Table 1.

Approaches to In Vivo Calibration of Microdialysis Probes

method	description	considerations	reference
extrapolation to zero flow rate	An estimate of analyte concentration surrounding the probe is obtained by extrapolating to zero flow rate, where analyte concentration in the tissue and in the probe would presumably be in equilibrium.	Requires determining the recovery of the probe at several flow rates with the analyte at a steady-state concentration in the matrix throughout the determinations.	35
use of very low flow rate	This extension of the aforementioned method carries out microdialysis sampling at a flow rate (ca. 50 nL/min) where the probe recovery is >95% so that assuming 100% extraction efficiency introduces negligible error.	Practical considerations include long sampling intervals to collect sufficient volume for analysis and problems manipulating small-volume samples such as evaporation.	26
zero net flux	With the analyte at a steady-state concentration in the tissue, the probe is perfused with a succession of solutions containing known concentrations of the analyte. A plot of the change in perfusate concentration versus the initial perfusate concentration should be linear with the slope being the inverse of the probe recovery and the y-intercept being the point of zero net flux; that is, the concentration of the analyte in the tissue.	Tissue concentration of the analyte must remain at steady state, whereas the EE of the probe is determined for several different initial analyte concentrations in the perfusate.	31, 32
retrodialysis	Uses the in vivo delivery of an internal standard (usually an analog or mimic of the analyte) in the perfusate as the basis for probe calibration. The method assumes that the in vivo recovery of the analyte has a constant known relationship to the in vivo delivery of internal standard. In some cases that relationship is determined in vitro and assumed to hold in vivo.	The advantage of this method is convenience, especially in terms of time. The experimentalist should consider that, in a biologically active matrix, delivery of an internal standard that binds to receptors or is metabolized in the same way as the analyte will introduce unwanted perturbations in the tissue area being sampled.	27-30, 36
in vivo delivery of the analyte	A variation of retrodialysis that overcomes the uncertainty introduced when an analog is used as the internal standard. Essentially, the in vivo delivery of the analyte to the target tissue is determined before the actual experiment. If possible, the delivery is determined again after completion of the experiment to confirm that the probe behavior has not changed.	The method is simple, convenient, and has a minimal time requirement. This method assumes that the extraction efficiency of the probe in the tissue of interest is constant regardless of the direction of the concentration gradient of the analyte. The assumption should be validated for analytes and tissues of interest.	15, 37