Craig E. Lunte and Dennis 0. Scott Department of Chemistry University of Kansas Lawrence, KS 66045
energy metabolism, and fermentation are among the fields in which monitoring chemical changes in the environment of living cells is crucial. The living systems of interest may reasonably be described as islands
Peter T. Kissinger Department of Chemistry Purdue University West Lafayette, IN 47907
A great deal of current research is focused on the behavior of low molecular weight substances in living syst e m s . Ph a r m ac ol ogy , molecular toxicology, neuroscience, nutrition, 0003-2700/91/0363-773A/$02.50/0 0 1991 American Chemical Society
full of life in a dead chemical sea, and the transport of substances to and from the islands can tell much about the activities therein. Pipes running through the sea frequently deliver and remove the chemical inventory.
Changes take place with time constants that range from less than a millisecond to more than a year. Th6 challenges for analytical chemists in measuring these changes are substantial. Microdialysis sampling is a power ful new technique for the study of in vivo pharmacokinetics and the metabolism of drugs. Interest in this area is burgeoning, as demonstrated by the large attendance at the second International Symposium on Microdialysis Sampling, which was held i n Indianapolis, IN, May 15-17, 1991. There were more than 80 presentations describing a variety of
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1,1991
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REPORT compounds a t a number of in vivo sampling sites. The abstracts from this meeting give an excellent overview of the state of the art ( I ) , and a monograph covering t h e neuro science applications is now in press
(2). In the microdialysis sampling approach, a hydrophilic pipe (a dialysis capillary) through which low molecu lar weight substances can easily diffuse is positioned in the living system. A fluid, the perfusion medium, whose macroscopic composition closely resembles that of the sea (the so-called extracellular space) is pumped through the pipe. Ideally, the ionic strength and pH of the perfusion medium will match those of the extracellular space. A precisely controlled flow rate allows chemicals to be predictably introduced into or removed from the extracellular space by establishment of a diffusional steady state across the membrane wall. Microdialysis sampling provides several advantages over conventional techniques, including a means of continuous sampling with no fluid loss. Samples can often be analyzed by LC without cleanup, and drugs can be separated from enzymes that might catalyze their degradation. The microdialysis process competes with or supplements other experimental protocols such as cell culturing, tissue slicing, tissue homogenizing, and conventional Sam pling of biological fluids (e.g., blood, urine, and spinal, lymphatic, sinoidal, and ocular fluids). The fact that no fluid is removed from or introduced into the system during the process is especially advantageous. In addition, it is possible to monitor multiple analytes by coupling other analytical techniques to the microdialysis system. Although LC is used most often, MS, immunoassay, luminometry, and electrochemistry are other possibilities. In these systems, the dialysis probe functions as a generic “biosensor” that can rapidly be employed for a variety of analytes; there is no need to engineer a new sensor for each one. Microdialysis can be accomplished using a variety of probe geometries. The most common design is the concentric tube illustrated in Figure 1. The body of the probe is usually constructed of stainless steel needle tubing, fused silica, or a combination of the two. The most common membrane materials are polycarbonate, polyacrylonitrile, and regenerated cellulose. Although the microdialysis concept 774 A
originated in the early 1970s, only recently has it had widespread application because of the commercial availability of the concentric probe design (3)and the ideal compatibility of microdialysis with the well-established liquid chromatographyelectrochemistry (LC-EC) techniques for neurotransmitter determinations (4). The neurochemical applications of microdialysis followed by LC - EC include detection of amino acids, glu-
cose, acetylcholinekholine, and biogenic amines. LC with UV absorbance detection is routinely used for lactate, pyruvate, purines, and some drugs. These experiments often are carried out with conscious animals. Figure 2 illustrates a fully automated system for neurochemical studies in a rat. A guide cannula is implanted under anesthesia, and the probe is introduced after the animal has re-
I
.
L
.
Figure 1. Concentric microdialysis probe.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, 1991
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REPORT gained consciousness. As shown in Figure 3, a chromatogram of purines from rat brain dialysate is comparable to that of a standard purine solution. Collected volumes for microdialysis are typically only a few microliters. With such volume-limited samples, small - diameter LC columns are clearly advantageous for minimizing the dilution of each analyte prior to detection (5,6). A few laboratories also perform microdialysis from the human brain. We must emphasize, however, that this work is highly experimental and it is not yet clear whether the technique will have routine diagnostic value for clinical situations. The neurochemical applications of microdialysis have been thoroughly reviewed (7-10). Applications to tissues other than the brain have been few, but they include the liver, adipose tissue, the eyes, and muscle. There are even a few applications to vegetable material, including our favorite, the banana. We believe that applications to fermentation systems, cell culture studies, and drug metabolism research show special promise.
Drug metabolism and pharmacokinetic investigations In vivo microdialysis sampling offers several advantages over conventional methods of studying xenobiotic metab olism and pharmacokinetics. Xenobiotic metabolism is most often studied using tissue preparations, which may not reflect actual in vivo metabolism. Determination of serum, urine, or fecal metabolites provides insight into the fmal products of metabolism but provides little or no information about the composition or site of intermediate metabolic processes. Microdialysis sampling techniques
are very useful for such studies because the integrity of the organism is not perturbed-all of the possible interactions between organs are kept in place. Actual physiological condi tions are maintained without the need to add cofactors for the operation of the system under study. Perturbations attributable to the homogenization of cells are eliminated. Because enzymes and cofactors are present in different compartments within the cell, they may or may not have access to a xenobiotic compound. Disruption of the compartmentalization by homogenization can have profound effects on the observed metabolism. The pharmacokinetics of a drug are typically determined by administering a known dose and withdrawing blood samples at timed intervals. These samples are then analyzed to derive a concentration-time curve. This approach can present problems with regard to both the determination of the drug concentration and the pharmacokinetic parameters derived from these data. Removal of blood causes a decrease in total blood volume, which may lead to alterations in the observed distribution and elimination of the drug. However, minimizing blood loss by decreasing the sampling frequency res u l t s i n lower resolution i n t h e concentration-time curve, and subtle pharmacokinetic changes may not be observed if sufficient resolution is not maintained. Because they contain protein that must be removed prior to LC, whole blood samples are also difficult to analyze. Procedures for protein removal can lead to the release of proteinbound drug, depending on the lability of the drug-protein interaction.
Pharmacokinetic parameters thus derived will correspond to the total drug concentration rather than to the more meaningful free drug concentration. A microdialysis probe can be used not only for sampling but also for administration of a compound. By using the probe for both dosing and Sampling, a specific area of an organ can be dosed and the metabolism within that area studied. Alternatively, a n animal can be given a systemic dose of a compound and microdialysis probes can be placed concurrently in the blood and in various tissues to determine the distribution of t h e compound and its metabolites. Acetaminophen pharmacokinetics. To demonstrate the utility of microdialysis for pharmacokinetic investigations, the kinetics of acetaminophen (APAP)were investigated in anesthetized rats (11, 12).The dialy-
Figure 3. Chromatogram of purines using a 254-mm biophase octyl column: 1, hypoxanthine; 2, inosine; 3, guanosine; 4, adenine; 5, adenosine. Figure 2. Automated microdialysis/LC system for neurochemical studies in a conscious rat. 776 A
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1,1991
(a) 1-pM purine standards (IO-pL injection), 0.02 AUFS and (b) rat brain dialysate (5-pL injection), 0.005 AUFS.
sis samples were analyzed by LC with W absorbance detection. This allowed the detection of APAP as well as its major metabolites, APAPsulfate and APAP-glucuronide. Because no fluid is removed from a n animal during a microdialysis exper iment, it is possible to sample continuously without disturbing the pharmacokinetics. Well-defined kinetic curves were constructed using a single experimental animal, and experiments on multiple animals were highly reproducible. Following an i.p. injection (100 mg/ kg dose), APAP rapidly distributes into the blood, reaching a peak concentration after approximately 1 h. The resulting concentration-time curve can be modeled using an open single -compartment model with first -order absorption and elimination according to Equation 1. C(t) = A s a f - Becot
ble II. Peak co tabolites following i.p
(1)
The half-lives of absorption and elimination were determined by fit ting a line described by Equation 1 to the data and determining a and 13, which correspond to the rate constants of elimination and absorption, respectively. The calculated half-life of absorption was 31.3 k 3.4 min and that of elimination was 36.0 k 2.0 min (n = 4). The metabolites of APAP appear a few minutes after APAP and follow its rise and fall (Figure 4). Validation of microdialysis sampling was performed by simultaneously collecting blood samples through an indwelling cannula implanted into the femoral vein. The pharmacokinetic curves obtained by both microdialysis and blood sampling confirmed that the results were equivalent when binding to plasma proteins was taken into account (Figure 5).
33.1 i2.7 2
APAP pharmacokinetics was also studied as a function of dose. The half-lives of absorption and elimination decreased with dose, as did the peak concentrations and the times required to reach the peak concentrations (Tables I and 11). Studies of the pharmacokinetics of
APAP were repeated using a n i.v. dose of 100 mgkg. After the i.v. injection, a peak concentration of APAP was immediately reached in the blood (Figure 6). Because the i.v. injection immediately introduces the entire dose into the blood, there is no absorption; therefore, the elimination of APAP is described by Equation 2, which has only one exponential term.
C(t) = A c a t
(2)
The half-life of elimination of APAP following a n i.v. dose was found to be 21.2 min, which is signif-
f/
APAP-sulfate 4PAP-glucuronide
I
Figure 4. Time course of APAP and its metabolites determined using microdialysis sampling.
Figure 5. Comparison of unbound APAP concentrations in blood determined by microdialysis and whole-blood sampling. Results for a typical experiment with a single animal. Bars represent microdialysis samples: diamonds represent whole-blood samples.
Figure 6. Time course of blood APAP concentration following an i.v. dose.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1,1991
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REPORT icantly shorter than the half-life determined using the same dose given by i.p. injection. The difference can be attributed to the fact that there is no absorption phase after a n i.v. dose. An absorption phase can lead to a longer observed half-life of elimination unless the half-life of absorption is much faster than the half-life of elimination. Therefore, i.v. dosing gives a more accurate estimation of the half-life of elimination than does i.p. dosing. This example, albeit for a well-established drug, demonstrates the potential usefulness of microdialysis sampling for pharmacokinetic studies. Acetylsalicylic acid pharmacokinetics. Acetylsalicylic acid (ASA) presents a particularly difficult problem for pharmacokinetic determinations because it is rapidly metabolized to salicylic acid (SA) in the blood. Simply removing blood samples from a n animal does not stop its metabolism, and, to minimize the continued metabolism of ASA, precautions must be taken to ensure that the enzymes responsible for the degradation are inactivated. These precautions are operator intensive and less than completely effective, and they complicate the procedure. Microdialysis sampling eliminates these problems. One of its major advantages over serial blood collections for metabolic and pharmacokinetic studies is that, because the enzymes responsible for metabolism are excluded from the sample by the dialysis membrane, no further metabo; . lism can occur. The hydrolysis of ASA in blood was initially investigated in vitro. Both freshly collected rat blood and water were spiked with ASA. The blood was sampled by microdialysis and by removing 100-pL aliquots and precipi -
tating protein with acetonitrile. The water sample was analyzed over the same time interval as the blood sample. Whereas the ASA in blood had completely hydrolyzed i n 40 min, that in the water sample was stable for over an hour (Figure 7). The hydrolysis of ASA could be followed equally well by microdialysis sampling and by removing aliquots of blood. By making repeated injections of the initial microdialysis sample over a 90-min period, it was shown
J,
that once the microdialysis sample was collected no further hydrolysis occurred. These results indicate that microdialysis sampling of ASA for pharmacokinetic studies eliminates the complication of continued enzyme hydrolysis i n a traditional blood sample. The pharmacokinetics of ASA in rats was studied following i.v. administration at doses of 100 m g k g and 200 mgkg (13). Figure 8 shows chromatograms for a blank, prior to dosing, and a sample of perfusate approximately 5 min after dosing at 100 mgkg. There are no interferences in the blank for either ASA or SA. Blood samples were also collect ed and analyzed for ASA and SA. Figure 9 is a concentration-time curve of ASA, corrected for protein binding, generated after a 100-mgkg dose using both blood sampling and microdialysis. A curve described by Equation 2 was fit to the data, and the half-life of elimination was determined. Although the half-lives of elimination are the same for blood sampling and microdialysis s a m pling, the actual concentrations measured in each sample are considerably different because of the binding of ASA to blood proteins. The extent of binding was determined in vitro using ultrafiltration. After correction for protein binding, ASA concentrations i n the blood samples were slightly lower t h a n those in the microdialysis samples. The lower concentration in blood may be a result of the continued metabolism in the blood sample after collection. For the 100-mgkg dose, the half-life of elimination was deter mined to be 6.6 f 2.3 min using microdialysis sampling and 7.8 f 1.0
Figure 8. Chromatograms of blood microdialysis samples: (a) blank, (b) 5 min after a lOO-mg/kg i.v. dose of ASA.
Figure 7. hydrolysis of ASA. (Adapted with permission from Reference 13.)
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Chromatographicconditions: mobile phase, 0.05 M ammonium phosphate, pH 2.5, with 20% (v/v) acetonitrile at a flow rate of 0.5 mL/min; two coupled 5-pm ODS (2.1 mm x 10 cm) columns; injection volume, 10 pL. (Adapted with permission from Reference 13.)
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1,1991
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Figure 9. Concentration-time curve for ASA. Bars represent microdialysissamples; circles represent blood samples. (Adapted with permission from Reference 13.)
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REPOR7 min using blood sampling. For the 200-mgkg dose, the half-life of elimination was determined to be 7.8 & 1.0 min by microdialysis and 11.1k 3.3 min by blood sampling. References (1) Abstracts of the Second International Symposium on Microdialysis, May 1517, 1991, Curr. Sep. 1991, 10, 66-118. (2)Microdialysis in the Neurosciences; Robinson, T. E.; Justice, J. B., Eds.; Elsevier: Amsterdam, in press. (3) Ungerstedt, C. U., U.S. Patent 4 694 832, 1987. (4) Kissinger, P. T. J. Chromatogr. Biomed. Appl. 1989, 488, 31-52. ( 5 ) Huang, T; Shoup, R. E.; Kissinger, P. T. Curr. Sep. 1990,9, 139-43. (6) Huang, T; Shoup, R. E.; Kissinger, P. T. Curr. Sep. 1990, 10, 16-18. (7) Westerink, B.H.C.; Damsma, G.; Rollema, H.; DeVries, J. B.; Horn, A. S. Life Sci. 1987, 41, 1763-75. (8) Kendrick, K. M. Methods in Enzymol. 1988, 168, 182-205. (9) Benveniste, H.; Huttemeier, P. C. Prog. Neurobiol. 1990,35, 195-215. (10) Tossman, U. LC-GC 1991, 9(1), 4652. (11) Scott, D. 0.;Sorensen, L. R.; Lunte, C. E. J. Chromatogr. 1990, 506, 41. (12) Scott, D. 0.;Steele, K. L.; Sorensen, L. R.; Lunte, C. E. J. Pharm. Res. 1991,8, 389. (13) Steele, K. L.; Scott, D. 0.;Lunte, C. E. Anal. Chim. Acta 1991,246, 181-86.
Craig E. Lunte (center) received his B.S. degree in chemistryfrom the Universityof Missouri-Rolla in 1979 and his Ph.D. fiom Purdue University in 1984. He is currently an assistant professor at the Universityof Kansas. His research interests include elucidating the relationship between electrochemical and biological activity in organic compounds, the development of analytical methods to study biochemical systems, and the application and theory of microdialysis pe&sion. Dennis 0. Scott (right) received his B.S. degree from the University of Arkansas at Fayetteville in 198% He is currently a Ph.D. candidate at the University of Kansas. His research interests include the development of bioanalytical methods, particularly for in vivo studies of drug metabolism, pharmacokinetics, and biologically important endogenous compounds. Peter T.Kissinger (left) received his B.S. degree from Union College (Schenectady, IO7 in 1966, and his Ph.D. fiom the Universityof North Carolina in 1970. He obtained a Fine Fellowship for postdoctoral research at the University of Kansas and began his teaching career at Michigan State University in 1972. He is professor of chemistry at Purdue University and president of Bioanalytical Systems, which he founded in 1974.
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