In vitro and in vivo microdialysis calibration using retrodialysis for the study of the cerebrospinal distribution of bupivacaine
Introduction
Local anesthetics are used via epidural routes for major pain control and for regional anesthesia. To reach the target neural tissue of the spinal cord, the drug has to cross the dura and arachnoid mater, to distribute in the cerebrospinal fluid (CSF), and then to cross the pia mater [1]. Because of the difficulty in serially sampling CSF, in vivo data on distribution transfer through the spinal meninges are scarce. In order to improve understanding of the transmeningal passage mechanisms, it is necessary to determine drug concentrations in CSF after epidural administration.
Microdialysis has been applied in pharmacological studies of endogenous compounds like brain neurotransmitters [2]. Currently, there is a growing interest in using this sampling technique for pharmacokinetic and biopharmaceutic studies of exogenous compounds 3, 4, 5. In the current study, microdialysis was chosen as the sampling technique to obtain samples without disturbing CSF flux. The main difficulty with microdialysis analysis is to obtain a reliable in vivo calibration allowing accurate in vivo concentrations to be established.
In the current study, retrodialysis (RT) was applied to calibrate microdialysis probes. This calibration technique is based on the principle that the relative loss (RL) of a carefully chosen internal standard, added to the perfusate, is related to the relative recovery (RR) of the substance of interest [6]. Bupivacaine and ropivacaine, which differ structurally by only one methyl group, were respectively utilized as substance of interest and as internal standard to study the disposition of bupivacaine after bolus epidural administration. The calibration was simultaneously validated through the use of the zero net flux method where recovery in vivo was estimated from dialysate concentrations in a wide range of concentrations, while maintaining the extracellular concentration at steady state [7]. Due to the toxicity of bupivacaine precluding the obtention of steady state concentration, the calibration zero net flux method was only performed in vitro. The K-factor, defined as the ratio between RL of internal standard and RL of substance of interest, was used to correct the dialysate concentrations and to determine the bupivacaine CSF concentrations. The extracellular concentration of bupivacaine was then calculated according to: C=Cdialysate×(K/RL) [8].
Section snippets
Chemicals
Bupivacaine (substance of interest), ropivacaine (internal standard of microdialysis) and etidocaine (external standard of HPLC) were supplied by Astra (Astra Pain Control, Sweden).
The composition of Ringer's solution was NaCl 8.6 g l−1, KCl 0.33 g l−1, CaCl2, 2 H2O 0.3 g l−1.
All other reagents were of analytical grade.
Microdialysis in vitro and in vivo
Microdialysis sampling was performed using a CMA/102 microinjection pump coupled to a microdialysis probe CMA/20 (membrane length 10 mm, 0.5 mm outer diameter, molecular weight
Results and discussion
The chromatograms in Fig. 1 displayed the selectivity (selectivity factor>1.2) of the separation of bupivacaine, ropivacaine and etidocaine in CSF dialysate and in plasma samples. The limits of quantification for bupivacaine in CSF dialysate and in plasma samples were 1 μg ml−1 and 2 ng ml−1, respectively.
In a first step, the influence of flow rate on RR of bupivacaine and on RL ropivacaine was studied. As shown in Fig. 2, the increase in flow rate (from 0.8 to 2 μl min−1) led to a twofold
Conclusion
The experimental results indicated that ropivacaine can be used as an internal standard for studying CSF bupivacaine kinetics. The microdialysis sampling technique allowed determination of CSF concentration of bupivacaine after epidural administration. This preliminary work has shown that microdialysis should be a promising tool to gain further insight into epidural disposition of drugs and to improve understanding of transmeningal diffusion mechanisms.
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