Anal. Chem. 1992, 64, 577-583
577
AC RES€ARCH
In Vivo Calibration of Microdialysis Probes for Exogenous Compounds Stanley Menacherry, Walt Hubert, and Joseph B. Justice, Jr.*
Department of Chemistry, Emory University, Atlanta, Georgia 30322
Several approaches for calibrating microdialysis probes for exogenous compounds in vivo are described whkh avoid the error Introduced by In vitro calibration. These methods are based on ostabikhlng a steady state of the exogenous compound by a contlnuour (zeroorder) iv infusion. The steadydate concentration Is estimated by three methods that directly determine the in VIVO concentration. The methods are (a) extrapolation of dialysate concentrations at varlous flow rates to the concentration at zero flow, (b) dialyds with concentrations of anaiyte added to the perfurion medium above and below the expected Concentration to determine the concentration at no net flux across the membrane, and (c) dlalyrk at a very slow perfdon rate (57 nUmln) where the recovery is expected to be better than 90%. Using these approaches, the recovery for cocaine in the brain was found to be (8.9 f OM)%, as compared to an In vttro recovery of (5.1 f 0.18)% at 24 O C and (7.4 f 0.18)% at 37 O C , at a perfusion rate of 1.2 pUmin through a 0.3- X 2mm microdialysis p". In vlvo concentratlon of cocaine in the rat brain for an intravenous dose of 0.3 mg/kg per mln was found to be 17.1 f 1.3 pM.
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INTRODUCTION Microdialysis is an in vivo sampling technique that has found extensive application in neurotransmitter research.14 Recently this technique has been extended to the monitoring of drugs and other exogenous compounds in the brain and blood of A recurrent problem with microdialysis is the lack of a reliable in vivo calibration method so that accurate in vivo concentrations can be established. The difficulty in estimating the in vivo concentration arises from the fact that microdialysis is a dynamic sampling method where analytes diffuse across a semipermeable membrane in the presence of a concentration gradient. They are then carried away by a perfusion medium that is constantly pumped through the microdialysis probe. The concentration of the analyte in the dialysate therefore is not at equilibrium with the external concentration. The difference in concentration between the dialysate and the external concentration is dependent on a number of factors, most important among them are the perfueion rate, surface area and molecular weight (MW)cutoff of the membrane, temperature, analyte species, and matrix properties of the external media.1° For a given 0003-2700/92/0364-0577$03.00/0
set of experimental conditions, the ratio between dialysate concentration and the external concentration is a measure of the efficiency or recovery of the probe. The in vitro recovery of a probe can easily be estimated using solutions of known concentration in the external medium and measuring the resulting dialysate concentration. In the past it was thought that the membrane was the limiting factor in determining recovery; therefore, in vitro recovery of a probe was used an index of in vivo probe efficiency. In recent work,11-14 it has been shown that in vitro recovery is not a reliable estimator of in vivo recovery because of factors in biological tissue that serve to either impede or enhance mass transport of the analyte from the external medium to the probe. In this context the membrane is not the limiting factor in determining recovery. Factors limiting free diffusion in vivo such as tortuosity and volume fraction have been discussed by Benveniste et al.,ll and in earlier work by Nicholson and Phillips16and Nicholson and Rice.16 These factors are dependent on the physical properties of brain tissue and therefore lead to a passivestate model of the recovery process. More recently, passive-state models have been modified to account for biological processes (active processes) such as transport to and from the brain by vascular circulation, metabolism, and active transport across membranes, including release and ~ p t a k e . ' ~The J ~ contribution of several of these factors has been demonstrated for neurotransmitter^.^^,^^,^^ To overcome the limitation of using an in vitro technique of probe calibration for endogenous compounds, two approaches have been proposed for estimating the concentration at steady state: (a) the extrapolation to zero flow rate methodlg and (b) the point of no net flux method.20.21These methods have been investigated in detail by Parsons and Justice14 and Farsons et al.17 who used these methods to estimate the basal concentration of dopamine (DA) in normal and cOcaine treated rats and the effect of bhydroxydopamine lesions on extracellular DA concentration and the concomitant recovery. The current paper describes an approach for estimating the in vivo concentration of exogenous compounds. This approach is based on establishing a steady-state concentration of the exogenous compound by constant intravenous (iv) infusion of the drug. The steady-state concentration is then determined using three methods: the two methods described above (point of no net flux and extrapolation to zero flow) and a third method that is based on a very slow perfusion rate. The in vivo concentration may then be used to establish the in vivo 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
recovery at any perfusion rate. Faster perfusion rates are necessary to monitor transient changes in the external concentration in a microdialysis experiment and generally more convenient to work with since larger sample volumes are obtained at faster perfusion rates. Two microdialysis perfusion rates that have been used for studying the Pharmacokinetics of cocaine were 0.622and 1.2 ~ L / m i n .In~ vivo ~ recovery was therefore determined a t these perfusion rates in the present experiments. Recovery was determined by setting the perfusion to these rates and measuring the dialywte concentration which was then compared to the in vivo concentration. In vivo recovery is reported as dialysate concentration divided by in vivo concentration and is expressed as percent recovery. In vitro recoveries were also measured, at room temperature (24 "C) and at the body temperature of the rat (37 "C), in order to assess the effect of temperature on recovery in vitro and for comparison with the observed in vivo recovery. In this study cocaine was used as the model compound because the pharmacokinetics of cocaine in the rat have been characterized after single infusions by Pan et aLp3and after multiple infusions by Menacherry et Multiple infusion kinetics indicated that cocaine given as a constant iv infusion will attain a steady-state concentration in the brain within 45 min after starting the infusion. The dose selected for constant infusion in these studies is relevant to behavioral studies of drug abuse in which subjects control their drug intake. The paradigm called self-administration is one of the primary behavioral methods for studying the abuse potential of d r ~ g s . Pettit ~~*~ and ~ J ~ s t i c emonitored ~ ~ , ~ ~ the neurochemical response to cocaine self-administration as expressed by dialysate levels of the neurotransmitter DA. However, the authors did not estimate the concentration of cocaine in the brain during self-administration. In the current study in vivo methods for estimating extracellular concentration were employed in order to obtain three independent estimates of the cocaine concentration in the brain after a constant iv infusion. The infusion dose was 0.3 mg/kg per min, which corresponds to the mean drug intake rate during self-administration reported by Pettit and Justice.28 EXPERIMENTAL SECTION Subjects and Surgery. Subjects were male Sprague-Dawley rats (300-350 g; Sasco King Inc.) housed in a temperature-controlled room under a 12-h light and dark cycle with food and water provided ad libitum. Experiments were conducted on anesthetized animals (400 mg/kg chloral hydrate sc supplemented by 0.1 mL of 200 mg/mL chloral hydrate given ip when required, as determined by response to tailpinch). Subjects were implanted with an iv catheter (3.5 cm) in the jugular vein. The catheter was connected to a Raze1 constant infusion pump. Surgical procedures for iv catheterization were that of Pettit et al.22 Sample size (n) in each group was 5,4, and 4 for the extrapolation to zero flow, point of no net flux, and the slow flow methods, respectively. Microdialysis. Sampling probes 2 mm x 300 pm (membrane: cellulosic tubing; MW cutoff 5ooo; Enka Glantzoff AG, Germany) constructed according to the method of Wages et al.l0 were implanted in the nucleus accumbens [AP +3.0 and L f1.7 from bregma; V +8.0 from the dura; incisor bar +5.0] according to coordinates of Pellegrino et a1.29and allowed to stabilize for 2 h at a perfusion rate of 0.6 pL/min. The perfusion medium was an artificial CSF solution (composition: 0.13 M NaC1,2.34 mM CaCl,, 2.65 mM KC1, 2.1 mM MgClp,0.1 mM ascorbic acid, and 10 mM dextrose in distilled water at a pH of 7.4). Chromatography. Cocaine in dialysate samples was determined by UV detection after separation on a microbore HPLC column, according to the procedure of Pettit et a1.22 The HPLC-UV system consisted of an ISCO LC-5000 pump (Lincoln, Nebraska), a Rheodyne injection valve (Model 7125; with a 5-pL sample loop), and a Perkin-Elmer UV/visible detector (LC-95) with a 1-pL cell. Injection volume was 6 pL. The column was 15 cm X 0.5 mm packed with 5-pm Spherisorb C18 phase. The absorption wavelength was 235 nm set to a response of 0.001
absorbance units full scale. The mobile phase consisted of 17% acetonitrile, 10% methanol, and 0.5% triethylamine (v/v) in 50 mM sodium phosphate buffer at a pH of 5.6 and a flow rate of 2 mL/h. The retention time of cocaine for these conditions was 5.2 min. The detection limit was 0.25 pM in the dialysate. The metabolites of cocaine (ecgonine and benzoylecgonine) did not interfere under these chromatographic conditions. Steady State. The pharmacokineticsof cocaine measured by the microdialysis technique have been reported by Pan et al.23 for single infusion and by Menacherry et for multiple infusions. These reports show that the pharmacokinetics of cocaine approximate to a two compartment model where compartment 1 is blood and compartment 2 is the brain and other lipogenous tissue. The pharmacokinetic constants reported by Menacherry et al." were first-order rate constants k12, kZ1,and kel;where k12 (blood to brain transport rate) was 0.33 0.09 mi&, kzl (brain to blood transport rate) was 0.18 0.045 min-l, and kel (the rate of elimination by metabolism and excretion) was 0.47 f 0.163 m i d (mean sd). The volume of distribution in the first compartment (vJ was 111 mL and in the second compartment (up) was 59 mL. Calculations using these constants suggested that a constant infusion would result in a steady-state concentration of cocaine in the brain within 45 min after starting the infusion (Figure 1). Cocaine solution adjusted for the body weight of the rat (3.75 mg/mL for a 300-g rat) was continuously infused at a rate of 24 pL/min to achieve a steady-state in vivo concentration. Infusions were administered through the jugular vein 2 h after surgery, and the concentration of cocaine was allowed to reach and maintain a steady state in the brain for 90 min before microdialysis sampling was begun. Microdialysis samples were then collected at 20-min intervals and steady-stateconcentrationwas confirmed by three consecutive samples having the same peak height ( 0.99). Therefore, no significant trends or changes in the steady-state concentration are apparent during the course of the experiment. Method 1. Extrapolation to Zero Flow. Figure 2 shows the application of the extrapolation to zero flow method for two solutions of cocaine a t room temperature. The concentrations of the solutions were 25 and 12.5 pM. The estimated C,'s when the two curves were simultaneously regressed with a common k (where k = FAin eq 1) were 25.3 and 12.3 fiM (n = 3), which agree well with the actual concentrations. Simultaneous solution of two flow w e s gives higher accuracy
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6,MARCH 15, 1992
580
.-
4
25 pM cocaine 12.5 pM cocaine
1
Table I. In Vivo Recoverya for a 2-mm Probe Estimated by t h e Three Methods expt no.
method extrapolation to zero flow
0.0
0.5
F,
1 .o 1.5 flow rote (pL/min)
2.0
mean f SEM
Flgure 2. Extrapolation to zero flow rate method in vitro. The plot shows the concentration of cocaine in the dialysate as a function of flow rate for two different solution concentrations of cocaine. The lower curve was obtained for a 12.5 pM solution at room temperatwe ( A M ckcles = data, dotted Une is the flt by nonilnear regression). The upper w e was obtained for a Concentration of 25 pM. The intercepts with the y-axis are C,,,the external solution concentrations. Concentrations of 12.3 and 25.3 pM were found from the lower and upper curve, respectively. The curves can be used to obtain the percent recovery at any flow rate. The common value of k (where k = rA in eq 1) estimated by nonilnear regression for the two curves was 0.09 mm/min. 25
,
1
2 3 4 5
I
I
- 20
i
no net flux
1
2 3 4
mean f SEM slow flow
1
2 3 4 mean f SEM
recovery, % 0.6 pL/min 1.2rL/min
18.8 30.0 14.7 6.1 21.3 18.1 f 3.0
10.3 10.3 7.7 3.9 12.8 9.0 f 1.5
18.2 18.5 17.4
12.1 6.8 9.8 10.4 18.0 f 0.33 9.8 f 1.1 18.3 17.8 11.6 20.0 16.9 f 1.8
7.9 8.5 6.1 8.6 7.8 f 0.58
ORecovery defined as concentration of cocaine in dialysate divided by the estimated in vivo concentration and expressed as percent.
Y
B 3 15 .-D
CI
I 15 3.
v
'
.E 10
1
.8
x -
5
0
0 0.0
Intercept = 17 pM
; 1 0 . Slope
0
1 .o
0.5
F,
1.J
2.0
2.5
flow rate (pL/min)
Flgurr 9. Extrapolation to zero flow rate method in a single subject. I n this rat, the estimate of C,,, the concentration in the extracellular fluid of the brain, was 19.9 pM. The value of k was 0.1 1 mm/min. The cocaine concentration is at the steady state during a continuous iv infusion at a dose of 0.3 mg/kg/per min.
than a single curve; however, working with two curves in vivo is not practical. When the in vitro curves were independently estimated with two determinations of k,then a greater error of estimation was observed; Co = 13.2 pM was obtained for the low concentrationand 23 pM for the higher concentration. The extrapolation to zero flow method works fairly well with good data; however, outliers tend to make the nonlinear regression unstable and in some cases the regression fails to converge satisfactorily. In such instances the estimate of k is seriously affected. Parsons et al." discuss the influence of k on the estimated zero flow concentration (Co). They found that k values lower than the optimum estimated by nonlinear regression tend to greatly overestimateCowhereas large values have less impact on the Co value. Figure 3 shows the application of this method in vivo for a single subject. The shape of the curve reveals a flow-rate dependence that is comparable to that observed in vitro. The estimate of k from the four trials was 0.12 f 0.03 mm/min (mean f SEM). In vivo concentration of cocaine in the brain using this method was 19.9, 17.4, 14.3, 27.3, and 8.75 pM for the five subjects which gives a mean of 17.5 f 3.04 (mean f SEM) for a constant infusion of 0.3 mg/kg/per min. The in vivo recovery a t 0.6 pL/min was (18.1 f 3.9)% and at 1.2 pL/min was (9.0 f O.66)% (mean f SEM). A comparison of recovery at these perfusion rates for each subject is presented in Table I. The average experimental time for one subject using this method was 12 h postsurgery. Method 2. Point of No Net Flux. The concentration of cocaine in the dialysate was measured for different concen-
0
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.
.-C
5 -
= 0.15
c
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0
v1
-
;-5
CI
0
20
40
60
SO
100
Cocaine conc. in perfusote (pM) Figure 4. Zero flux method in vivo in a single subject. Difference between analyte concentration in the dialysate and in the perfusate is plotted as a function of the perfusate concentration. Dotted line is the linear regression to data and wild line is the zero line of the y-axis. The intercept of the two lines is the point of no net flux. In this rat this point is estimated to be at 17 pM cocaine in the perfusate. The slope of the regression line is an estimate of recovery, which is 15% for this subject.
tration of cocaine in the perfusate. Figure 4 shows the effect in vivo of varying cocaine concentrations in the perfusate on the net gain or loss of cocaine in dialysate. The difference in concentration between dialysate and perfusate is a measure of the net transport of analyte across the membrane. The estimated in vivo concentration is the perfusate concentration corresponding to the intercept of the first-order regression and the zero point on the y-axis(x-axis intercept). T h e estimated in vivo concentrationof cocaine in the brain using this method for a constant infusion of 0.3 mg/kg/per min was found to be 16.2 f 1.5 pM (mean f SEM), based on individual animal values of 17.0, 12.1, 17.0, and 18.8 p M . The in vivo recovery at 0.6 pL/min was (18.0 f 0.33)% and at 1.2 pL/min was (9.8 f L l ) % (mean f SEM). In vivo recovery in this method is also given by the slope of the regression line. Since these experiments were conducted at a perfusion rate of 0.6 pL/min, the recovery estimated by the slope can be compared to the recovery estimated from the ratio of dialysate concentration at 0.6 pL/min and the in vivo concentration. The average recovery at 0.6 pL/min from the slope was found to be (16.6 f 1.61%. This estimate is not statistically different from the estimate of 18.0% from dia-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
lysate and in vivo concentration. The average experimental time for one subject using this method was 13 h postsurgery. A variation of this method involves perfusing with the drug of interest and measuring the loss from transport out of the probe.3o This is conceptually equivalent to the upper half of the curve in Figure 4, though only a single concentration is used. A signi!icant advantage is that one need not systemically administer the drug, thereby avoiding additional surgery. Method 3. Slow Perfusion Rate. The expected high recovery with a slow perfusion rate was based on a plot of the dependence of recovery on perfusion rate (Figure 3). From the graph it is apparent that the recovery of cocaine is better than 90% for perfusion rates of around 50 nL/min. The actual perfusion rate used in these experiments was 57 nL/ min. The sampling interval was 30 min at this perfusion rate. The dialysate was collected into 3.0 pL of artificial CSF in a 5-mL plastic vial. Using this method the estimate of in vivo concentration was found to be 17.4 f 1.5 r M (mean f $EM) based on 15.3,17.6, 15.0, and 21.6 pM for four subjects. The in vivo recovery at 0.6 rL/min was (16.9 f 1.8)% and at 1.2 pL/min was (7.8 f 0.58)% (mean f SEM). The slow perfusion rate method was also teated in vitro with a 10 pM standard solution of cocaine. The concentration in vitro estimated by this method was 9.88 f 0.68 pM (mean f SEM) which is in good agreement with the expected concentration. No significant differences in estimates were seen using the method in vitro a t room temperature (24 "C) and at 37 "C. The average experimental time for the slow perfusion rate method was 4 h postsurgery. A potential improvement in this method is to use refrigerated sample collection to reduce evaporative loss. The estimates of in vivo concentration in the brain by the three different methods were not significantly different (F(2,10) = 0.09; p > 0.9). The average in vivo concentration of cocaine estimated by all three methods was 17.1 f 1.3 pM (mean f SEM). In vivo recovery estimates as measured by the three methods are shown in table I for comparison. There was no significant difference in recovery by the three methods. In vivo recovery at 0.6 pL/min was (17.7 f 1.61% and was (8.9 f 0.68)% a t 1.2 pL/min (mean f SEM of estimates by all three methods). All of the three in vivo methods described gave comparable recovery and concentration values. However, these methods require a prohibitive amount of time to be used as a routine calibration procedure with every microdialysis probe. The methodology described above can be used to characterize in vivo recovery for a given analyte and set of experimental parameters. Nonuiformity among probes used in a particular experiment can then be detected on a routine basis by in vitro methods. While all three of the above methods gave the same results for both the in vivo brain concentration of cocaine and the in vivo recoveries at 0.6 and 1.2 pL/min, the slow perfusion rate method took the least amount of time to conduct. It is easier to maintain the steady-state concentration of the exogenous compound for the shorter experimental time required by this method. Recovery in Vivo and in Vitro. Figure 5 shows the comparison between in vivo recovery and in vitro recoveries at room temperature (24 "C) and at 37 "C. Recoveries at 0.6 pL/min were (9.6 f 0.98)% (n = 4), (13.0 f 0.44)% (n = 41, for 24 "C and 37 OC, respectively, while recovery was (17.8 f 1.63)% (n = 13) in vivo (mean f SEM). Recoveries a t 1.2 pL/min were (5.1 f 0.181% (n = 4) and (7.4 f 0.18)% (n = 4) for 24 "C and 37 "C,respectively, while it was (8.9 f 0.68)% (n = 12) in vivo. Statistical analysis using the t test (Crunch 4.0, C m c h Software, Inc.) indicated that at 1.2 pL/min the
581
20
0 0.6 uL/min m 1.2 uL/min
8
p 10 0 0
0)
E
5
0 in vitro 24' C
in vitro 37oc
in vivo
Flgure 5. Comparison of recovery In vltro and In vivo. Recovery in vitro Is higher at 37 "C than 24 OC. Recovery In vivo Is higher than recovery in vitro. Numerical values in text.
in vivo recovery was significantlyhigher than in vitro at both 24 OC (p < 0.02) and 37 "C 0, < 0.02). The in vitro recoveriea were also different from each other (p < 0.02) at this flow rate. At 0.6 pL/min, the in vivo recovery was significantly higher than in vitro at 24 "C (p < O.OOOl), while the difference with 37 OC in vitro did not quite reach statistical significance (p < 0.058). The difference between in vitro recoveries at the two temperatures for this flow rate was significant (p < 0.Oool). In the current experiments, the increase in vitro recovery of cocaine on going from a temperature of 24 "C to 37 "C was 35% at a perfusion rate of 0.6 pL/min and 45% at 1.2 rL/min. This effect of temperature on recovery is consistent with the temperature dependence of diffusion reported by Krnjevic and MitchelPl and with earlier work that reported a 40% increase in the recovery of sucrose at perfusion rates of 2,5, and 10 pL/min21 and 34% for Ca2+ at a flow rate of 2.5 ~L/min.~~ As indicated in Figure 5, the increased recovery in vivo cannot be attributed to the effect of temperature on diffusion alone. Various mathematical models of the mass transport to a probe have been pr0ped11J2J193233 that consider barriers to free diffusion present in a structured medium such as the brain. Barriers to free diffusion in the brain result from the geometry of the extracellular space affecting analyte movement in two ways: (a) through tortuosity, A, and (b) through the volume fraction, a. (a) Tortuosity refers to the increased path length that a molecule traverses to reach the probe because it is restricted to the irregular channels that comprise the extracellular space. (b) Volume fraction refers to restriction of analyte to the extracellular fluid, which comprises about 20% of the total volume. Some molecules cross the cell membrane more readily than others, thus altering the effect of tortuosity and volume fraction. Microdialysis samples the extracellular fluid in vivo and therefore only has access to the analyte present in a smaller volume as compared to a homogeneous external medium. The combination of reduced volume fraction and increased effective path length should lead to a reduced recovery in vivo. From theoretical considerations it has been shown that the net effect of these two factors is to reduce the in vitro diffusion coefficient by a factor of X2/afor in vivo condition^.^' Using the average values of a and X reported by Nicholson and Rice,lGa reduction in recovery by a factor of 10 is to be expected. It is clear from Figure 5 that this does not happen. The enhanced recovery seen experimentally demonstrates that additional factors must be involved in the mass transport of material to a microdialysis probe. An improved model that takes into account the effect of microvasculature transport has been suggested by Morrison et al.13 The additional considerations which have been implemented in a computer simulation of the model include the rate constant for irreversible metabolism, the blood/brain and brain/blood transport coefficients, a species generation rate,
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992 lB1 Diffusion +TransDort
Unexpectedly high recoveries are also seen fro neurotransmitters.14J8 For endogenous materials such as neurotransmitters, the processes of release and uptake play the same role that microvascular transport and metabolism do for exogenous compounds. It seems likely that the same effeds on mase transport to and from the probe OCCUT in the surroundjng tissue. Neurotransmitter release into the extracellular fluid of the depletion zone defies a steeper gradient for transport to the probe, while removal from the extracellular fluid by uptake (for catecholamines) or metabolism (for acetylcholine) creates a steeper gradient for transport from the probe. Elimination of these processes reduces the recovery."
Distance From Probe
Flfpe 6. Effect of bkod/breln transport on the concentration graMnt around a probe. The figure is computed from a finite element model
of the microdialysis probe.34 The graph represents concentration gradients around a probe after a fixed period of analyte removal by microdlatysls. Curve A shows the concentration gradlent when mass transport is by diffusion from the surrounding region only. Curve B show the gradlent when mass transport occurs by both diffusion from suroundbrgtlssoe and by kcel supply of materlai in the depletkn regIan around the probe. the total binding, and the partitioning between the extracellular and intracellular compartments. The analytic solution of the equations describing these processes is very involved. However, the effect of the inclusion of active processes to the passive diffusion model can be visualized by a fiiite element digital simulation." The finite element simulation considered only transport by diffusion and transport across the blood/ brain barrier, using the rate constanta of blood/brain transport for cocaine (k12 = 0.33 and kzl = 0.18 mi^'^^). Since all the relevant factors have not been considered, the simulation is more qualitative then quantitative but serves to illustrate the basic principle of the effect of microvasculature transport. The results of a simulation of the concentration profiles around a probe after a fixed period of dialysis are shown in Figure 6. Shown in the figure is the depletion zone and the associated concentration gradient that is formed around a probe as analyte is removed by the probe. For any given effective diffusion coefficient, the recovery to the probe is determined by the magnitude of the concentration gradient. When the gradient is defined by diffusion only, curve A, the depletion zone is large and consequently the concentration gradient is small, leading to a low recovery. If the depletion zone is replenished by blood/brain transport, curve B, a larger concentration gradient is created which leads to higher probe recovery. The inverse figure, with concentrationdecreasing from the probe surface into the tissue, is created for transport from the probe to the tissue, as in the upper part of Figure 4, where the concentration of drug is higher in the probe than in the tissue. The drug diffUses out of the probe into the surrounding tissue and is removed from the tissue surrounding the probe by diffusion, by microvascular transport, and by metabolism. When the combined effects of transport and metabolism are slow, a gradual decline in concentration is established as a function of distance from the probe as the drug diffuses out into the tissue. The faster a drug is metabolized in the tissue or is removed by microvascular transport, the steeper the gradient that is established, and the higher the rate of transport from the probe. The experimental data are summarizedin Figure 5, and this qualitative simulation supports the theoretical work of Morrison et al.,13 vis., mass transport by the microvasculature in the brain is a strong determinant of in vivo recovery for exogenous compounds. These effects are apparently strong enough to overcome the effects of tortuosity and volume fraction which tend to reduce recovery in vivo relative to that in free solution.
CONCLUSION In vivo concentrations and recoveries of exogenous compounds at steady state can be estimated by several microdialysis techniques. Using three in vivo techniques the concentration of cocaine in the brain was found to be 17.1 pM for a dose that is commonly used in self-adminkbationstudies. In vivo recovery was found to be higher than in vitro recovery. This cannot be explained by a passive diffusion model but is accounted for in a model that incorporates active processes. The extent that the active processes of microvasculature transport, metabolism, release, and uptake affect the in vivo recovery will be different for each exogenous compound and neurotransmitter. ACKNOWLEDGMENT This work was supported by NSF Grant BNS-8812768 and a grant from the National Institute of Drug Abuse (no. DA05827). Registry No. Cocaine, 50-36-2. REFERENCES Church, W. H.; Justice, J. B.. Jr. Anal. Chem. 1987, 59, 712-716. Justice, J. B., Jr.; Parsons, L. H. I n Chedcal Sensor T w ; Yamazoe, N., Ed.: Elsevier, Amsterdam, 1990; pp 249-262. , Ungerstedt, U.; Haiistrom. A. Llfe Sd.1987, 41, 860-864. (4) Zetterstrbm, T.; Sharp, U.; Ungerstedt, U. Ew. J. phennecd. 1985, 106, 27-37. (5) Nicoleysen, C. L.; Pan, H. T.; Justice, J. B.. Jr. &a/n Res. 1987, 456, 317-323. (6) Hwd, Y. L.; Kehr, J.; Ungerstedt, U. J . Newochem. 1988, 57, 1314- 1316. (7) Caprioli, R. M.; Lin, S. N. Roc. Net/. Aced. Sei. U . S . A . 1990, 8 7 , 240-243. (8) Menacherry, S. D.; Justice, J. B., Jr. Anal. Chem. 1990, 62, 597-601. (9) 8Brock, 12-6 13. J. B.; Justice, J. B., Jr. Ann. N.Y. Aced. Scl. 1990, 604, - .- - .-. (10) Wages, S. A.; Church, W. H.;Justice, J. B., Jr. Anal. Chem. 1988, 58. 1649-1658. Benveniste. H.i Hansen, Anker, J.; Ottosen, N. S. J. Neuachem. 1989, 52, 1741-1750. Bungay, Peter M.; Morrison, P. F.; W i c k , R. L. LMe Sei. 1990, 46, 105-1 19. Morrison, P. F.; Bungay, P. M.; Hslao, J. K.; Ball, B. A.; Mafford, I . N.; Dedrick, R. L. In M/cfOd&lyslsln the Neuroscknces; Robinson, T. E.: Justice, J. B., Jr., Eds.; Elsevier: Amsterdam, 1991; pp 47-80. Parsons, L. H.; Justice, J. B., Jr. J . Neuroahem. 1992, 58, 212-218. Nicholson, C.; Fhiilips, J. M. J. phlsol. (Londrxr) 1981, 327. 225-257. Nicholson, C.; Rice, M. Ann. N . Y . Aced. Scl. 1988, 487, 55-71. Parsons, L. H.: Smith, A. D.; Justice, J. B., Jr. J. Newoscl. Meth. 1901, 40, 139-147. Parsons, L. H.; Smth, A. D.; Justice, J. B., Jr. Synapse 1991, 9, 60-65. Jacobson, I.; Sandberg, M.; Hamberger, A. J. Neurosci. Meth. 1985. 15, 263-268. Lonnroth, P.; Jannson, P. A.; Smith, U. Am. J . physld. 1987, 256. E250-E255. Lindefors, N.; Ambera, G.: Unoerstedt. U. J. Phermacd. Meth. 1989. 22, 141-156. Pettt, H. 0.; Pan, H. T.; Parsons, L. H.; Justice, J. B., Jr. J. New. " te 1000. 55. 798484. Pan. H. T.: Menacherw. S. D.: Justice. J. 6.. Jr. J . Netrod".1991. 56, 1299-1306. Menacheny, S. D.; Brock, J. W.; Justice, J. B., Jr. J. Nevosci. Meth., submitted for publication. Pickens, R.; Thompson, T. cherecterlstles of s U " t &ug rdnfarcemflt Stlmulus w o m s of &w; New - ApDleton-CenturVCrofts: .. York, 1971; pp.177-192. Baister, R. L.; Schuster, C. R. J . Exp. Anal. Behev. 1973, 20, 119-129. ~
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M, H. 0.; Jwtke, J. B., Jr. phermecd. Bkchem. Behev. 1989, 34, 809-904. (28) Pettlt, H. 0.;Justice, J. B., Jr. &ah Res. 1991, 539, 94-102. (29) pdk9rin0, L.; Memino, A.; Cuahman, A. A Stetuotaxk A L s of the Fbt h; pknum:-New York, 1979. (27)
(30) Ute. *' J. B., In Jr. Eda.; Elsevlwthe Amsterdam. Nwasclences; 1991;Robhmo pp 155-174. T* (31) Kmjevic, K.; M l t C h d , J. F. J . physkl. 1 0 0 , 153, 562-572.
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(32) Benvenlste, H. J . Ne". 1989, 52, 1667-1679. (33) Amberg. b.; Undefors, N. J . phennecd. Mth. 1989, 22, 157-183. (34) Menacheny, S. D., Ph.D. TheSlS, EITIOIY Unhrerslty, 1991; pp 111-131.
RECEIVED for review August 14,1991. Accepted December 17, 1991.
Separation and Detection of Aliphatic Anionic Surfactants Using a Weak Anion Exchange Column with Indirect Photometric and Indirect Conductivity Detection Shahbaz A. Maki, Julie Wangsa, and Neil D. Danielson* Department of Chemistry, Miami University, Oxford, Ohio 45056
of 0.2-pm cation exchange particles in the hydrogen form with Naphthrkmdkulfonateacetrcaonnrlkas a moblie phase alone w l l h a ~ w l ~ r b o n p o l y n n r d l c c r c o k m n the eluent to convert the tetrabutylammonium form of anionic surfactants into the more conductive free acids has been rehm drown to k an df.ctive combhatkn for the mparatlon ported.6 A phenyl column with a 75% methanol-25% 0.1 M of allphatk anknlc wrfactants. Indlrod conductlvlty and NaN03 mobile phase could separate long-chain (C12-C19) photomtrlc dotectbn mockr are used to modtor those anaalkanesulfonate surfactants with refractive index detection.6 lytw. Tho rotontkn of these surfactants k found to depend The evaporative light scattering detector has also been onboththe knlcrb.ng#landtheorganksolvont content of evaluated for the HPLC quantitation of surfactants down to tho moblk phase. Tho mochadam of rotontbn k k l k v e d to 20 nm01.7 k a comblnatkn of both reversed phase and Ion exchange Ion-interaction chromatography utilizing a RP column with p"a8. 8rkctlvo wparatbn of both C&, alkanean aromatic ion-pairing agent has been a popular approach Wnonatw and ahyi sulfates can be achleved In less than @ for the separation and detection of aliphatic anionic surfacmln. DotocUon htb are as low as 5 ng for mort analytw, tants. Alkyl surfactants such as C6-C8sulfonates were sepO X t m hwn at kaSt 500 ppm t0 the WRh knr arated on a Bondapak C18 column with a 6040 methanolwb-ppm kvel. water mobile phase containing 0.2 mM cetylpyridinium INTRODUCTION Limited work has been cited in the literature on the liquid chromatographic separation and detection of the industrially and environmentally important class of surfactants, namely, the long-chain alkanesulfonates and alkyl sulfates as well as other aliphatic anionic surfactants. Their separation and detection is somewhat troublesome because of the dual hydrophobic and ionic nature of the compounds as well as their lack of UV absorbance and small equivalent conductance. However, some HPLC methods with direct detection of the separated aliphatic anionic Surfactants have been published. For the analysis of C8-C18alkanesulfonates, Smedes et. all fluorometridy detected the extractad acridiniumaurfactant ion pair in chloroform after reversed phase (RP) separation with 40-60% acetone/water in sodium dihydrogen phosphate. To eliminate the need for on-line extraction, prederivatization of alkyl sulfates with 4-(diazomethyl)-N,N-dimethylbenzenesulfonamide permitted HPLC detection at 240 nm.2 However, the reaction had to occur in 100% methanol with the surfactants in the acidic form. Williams3 reported the separation of C& and C6-C8 sulfonates, in 20 and 12 min using 0.001 and 0.003 M bicarbonate/carbonate buffers, respectively, by anion exchange with suppressed conductivity detection. Weiss was able to separate C6-C8sulfonates in 16 min on a polystyrendivinylbenzene column using 28% CH&N/water in 0.002 M tetramethylammonium hydroxide using suppressed conductivity detection.' Recently, a paper describing the postcolumn mixing of an aqueous suspension 0003-2700/92/0364-0583$03.00/0
chloride acting as both the ion-pairingreagent and the means for UV detection at 254 mm?s9 The same conditions were used, but with a different ion-pairing reagent (phenethylammonium ion) for the quantitation of pentanesulfonate and hexanesulfonate from concentrations of 1.6 to 25 mM.'O Ion-interaction chromatography on a polymeric column with iron-l,l0-phenanthroline and indirect photometric detection has also been reported for the separation and detection of alkanesulfonates and alkyl sulfates." N-Methylpyridinium chloride or copper sulfate were used as the ion-interaction reagents for the separation and detection of positionally isomeric alkanesulfonates.12 Iodide and nitrate which absorb light in the UV have been used as spectator ions to provide the means of indirect photometric detection of even numbered Ca-Ci4 alkyl sulfates. Both isocratic and methanol gradient elution (from 30 to 90%)were employed for the RP separation of these surfactanb in about 14 min with detection limits in 3-5-pg range.13 In all these methods, the mobile-phase concentration must be optimized carefully since it controls both the ion exchange column capacity and detection ability. Anion exchange chromatography with indirect detection is not a common approach for aliphatic sulfonates although it is mechanistically a simpler method than ion-interaction chromatography. Lar50n'~used biphthalate, sulfosalicylicacid, and m-sulfobenzoic acid in 6040 acetonitrilewater as mobile phases to separate C2-C8sulfonates on a strong cation exchange column with indirect UV detection at 297, 320, and 298 nm, respectively. Detection limits at the 0.3-pg level and a linear response ranging from 10 to lo00 ppm were reported. Lack of suitable columns and competitive ion exchange eluents 0 1992 American Chemical Society
