Microdialysis Probe Recovery

A major concern in using microdialysis as a tool for the determination of unbound drug concentrations in the in vivo as well as in vitro settings is the

Figure 1 Custom-designed microdialysis probe for posterior or anterior chamber placement into the rabbit eye.

assessment of recovery. Recovery may be defined as the proportion of solute extracted from the medium surrounding the probe (55). Recovery is dependent on the following parameters: dialysis membrane length, perfusion flow rate, diffusion rate of the solute through the compartment (the usual rate-limiting step in the process), and membrane properties (47). Recovery can

Figure 2 Rabbit eye with a microdialysis probe in the anterior chamber (magnification ~ 4x).

also be time- and temperature-dependent. Typical recovery values observed in the literature range from a low of ~ 10 up to 100%. By maximizing the dialysis membrane length, significant increases in recovery can be realized. Decreases in perfusion flow rate also increase the relative recovery (although they also decrease the available sample volume). The recovery of solute can be difficult to ascertain. Ideally, probe perfusate composition should closely match the environment of the medium in which it is placed. The probe also can create a microenvironment near the probe surface, which may be different than the medium more distant from the membrane (47). Several different types of recoveries are evaluated in microdialysis studies; these include relative recovery (or concentration recovery) and absolute recovery (mass recovery). Relative recovery is the fraction of solute obtained in the dialysate relative to the actual concentration in the medium in which the probe is placed. Absolute recovery is the total amount of solute collected over a specified time period. A number of approaches are used to estimate the recovery of a solute by the microdialysis probe, including water recovery, no-net-flux (or difference method), perfusion rate, and relative loss (55,56).

The water-recovery method is of limited use for in vivo settings because drug diffusion characteristics are usually different in artificial aqueous physiological buffers or solutions than in the dynamic in vivo environment. Where a solid in vitro-to-in vivo correlation is established, the water-recovery method has utility. For this method, the microdialysis probe is placed in a reservoir (usually stirred) containing a known concentration of solute. The perfusion medium, an aqueous solution of similar composition to the medium in which it is placed but without solute, is delivered through the probe at a constant rate. Dialysate is collected and the amount of solute determined via appropriate analytical methods. The ratio of the dialysate concentration of the known concentration of the medium in which the probe is placed is the relative recovery. This method is known to underestimate the concentration in the medium sampled (55).

The point of no-net-flux or difference method is used for in vitro and in vivo studies. By varying the concentration of solute in the perfusion medium and fixing the solute concentration in the surrounding medium, the dialysate solute concentration is assessed. The direction of the concentration gradient of solute depends on whether the concentration in the perfusion medium is higher or lower than the concentration in the surrounding medium (55). A plot of the perfusate solute concentration versus the difference in concentration between perfusate and dialysate is constructed; the x-intercept identifies the concentration at which no net flux of solute occurs (55). In theory, this value will be the concentration of the surrounding medium. This method is very time-consuming.

The perfusion rate method is based on the principle that recovery is dependent on the rate of perfusate transit through the probe. With an increase in perfusate transit, there is a corresponding decrease in relative recovery. Conversely, the lower the perfusion rate, the higher the relative recovery. For the perfusion rate method, the initial surrounding medium contains no solute and the probe is perfused with a fixed concentration of solute (55). This method is the most exhaustive in that several different surrounding media concentrations must be assessed separately, each at different perfusion rates (in vitro). A typical experiment might evaluate four different concentrations for the medium at three different perfusion rates each over an extended period. Frequently regression models are employed to provide the best estimates of probe performance. Typically for in vivo determinations, the lowest possible perfusion rate (e.g., 0.1 mL/min) is selected. This maximizes the relative recovery to nearly 100% in some cases. At such low flow rates, longer collection times are required to obtain sufficient sample for further analysis.

The relative loss method is similar to the water-recovery method, but is operated in reverse. Rather than placing a known concentration of solute in the medium surrounding the probe, the solute concentration of the perfusate is fixed. The surrounding medium, which in most situations contains small quantities of solute (i.e., sink conditions), then provides a negative concentration gradient of the solute. The net loss of solute reflects the relative loss of solute to medium. This method, which is based on the premise that recovery is the same in both directions across the membrane (47), is by far the simplest to use in the in vivo setting and provides a reliable estimate of recovery. Relative loss is the ratio of the difference in perfusate to dialy-sate solute concentrations to the perfusate concentration (56). This method is often referred to as retrodialysis recovery. Under nonsink conditions of the surrounding medium, an internal standard is sometimes employed.

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