Perfusion flow rate, analyte recovery

Perfusion flow-rates ranges between 0.1 and 5 til/min. The tendency is to use lower flow-rates as this may increase recovery, provided that an analytical technique is available to deal with the smaller sized samples. 

A number of variables, such as the perfusion flow rate, the membrane surface area and geometry, the MWCO of the membrane, the diffusion characteristics of the collected analyte, the composition of the perfusion medium, and the temperature, influence recovery parameters. When the microdialysis is carried out in vivo, the recovery can also be affected by some tissue properties, including tissue tortuosity, the extracellular space volume, the tissue blood fluid and the tissue metabolism of the substance. 

In microdialysis, the recovery of analyte from the sample depends on a number of factors, including the chemistry of the analyte, temperature, perfusion rate, membrane surface area, membrane characteristics, and the nature of the sample (including its fluid volume percentage and whether it is in motion). MiCTodialysis is typically done at low perfusion flow rates (0.5 to 2 /d/min). As flow rate increases, relative recovery decreases, but absolute recovery increases. Relative recoveries from small membrane probes are in the 1-20% range. SubstantiaUy higher recoveries in the 50-80% range can be obtained with longer loop-type probes. 

This is a method enabling the estimation of the in vivo recovery of an analyte of interest introduced by Loimroth in 1987. The procedure involves adding the anal3 te of interest into the perfusion solution at different concentrations at a fixed perfusate flow rate and measuring the difference between perfusate and dialysate analyte concentration. The recovery represented by the left term of Eq. 3 can be assumed to be an unknown value P since the perfusate flow rate and resistance are left unchanged. When it is rearranged... 

As the relative recovery will never reach 100%, the dialysate concentrations are only a fraction of the true concentration of the analyte of interest in the surrounding fluid. Before using a microdialysis probe for continuous sampling or monitoring, the trae concentration of the analyte of interest in the surrounding environment and the recoveries at certain perfusate flow rates have to be obtained. There are many different methods for calibration, which are discussed in the following [1]. 

One of the most important issues to consider when making microdialysis measurements is the recovery of the analyte from the dialysis probe and the numerous factors that can influence recovery. The factor that the experimenter has the most control over is perfusion flow rate, which can regulate percent recovery, sample volume, and throughput capabilities related to the temporal resolution of the method. Employing a low perfusion flow rate (<1 pl/min) results in enhanced relative recovery but a concurrent decrease in absolute recovery. Figure 20.3(b). Relative recovery is the concentration of the analyte in the dialysate sample divided by the concentration in the sample media [6]. Absolute recovery is defined as the mass of analyte transport... 

At typical flow rates, the concentration in the dialysate, Cout, is less than the actual concentration in the extracellular fluid, Cext (23). The ratio of Cout/Cext is defined as relative recovery, R, and must be considered for probe calibration and sampling optimization. In vitro, R is easily calculated because the dialysate and the extracellular fluid are homogenous therefore, probe calibration is easily obtained. However, in in vivo studies, calculation of R is difficult because of the active removal of neurotransmitters by uptake and tortuosity. Movement of analytes is impeded by tissue that surrounds the probe, and this movement cannot be easily accounted for with in vitro calibrations. Therefore, the most common method to determine concentrations in vivo is the zero-net flux method, in which known analyte concentrations are added to the perfusate (Cin), and then the analyte concentration is measured at the probe outlet (Cout)- The difference between analyte concentration at the inlet and outlet is used to establish the actual analyte concentration in the tissue, and the relative recovery rate can be calculated. This calibration method can be used to estimate basal levels of neurotransmitters. For example, the zero-net flux method has been used to determine that basal concentrations of dopamine are approximately 1-3.5 nM (24, 25). Although basal level concentrations... 

When experiments are performed in vivo, previously independent components of the model, the sampling, and the analytical method become interdependent. The conditions that were optimal for each independent component must now be considered in relationship to the other components. For example, if the method requires a large sample volume the flow rate of the perfusate must be increased. If the flow rate is increased, the extraction efficiency will be decreased. The result is that the analytical method will require better limits of detection. On the other hand, if the flow rate is decreased to increase the extraction efficiency, the temporal resolution will be compromised [5]. The increased recovery can also deplete compounds of low molecular mass in the tissue near the probe, thereby perturbing the experimental conditions. 

In ultrafiltration, analyte molecules are basically carried along with the flow of water and electrolytes. The factors determining recovery in ultrafiltration are membrane characteristics, temperatme, and chemistry of the analyte. Unlike microdialysis, recovery is not dependent on flow rate, membrane surface area, or probe size. Recovery tends to be higher than for dialysis, since there is no perfusion medium to dilute the collected analyte. Ultrafiltration recovery rates are typically in the 90-100% range. This high recovery rate simplifies determination of in vivo analyte concentrations. Table I illustrates some in vitro recoveries obtainable with ultrafiltration probes. 

Concentric Microdialysis Probe When a microdialysis probe with perfusion fluid flowing at a flow rate of is placed in a bath of analyte of concentration Cr, the microdialysis recovery, also known as dialysate extraction fraction, is described by a balance of the diffusion of the analyte across the microdialysis membrane into the perfusion fluid with the convective flux due to the perfusion flow ... 

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