discussed by various groups [44-45]. The advantages of triple-quadrupole MS-MS spectra at ion-trap sensitivity as well as the enhanced sensitivity and speed in DDA experiments with the (J-LIT was demonstrated for the collagenase inhibitor trocade [44]. Various enhanced scan functions of the Q-LlT were applied in the identification of 6-aminobutylphthalide metabolites in rat brains, using microdialysis and LC-MS-MS [45]. Simultaneous quantification of a parent compound and screening for its... 

Zhou and Johnston [55] reported protein characterization by capillary isoelectric focussing (CIEF) on-hne coupled to RPLC-MS. Direct coupling of CIEF to ESl-MS is limited by interferences by the ampholytes. Inserting RPLC in-between can help removing these interferences. CIEF is performed in combination with a microdialysis membrane-based cathodic cell to remove the ampholyte and to collect protein fractions by stop-and-go CIEF prior to transfer to a 5><0.3-pm-ID C,8 trapping colunm and RPLC separation on a 50><0.3-pm-ID C4 column. The separation is performed using an acetonitrile-water gradient (0.1% acetic acid). ESI-MS is performed on a quadrupole-TOF hybrid (Q-TOF) instrument. 

The subsequent chapters describe the role of mass spectrometry in the drug discovery process in greater detail. Initial chapters deal more with instrumentation and sample preparation as they relate to mass spectrometry. The remaining chapters describe the utility of mass spectrometry in different areas of the drug discovery process, such as combinatorial chemistry, drug transport, drug metabolism, pharmacokinetics, and microdialysis. 

Microdialysis coupled to mass spectrometry is a powerful technique for on-and off-line analysis, providing information on pharmacokinetics, drug transport, and metabolite formation. With the widespread availability of LC/ESI/MS instruments there has been a shift toward liquid chromatography and away from gas chromatography and flow injection. Electrospray is the ionization method of choice for most applications and tandem mass spectrometry has grown in popularity. Thermospray and cfFAB applications have been used with microdialysis, but are older, obsolete techniques. 

In our work, an improved CIEF-RPLC-MS system has been developed for protein separation and characterization." A microdialysis membrane separates the cathodic cell from the capillary column. During the focusing step, catholyte is able to traverse the membrane but protein molecules cannot. After focusing, proteins are hydrodynamically pushed past the membrane to a microselection valve that collects and transfers fractions to the RPLC system for further separation. This configuration permits a linear pH gradient to be maintained in the separation capillary with no voltage applied to the microselsection valve. The cathodic cell and microselection valve can easily and safely be retrofitted into commercial instruments. 

The Kennedy lab has been at the forefront of using rapid capillary electrophoresis to measure neurochemical changes. Optically gated injection of OPA-derivatized amino acids has been achieved in less than 2 s. However, the high salt concentrations of physiological samples, such as cerebral spinal fluid, can reduce EOF, and make separations slower. Still, 10 s temporal resolution for online monitoring of directly sampled and microdialysis samples were obtained. An instrument has been developed in the Kennedy lab for online analysis of microdialysis samples after precolumn... 

Rapid capillary electrophoresis measurements in general require small i.d., short length capillaries with fast injection and sensitive detection techniques. One example of a successful implementation of all these principles is a rapid capillary electrophoresis instrument developed by Bowser and Kennedy to analyze online microdialysis samples for in vivo monitoring (Figure 15.4). This instrument used small, 10 p,m i.d. capillaries that were 10 cm long. Applied voltages were 20,000 V, or 2000 V cm-1. 

While the vast majority of microdialysis sampling to date has been achieved in conjunction with conventional systems, microdialysis has also been coupled online with biosensors and, more recently, to microchip-based separation devices. These devices can be fabricated using low cost materials and are amenable to mass production [11]. The smaller dimensions of microchip systems minimize sample and reagent volume requirements and allow placement of the analysis system closer to the aqueous sample or animal being sampled. Due to the planar nature of microchip devices, additional sample handling procedures such as preconcentration, mixing, extraction, or derivatization can also be carried out on the same platform [12-15]. Considerations for coupling microdialysis to microchip instrumentation systems will be discussed in Section 48.5. 

Weiss, D. J., et al.. Applications of microdialysis/mass spectrometry to drug discovery, in The Mass Spectrometer in Drug Discovery A Practical Guide to Instrumentation, Experimental Strategies and Applications, Rossi, D. T. and Sinz, M. W. eds.. Chapter 12, Marcel Dekker, New York, 2001, pp. 377-397. 

In order to conduct microdialysis experiments, several other components are required. Syringe pumps are often used to control the perfusate flow rate. The pump has to be able to deliver flow rates precisely in the microliter per minute range. Tubing is needed to connect between the probe and the pump which drives the perfusion flow and, in some cases, between the probe and a sample collector as well. The total dead volume of tubing should also be maintained as small as possible to have better time resolution. The perfusion fluid is a medium resembling the composition of extracellular fluid with minimal or zero concentration of the molecules of interest. Dial-ysate exiting from the outlet of the microdialysis probe is usually collected in a vial for later analysis. It is also possible to coimect the outlet directly to an analysis instrument without using a collector, which is usually preferred, if possible, for its convenience and usually faster analysis results. 

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