Anal. Chem. 1986, 58,1649-1656
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Sampling Considerations for On-Line Microbore Liquid Chromatography of Brain Dialysate S. A. Wages, W. H. Church, and J. B. Justice, Jr.* Department of Chemistry, Emory University, Atlanta, Georgia 30322 A push-pull dlalysls perfusion system for monitoring neurotrhnsmltter release in deep structures of the brain of freely moving anlmals Is characterized. The internal volume of the system has been reduced so that very slow perfuslon rates can be used. These slow rates yield hlgh recoverles and small sample volumes, allowlng mlcrobore columns wlth their inherent increase in mass sensltlvlty to be incorporated Into the chromatographic system. Recovery of sample exceeded 90% at 0.1 pUmln at 37 OC. The maxbnum sample recovery per mlnute occurred at approxlmately 2 pL/mln. The contrlbutlon of the perfuslon system to sample dispersion was found to be independent of perfuslon rate above 0.1 pL/mln. The detection llmH for the MHlrolransmtHer dopamine was 300 fg with a S / N of 2. The method Is suitable for sampilng low-molecular-weight molecules in most brain structures.
Chemical analysis inside the living brain has long been a difficult problem. Obtaining chemical information with minimal disruption of the cellular organization of the tissue creates some unique sampling difficulties. Nevertheless, several recent approaches, voltammetry and dialysis perfusion, have shown promise for monitoring the dynamic chemistry of the central nervous system. To aid in the interpretation of voltammetric data obtained in vivo, a series of experiments were initiated to obtain extracellular fluid (ECF) from local structures in the brain. The various electroactive species were separated by high-performance liquid chromatography (HPLC) and quantitated with electrochemical detection (1-3). It soon became evident that chromatographic analysis of the ECF was a method with utility beyond the original purpose of validation of voltammetry in vivo. Further characterization of the sampling and chromatography was therefore undertaken. The push-pull perfusion method was used in our initial experiments to obtain extracellular fluid (1). Push-pull perfusion for monitoring extracellular neurochemicals was first developed by Gaddum in 1961 (4). In Gaddum’s design for push-pull perfusion, two concentric stainless-steel tubes are placed directly in the brain and the extracellular fluid is perfused with artificial cerebrospinal fluid (CSF). Neurotransmitters and metabolites diffuse into the perfusion flow and are removed from the extracellular fluid via the flowing liquid. This technique has undergone many improvements since the first report ( 5 , 6 ) and has been used extensively to monitor the release of neurotransmitters in a variety of deep structures within the brain (7-9). The open design has several inherent problems including excessive tissue damage a t the tip of the cannula and relatively low recoveries from the extracellular medium. Also, because the flow in and out of the cannula is open to the surrounding tissue, there is a need to “cleanup” the perfusate before the samples can be injected onto a high-performance liquid chromatography system. Brain microdialysis, which was first introduced by Tossman et al. in 1981 (IO),eliminated many of the problems associated with push-pull perfusion and demonstrated the feasibility of dialysis of the extracellular fluid of the brain. The dialysis membrane, with a molecular weight cutoff of 5000, eliminates 0003-2700/86/0358-1649$01.50/0
the need to cleanup the sample before the HPLC injection. It also minimizes tissue damage because the flow and associated turbulence are maintained within the dialysis tube. It has been shown that intracerebral dialysis can be used for monitoring changes in dopamine release following administration of d-amphetamine (11) and apomorphine (12). A dialysis cannula similar to the one reported by Tossman et al. (1981) has been designed and characterized in this lab (13). Ungerstedt has written a thorough discussion of dialysis perfusion (14). Microdialysis is currently used in this lab to quantitate extracellular neurotransmitter metabolites and to correlate changes in their concentrations with behavior. On-line high-performance liquid chromatography with electrochemical detection is used to separate and quantify the compounds of interest. The dopamine metabolites, 3,+dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite, 5-hydroxyindole-3-aceticacid (5-HIAA), have been monitored in the striatum of freely moving rats following neuroleptic administration (2). Changes in the extracellular concentration of the metabolites, DOPAC and HVA, have been used as an index of dopaminergic activity. Although useful, leveh of metabolites do not give the temporal information on neurotransmitter release desired for behavioral studies. With the system used previously in this laboratory, dopamine was not detected because its extracellular concentration is approximately 1%or less of the metabolite concentration and was below the detection limit of the system. Other dialysis techniques presently used for sampling dopamine from the ECF (11,12,15) differ somewhat from the method described here. Sample collection is performed offline, using a small tube to collect the perfusate as it is pushed out of the brain. Samples are collected at frequent intervals (usually 20 min) during a given experiment, and each sample collection requires contact with the research animal. This poses no problems for most pharmacological studies but can be detrimental to behavioral research. A second difference in this and other dialysis techniques is the design of the dialysis probe (cannula). Both the loop (11, 12) and the transstriatal dialysis designs (15) have a greater membrane surface area in contact with the ECF. This increase in surface area has been shown to increase recovery across the membrane (13). The purpose of this study was not to replace these techniques that have produced much useful information concerning extracellular dynamics, but to approach the detection of these low-concentration compounds in a different manner and to make this sampling procedure useful for behavioral as well as pharmacological studies. Accordingly, we have modified the dialyzed sample collection procedure and chromatography to handle very small samples and to permit detection of dopamine in the extracellular fluid with on-line microbore liquid chromatography. The present system design is a modification of our previous push-pull perfusion system (16). The dialysis design has been improved through reduction of the internal volume of the sampling tubing and sample size by approximately 2 orders of magnitude. This reduction offers two advantages. First, it allows for the perfusion flow rate to be slowed considerably. The dialysis technique previously used in this lab had a 0 1986 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
perfusion flow rate of 4 pL/min (3). The associated recovery at this flow rate in brain tissue has been estimated to be approximately 2-10%. It was shown in model studies for this technique (13) that as the perfusion flow rate slowed, the recovery rate across the dialysis tubing increased nonlinearly. This increase in recovery across the membrane allows for more concentrated samples to be removed from the extracellular fluid via the dialysis device. I t has been difficult in the past to perfuse at these very slow perfusion rates due to the small volumes that must be manipulated and the time required to collect these samples. This new design solves these problems. Second, the new design enables the use of microbore HPLC columns with increased sensitivity, which more than compensates for the reduced sample size. Microbore chromatography, which was first introduced by Scott and Kucera in 1976 ( l a ,offers several advantages over conventional chromatography. An attribute of microbore columns that is especially important to the present study is high mass sensitivity. High mass sensitivity is necessary when working with very small biological samples that contain limited quantities of the solute to be analyzed. This sensitivity enhancement is due to reduced sample dilution relative to conventional columns (4.6 mm i.d.) and is proportional to the ratio of the squares of the column radii. Microbore chromatography using amperometric detection and 1-mm-i.d. columns has been shown to yield a 20-fold increase in sensitivity compared to 4.6-mm-i.d. columns (18). In the present study, the microdialysis system is characterized and the results are used to obtain suitable working conditions for several ranges of sample concentrations and sampling frequencies. This includes the determination of compounds that are present in concentrations well above the detection limit of the system (metabolites) as well as compounds with relatively low extracellular concentrations (neurotransmitters), which require that the detection limit be minimized. In general, the concentration and the sampling frequency define four categories of analyses, based on high or low concentration and high or low sampling frequency. All four cases are discussed using the monitoring of the neurotransmitter dopamine and its metabolites as an example.
EXPERIMENTAL SECTION Cannula Construction. The dialysis cannula, as seen in Figure 1, is constructed by inserting two lengths of fused silica tubing (0.025mm i.d., Anspec Co., Ann Arbor, MI) into a 20-mm section of dialysis tubing (Fisher Scientific) that has been sealed at one end with a cyanoacrylate adhesive. One piece of the silica tubing extends approximately 40 cm out of the dialysis tubing and is fitted directly into the Rheodyne injection port (Rheodyne, Inc., Cotati, CA) while the second piece of tubing is adapted to fit the push line of the syringe pump. The connection to the injection valve is accomplished by sealing the fused silica tubing inside a short length of stainless-steel tubing with cyanoacrylate so that the end of the fused silica is flush with the end of the stainless-steel tubing. The stainless-steel tubing is then connected to the injection valve in the usual manner using a Rheodyne bushing and ferrule. The distance between the two ends of the fused silica tubes inside the dialysis tubing is adjusted to about 5 mm. This distance is governed by the size of the brain structure being perfused. The top of the dialysis cannula is sealed with the adhesive and allowed to dry. The cannula is inserted into the center of a threaded cannula base from Plastic Products (Roanoke, VA) for work in behaving animals or is simply held in a stereotaxic holder for work with anesthetized animals. Column Preparation. The packing procedure for the microbore columns is that of Myer and Hartwick (19). Briefly, the columns were packed in the constant-pressure mode with a 65 mg/mL isopropyl alcohol (IPA) slurry at 9OOO psi. Methanol was used as the packing solvent. All solvents were thoroughly filtered and degassed prior to use. The slurry reservoir was constructed from an old column (4.5 mm X 25 cm) that was emptied of packing material and cleaned extensively. A 5-mm section of stainless-steel
[
DIALYSIS C A N N U L A
i
I
DIALYSIS TUBING 0.025mm ID FUSED S I L I C A TUBING
-yC,
3mmk
Flgure 1. Details of dialysis cannula. Diameter of cannula is ap-
proximately 300 pM. Length can be adjusted depending on region to be perfused. Fused silica tubing inside dialysis cannula goes directly into injection port. tubing (1mm i.d.) was used as an adapter to connect the reservoir to the microbore column. The reservoir and adapter were sonicated in methanol after use to remove all packing material. All newly packed columns were washed with 5050 methanol/water prior to use. Chromatography. A Waters Model 590 programmable solvent delivery module was used with an IBM Model 230 LC detector. The microprocessor in the module was programmed to actuate injections pneumatically at preset intervals. The working electrode was a glassy carbon electrode from Bioanalytical Systems (BAS, West Lafayette, IN) held-at a potential of +0.75 V vs. a Ag/AgCl reference electrode. Analyses were done on a Rainin Short-one column (4.6 mm X 10 cm) with 3-pm CISpacking, or with 1.0-mm X 10-cm microbore columns packed with 3-pm CIS packing. Microbore columns were kindly supplied by Ivan Mefford, Department of Chemistry, Boston College, Chestnut Hill, MA, or packed in this lab using a column packing pump from Haskel, Inc. (Model 29426). The mobile phase consisted of 0.1 M citric acid, 0.17 mM sodium hexyl sulfate, 0.06% diethylamine, 0.05 mM EDTA, and 7% acetonitrile at a pH of 3 (20). The flow rate of the solvent was 1.2 mL/min and from 80 to 100 pL/min for the conventional and microbore columns, respectively. Samples were injected with a Rheodyne 7413 injection valve with the 0.5-, 1-,or 5-pL sample loop in place. This injection valve was used rather than a Rheodyne 7410 to maintain perfusion flow in the dialysis cannula during sample injection. If the perfusion flow is interrupted (e.g., with a 7410 in inject mode), the perfusion medium flowing into the cannula is forced out of the dialysis membrane rather than through the pull line, thus creating an increase in pressure on surrounding brain tissue. The artificial CSF is prepared by adding 7.46 g of NaC1,0.190 g of KCl, 0.140 g of CaCl,, and 0.189 g of MgCl, to 1 L of distilled water (21). The injection port is in-line with the dialysis cannula and the pull syringe of the perfusion pump. With this arrangement, perfusion fluid from the cannula continuously fills the sample loop. Samples are automatically injected at preset intervals and the valve returned to the load position to begin filling with the next sample while the previous sample is eluting. The complete system is illustrated in Figure 2. Sampling. Samples were collected with a Harvard Apparatus compact infusion pump, Model 2274 (Harvard Apparatus, South Natick, MA), which was modified to push and pull equal volumes of solution using two 250-pL gas-tight syringes (Hamilton, Inc.). The first group of experiments was performed to determine the conditions that maximize the amount of sample collected per unit volume (concentration) or per unit time (absolute amount) for injection onto the HPLC. In order to characterize the relationship between the absolute amount of material recovered per minute as a function of the perfusion rate and recovery across the dialysis membrane at that
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
AMPE ROMETR IC DETECTOR
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Figure 2. Monitoring system for chromatographic analysis of dlalyzed perfusate. The pull side of the flow passes through the sample loop of the HPLC system.
rate, a graph of perfusion rate vs. the amount of material recovered per minute was constructed. Calculations of amount recovered were made assuming a concentration of 1.0 pM outside the membrane. The amount of material per minute was calculated at a particular perfusion flow rate according to the following equation: A = CPR
(1)
where A equals the amount of material recovered per minute (pg/min), C is the external concentration (pg/pL), P is the perfusion flow rate (pL/min), and R is the recovery at that flow (%).
Separate experiments were carried out to determine the recovery across the dialysis membrane as a function of temperature at these very slow perfusion rates. The perfusion rates used were 0.08,0.11,0.15, and 0.21 pL/min, and the solutions were maintained at 23 or 37 "C using a Haake E-12 constant-temperature water bath (Haake, Inc., Saddle Brook, NJ). DOPAC (1ng/pL) was used for these recovery studies. The solution was continually purged with helium to prevent oxidation. A 5-pL injection loop was used for sample injection. To determine whether the recovery across the dialysis membrane remains constant with time, the dialysis cannula was placed in a solution containing 100 pg of dopamine and 20 ng of ascorbic acid per 5-pL sample. The solution was continuously purged with helium and maintained at 37 "C.The ascorbic acid and helium were present to prevent oxidation of dopamine. This solution was then sampled repeatedly. Samples were pulled into the 5-pL HPLC sample loop continuously at a perfusion flow rate of 0.42 pL/min for 8 h. Samples were injected onto the column at 15-min intervals using the microprocessor-controlledpneumatic actuator. To characterize sample dispersion at these slow perfusion rates, two sources were examined for their contribution to the sample variance: (1)collection and transport to the chromatograph and (2) the injector and associated connections. To study the volume variances resulting from transport in the microdialysis system, the outflow line of the dialysis cannula was connected directly to the stainless-steel block of an amperometric detector cell. Sample volumes were introduced by placing the cannula into a beaker of 0.1 mM dopamine in artificial CSF for a specified amount of time and then returning the cannula to a beaker of CSF. Several volumes were used for each perfusion rate. The peak variance was determined by measuring the peak width at half-height. The experimental data were fit to the expression u2tot =
( A 2 / F+ ) B2
(2)
using the least-squares method. The above equation states that the totalvariance is the sum of the volume variance, A (normalized and the square of detector by the square of the flow rate, 0, response time, B (22). To examine the contribution from the injection valve and associated connections to system response time, samples were taken at 5-min intervals and the peak heights measured. A step change was made in concentration by moving the dialysis cannula from one beaker to another and observing the resulting peak
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heights at 5-min intervals. The time needed to reach a steady-state response at the detector was determined. Steady-state response was defined to be three consecutive samples showing less than a 5% variation in peak height. Once a steady state was achieved, the cannula was replaced in the CSF solution and samples were taken until the peak disappeared. To determine whether the internal volume of the dialysis probe contributed to the time delay, this same procedure was done without the dialysis membrane in place. Chromatographic Response. After the conditions that produce the greatest amount of sample per minute were determined, the conditions that increase the chromatographic response (signal) were also examined. Factors that influence response include (1)column diameter and (2) detector volume. Therefore, after the initial characterization of the system was complete, the 4.6-mm4.d. column was replaced with a 1-mm4.d. microbore column. Various detector volumes were also examined. To minimize dead volume for this system, the column inlet was connected directly to the injection port of the HPLC and the outlet directly into the stainless-steel block of the electrochemical detector, eliminating all connecting tubing. Also, in order to retain column efficiency for early eluting peaks on these columns, the detector cell volume was reduced relative to that for a larger column. To compare the detection limits of the 4.6-mm- and 1.0-mm4.d. columns, calibration curves were constructed for dopamine on both column types. The concentration range used for calibration of the conventional column (10 cm X 4.6 mm) was 0-200 pg while that of the microbore (10 cm x 1mm) was 0-10 pg. The dopamine samples were diluted in artificial CSF. The detection limit was taken as the amount corresponding to a SIN of 2.
RESULTS AND DISCUSSION With the dialysis perfusion method described previously (2,3),dopamine recovered from the extracellular fluid of the striatum had been below the detection limit of the system. One of the goals of the current microdialysis system was to make the on-line detection of dopamine feasible. Improving the detection limit of the system involved addressing two problems: obtaining as much material as possible considering the biological and chromatographic constraints and obtaining as large a signal as possible from that sample. The major biological constraint is minimizing perturbation of the neural environment. The chromatographic constraints follow, in part, from the biological constraint in that the analysis is very sample limited. Because dopamine is found in relatively low extracellular concentrations, experiments were conducted to examine the factors affecting the concentration of the neurotransmitter collected in each sample. Additional experiments were aimed a t improving the sensitivity of the chromatographic system. The conditions for maximizing sensitivity of the system involve many factors, some of which are opposing. For example, slower perfusion rates give higher recovery but also give larger sample dispersion in transport from the brain to the chromatograph, as well as smaller sample volumes per unit time. Although larger sample volumes appear to be a good way of maximizing sample size, their use precludes the use of microbore columns with the concomitant increase in sensitivity. Therefore, compromises must be made when determining the best working conditions for a given analysis. The operating parameters for the analysis are dependent on the experimental design and desired results. For example, pharmacological experiments do not usually require the high sampling frequency of behavioral studies. Also, analyses of metabolites present in relatively high concentrations do not require the sensitivity needed for measurement of low-concentration neurotransmitters. The present experiments use a sample volume of 0.5 pL. It is possible to analyze far smaller samples by liquid chromatography with electrochemical detection. Knecht et al. have demonstrated the detection of a femtomole in less than a nanoliter using open tubular columns of 15 pm i.d. with a
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ANALY ICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986 100
90
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0
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60
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50 .04
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Figure 3. Recovery across dialysis membrane vs. temperature at various perfuslon flow rates. Sample used was 1 ng of DoPAC11 pL of mobile phase continually purged with helium.
single graphite fiber electrode for detection (23). Recovery. Recovery of material from the extracellular fluid of the brain is dependent on a number of factors, including perfusion rate, temperature, concentration, molecular weight, molecular shape, and ionic state, as well as chemical interactions with the dialysis membrane. The maximum molecular weight of a compound recovered is controlled by membrane pore size. The present membrane has a molecular weight cutoff of 5000 while the compounds of interest are less than 300. Therefore, there should be little effect of molecular weight on the recovery of these compounds. The relatively slight effect of concentration has been examined previously (13). Of the remaining factors, perfusion rate and temperature are the major influences on recovery. Figure 3 shows the effect of varying temperature and perfusion rate on the recovery of DOPAC from solution. Similar behavior was seen with the other compounds. The recovery at 37 "C was considerably greater than that a t 23 "C. This is probably due to a 1-2% increase per degree in the diffusion coefficients (24).At physiological temperature (37 "C), recovery ranged from 84% at 0.21 pL/min to 93% a t 0.078 pL/min. Recovery from brain tissue is expected to be lower than from solution due to reduced mass transport to the dialysis cannula in the brain (25). This discrepancy can lead to error in estimating actual extracellular concentrations. This error should be reduced by perfusing a t very slow flow rates where recovery approaches 100%. For example, a t 40 nL/min the recovery of dopamine is greater than 95% a t room temperature. Under the same conditions, the metabolites DOPAC, HVA, and 5-HIM had recoveries of 95, 90, and 8870,respectively, while ascorbic acid and uric acid had recoveries of 88 and 91 YO. At 10 nL/min, the recoveries were even higher. At these very slow perfusion rates, the neural environment is perturbed less because the rate of removal of material from the extracellular fluid is less, thus suggesting a more accurate representation of extracellular concentrations. The effect of perfusion flow rate over a wider range of flow rates is illustrated in Figure 4, which shows the recovery as a function of perfusion rate between 0 and 10 pL/min. The recovery of sample can be expressed as a function of concentration or as a function of time. Relative recovery may be defined as the concentration of the perfused sample relative to the concentration of the external solution. Absolute recovery is defined as the amount in the perfusion medium per unit time. Both expressions of sample recovery are plotted in Figure 4. The relative recovery is low at the higher perfusion rates but approaches 100% (equilibrium) as the perfusion rate approaches zero. The absolute recovery is zero at zero flow, reaches a broad maximum at about 2-3 pL/min, and declines at the higher flow rates. Clearly, for maximum
0
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Figure 4. Amount of sample collected per minute as a function of perfusion flow rate (eq 1) uslng dialysis (0).Calculations were made assuming an external concentratlon of 1.O pM and adjusting for the recovery (A)across the &lysis membrane at each perfusion flow rate.
n l l
CI Figure 5. Sample dispersion of 5-min samples of 0.1 pM dopamine in 10 cm of 0.025-mm fused silica tubing at perfusion rates of 56, 109, and 300 nL/mln.
amount of material recovered, operation at a perfusion rate of about 2 rL/min is best. System Response. The sampling methodology should reproduce as accurately as possible any changes occurring in the extracellular fluid. An understanding of any distortion caused by the sampling process is important for accurate representation of the dynamics of the extracellular environment. Major concerns of the present work have been the effect of sampling methodology on the integrity of the sample with respect to dispersion and to the time response of the system. More specifically,the extent of the contribution of the various sampling components (perfusion system and injection valve) to sample dispersion and system response has been investigated. The extent that a 5-min sample is dispersed at three different perfusion rates is shown in Figure 5. At 300 nL/min, there is very little spread of the sample as it moves through the 0.025-mm tubing. At 109 nL/min the sample still retains its basic shape, but when the perfusion rate is lowered to 56 nL/min, serious dispersion of the sample occurs. If very
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
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Flgure 6. Sample dispersion as measured by volume variance for the fused silica tubing as a function of pertusion flow rate. Sample used was 0.1 pM dopamine. Tublng length from cannula to detector was 10 cm with an Internal diameter of 0.025 mm.
narrow samples are used for the characterization, for example, 5 s, the dispersion assumes a Gaussian shape. For this case, a good measure of sample dispersion is the sample volume variance (eq 2). The contribution of the dialysis cannula and the fused silica tubing to the sample volume variance as a function of perfusion rate is shown in Figure 6. From these data it is possible to determine the extent to which the sample is dispersed from the time it leaves the brain until it reaches the injection valve. It is important to minimize the dispersion of the sample zone as it moves through the perfusion system so that there is minimal overlap of consecutive samples. It is well-known that the use of air segmentation in a flowing stream restricts sample dispersion most effectively. Due to the nature of the dialysis cannula and the fact that air bubbles present in the flow may get caught inside the dialysis tubing, causing variations in recovery, air segmentation is not feasible with this sampling method. From Figures 5 and 6 it can be seen that at perfusion rates above 100 nL/min, sample dispersion is m i n i and c o n s b t . For a 5min sample obtained a t these flow rates, the sample width is increased approximately 28 %. This value was determined by measuring the peak width at 5 % peak height (26),where effects of tailing are readily apparent. For sampling intervals greater than 5 min, the contribution of the perfusion system to temporal distortion in sample handling can probably be ignored. For the 25-pm-diameter tubing used, sample dispersion increased dramatically a t perfusion rates slower than 100 nL/min. If very slow perfusion rates are required, then smaller diameter fused silica tubing can be used to decrease dispersion. Aside from the dispersion of the sample occurring during transport from the brain to the chromatograph, the effect of the chromatograph on system response must also be considered. The contribution of the injection valve and its connections to the system response can be seen in Figure 7. This figure shows the time delay for a steady-state response to be established as a function of various perfusion rates following a step change in concentration of sample. The time delay is not due to the injection loop itself. The loop always starts with pure mobile phase before each sample begins to flow into the loop. For one sampling volume, the fraction of mobile phme replaced by sample is a reproducible fixed amount for a given perfusion rate. The fraction can be increased and made to approach 1by decreasing sampling frequency. We have focused on using no interval between samples. This approach sacrifices a fraction of the signal for increased sampling frequency. The observed time delay is attributed to dead volume between the end of the fused silica tubing and the sample loop. This volume is present in the channel that
0
5
IO
15 20 25 TIME (MINUTES)
X,
35
40
Flgure 7. Complete system response as a function of perfusion flow rate. Distllled water and a DOPAC solution (10 pg/pL) were perfused with samples injected at 5-min intervals. Temperature was maintained at 37 O C .
connects the two and is due to the finite wall depth of the stainless-steel injection valve stator between the end of the fused silica tubing and the rotor. The volume of this channel is about 100 nL. At the high perfusion rates, the signal achieves steady state in the first 5-min sample interval, while at the slower perfusion rates, several injections are needed to wash out the dead volume before allowing the signal to reach a maximum. For example, a flow rate of 1.15 pL/min resulted in no time delay for a maximal response to be seen, whereas at a perfusion rate of 0.15 pL/min, the time delay seen until steady-state response was achieved was 20 min. This experiment was repeated without the dialysis membrane. No changes in the results were noted, indicating that the membrane does not contribute significantly to the observed time delay. By use of these data and the results from the perfusion system analysis, it appears that the major contribution to delay in the system response results from the volume present in the injection valve between the end of the fused silica tubing and the rotor of the injection valve. Chromatographic Response. The purpose of this work was to characterize the sampling methodology of on-line microdialysis. One of the most important parameters in such a characterization is the detection limit. In the present analysis, one is sample limited, particularly if a high sampling frequency is desired. Accordingly, the utility of microbore chromatography was examined. Both conventional and microbore chromatography can be used in conjunction with dialysis perfusion. The sensitivity requirements of the analysis dictate the mode used. The detection limit for dopamine with the conventional column was approximately 5 pg at a S I N of 2 while the detection limit on the microbore column was 300 fg with the same SIN. Both gave a linear response over the concentration range of interest. The small-bore column offers about a 16-fold sensitivity enhancement over the 4.6mm-i.d. column, indicating that microbore chromatography should be used in situations where high sensitivity is desired. Conventional chromatography offers sufficient sensitivity for many analyses (e.g., metabolites) without the more stringent demand of low system dead volume required with microbore columns. Sample volume is an important consideration when sampling on-line. Although the absolute sample size can be increased by increasing the sample volume, increasing sample size by injecting larger sample volumes has minimal utility when using microbore chromatography. To benefit fully from the sensitivity enhancement of microbore chromatography,
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the sample volume should be 5 pL or less (preferably less than 1p L to maintain high efficiency). The same is true for short, high-speed (10 cm with 3-pm packing) conventional columns. Excessive detector cell volumes can be detrimental to the response observed when using microbore columns. It has been recommended that the cell volume be reduced to approximately 0.1 the volume of the eluting peak to minimize dispersion (27). Also, Weber and Purdy have predicted that the current produced by an oxidizable species increases as the volume of the electrochemical cell decreases (28).Accordingly, the 51-pm spacer of the electrochemical cell was replaced with a spacer of 20 pm thickness. The channel width was also decreased from 5 mm to 3 mm, reducing the internal volume by a factor of about 4 to give a cell volume of 0.96 pL. This cell volume gave a signal 2.5 times larger than a cell of 1.4 pL. Choice of Sampling Parameters. By use of the results obtained in the characterization of this system, the working conditions for several different categories of analysis can be established. The sampling parameters given for the various categories are not necessarily optimal, but are intended to give a better understanding of how these parameters affect sampling. Trade-offs must be made for a given application. The sampling problem can be divided into four categories defined by two considerations, the concentration of the analyte and the frequency of sampling. For the purposes of this categorization, we are taking high concentration to mean greater than 1pM. High concentration and low sampling frequency is the simplest category. A requirement of high sampling frequency with high concentration creates an additional demand on the system. The third category is low concentration, sampled a t a low frequency. The last category is the most demanding, that of low concentration sampled at a high frequency. For each category, the amount of material recovered relative to the perfusion rate, sampling interval, sample loop volume, and mode of chromatography will be considered, along with possible applications for in vivo analysis. The first two categories involve the detection of compounds that are present in relatively high external concentrations. In the striatum of the rat, these are the neurotransmitter metabolites DOPAC and HVA, from dopamine, and 5-HIAA from serotonin, as well as ascorbic acid and uric acid. The extracellular concentrations of the neurotransmitter metabolites range from 5 to 20 pM. Thus, conventional chromatography provides sufficient sensitivity for their detection, particularly at low sampling frequencies. In these cases, sampling frequency is limited by the time required for the chromatography. Therefore, a column that separates in the desired time should be used. A high-efficiency short column can be used to shorten the time for separation of the metabolites. Although microbore chromatography may be used for the separation, the more stringent dead volume requirements must be considered. Figure 8 illustrates the automated sampling and chromatography of the dopamine metabolites, DOPAC and HVA, and the serotonin metabolite, 5-HIAA, using a 13-cm microbore column. As stated earlier, these columns perform best with sample volumes of 5 pL or less. After the sample volume is set, the perfusion flow is defined by the desired sampling frequency. An example of sampling parmeters for this category is the use of a short, high-efficiency conventional column with a 5-pL loop, a 10-min sampling interval, and a perfusion flow of 0.5 pL/min. This obviously assumes the separation is complete in 10 min. These conditions are appropriate for monitoring the dopamine metabolites during behavior. In general, behavioral studies require more frequent sampling to monitor small transient fluctuations in the extracellular concentrations, as compared to pharmacological manipulations which usually cause intense
I
I
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I
Flgure 8. Series of chromatograms of dialyzed perfusate from the anterior striatum of an anesthetized rat. Sampling of the ECF is continuous. The sample loop is filled as the previous sample is chromatographed. The elution order is DOPAC, 5-HIAA, and HVA. Conditions are described in text.
longer lasting changes. However, even for behavioral studies, the sampling frequency for metabolites need not be very high, while as high a sampling frequency as possible is needed for monitoring the neurotransmitters themselves. This difference arises because an abrupt change in neurotransmitter release results in only a very slow change in extracellular metabolites. A 1-min period of increased dopamine release generates a relatively slowly increasing concentration of DOPAC, which reaches a maximum 20 min after release and which lasts over an hour (29). The metabolites of dopamine and serotonin have been monitored in this lab with conventional chromatography at 30-min intervals using a 100-rL sample loop (2, 3). The third category to be examined is that of sampling compounds with low external concentrations, but with long sampling intervals. Without the need for a high sampling frequency, sample volumes can be larger. High-efficiency conventional or microbore columns can be used for this category of analysis (15). Figure 9 illustrates the detection of dopamine in vivo using a microbore column. A 1Bmin sampling interval was used with a perfusion flow of 0.42 pL/min and a 5-WLsample loop. There are cases of analyses in this category where microbore chromatography has advantages. The most notable case is that of determining the absolute extracellular concentration of a species in the extracellular fluid. Although significant dispersion of the sample occurs a t perfusion rates less than 0.1 pL/min, these very slow perfusion rates can be used for sample collection when sampling the steady-state resting level. Because of dispersion, rapid fluctuations in the extracellular environment would not be followed accurately, but that is not a problem when sampling the steady state. These slow perfusion flows can be used to determine basal concentration levels of the neurotransmitters and metabolites in the striatum of anesthetized rats. Previously, basal concentration levels have been estimated from data using flow rates of 2 pL/min with associated recoveries of approximately 20% per compound (11). The recovery of these compounds by the dialysis device is somewhat lower in vivo, creating some uncertainty in these estimates. An advantage of using the slower flows (10-40 nL/min) is that the recovery across the dialysis membrane approaches loo%, while removing significantly less material from the extracellular fluid, thus reducing the perturbation to the neural environment and making estimation of external concentrations more accurate. A 0.5-j~Lsample loop, a perfusion rate of 30 nL/min, and a 1-h sampling interval are currently being used for the determination of these basal concentration levels. The recovery across the dialysis device a t this flow is 95% or greater depending on the particular cannula. The analysis of compounds with relatively low external
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
0 4 8 TIME (rnins)
Figure 9. Chromatogram of dialyzed perfusate from the anterior striatum of an anesthetlzed rat. Dopamine elutes at 5 min and is the only peak on scale using this recorder. The peak height represents 25 pg in 5 pL of extracellular fluid as determined by comparison wth a 50-pg perfused standard at 37 O C . Conditions are described in the text. concentrations at a high sampling frequency is the most demanding of the four categories. An example of this category is the analysis of neurotransmitters in relationship to behavior. The extracellular concentrations are in the 10-50 nM range. While as high a sampling frequency as possible is desired, obviously, the sampling frequency can be no higher than that which gives an adequate signal for quantitative purposes. The sampling frequency is ultimately limited by the time required for the chromatography. In deciding how frequently the neurotransmitters must be sampled, the frequency components of the changing extracellular concentration are an important consideration. It is only necessary to sample at twice the highest frequency of interest, but the nature of these fluctuations for extracellular dopamine in behaving animals is unknown. In this last category the slower perfusion rates are useful for creating samples of higher concentration in a smaller volume. However, a t perfusion rates less than 0.1 pL/min sample dispersion in the fused silica tubing becomes a major concern (Figures 5 and 6). Dispersion of the sample as it is transported from the brain to the injection valve can make temporal resolution of external concentration difficult. Perfusion rates less than 0.1 pL/min are not satisfactory for this particular case without reducing the diameter of the tubing transporting the sample from the brain to the chromatograph. Reduction of the internal diameter of the fused silica tubing to 5 or 10 pm may allow these very slow perfusion rates to be used without excessive dispersion. The results obtained in this work are useful for planning on-line analyses. As an example, consider an application such as monitoring the neurotransmitter dopamine with a 5-min
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sampling interval. By use of the perfusion rate that maximized recovery per unit time, 2 pL/min, 10 pL would be collected. Extracellular dopamine, at a concentration of 10 nM, recovered at an exchange rate of about 20%, would result in 20 fmol, or 3 pg/sample. Thii could possibly be analyzed with a conventional short, high-efficiency column. On the other hand, using a perfusion rate of 0.1 pL/min produces a recovery of 90% in a volume of 0.5 pL, or 4.5 fmol(0.7 pg). The amount of dopamine is 5 times less, but the factor of 16 increase in sensitivity of the microbore column more than makes up for the difference. Also, the perturbation to the neural environment caused by removal of low-molecular-weight compounds is 5 times less. The four categories examined above present varying degrees of difficulty for the analyst. While the sampling of metabolites with long sampling intervals is relatively straightforward (2, 3), frequent sampling of the neurotransmitters per se is much more difficult. Smaller bore columns (less than 1 mm) can be used for further sensitivity enhancement of these relatively small samples. Currently, 0.1-0.5-mm4.d. columns are being investigated for this purpose. The use of these smaller inner diameter columns requires much more strigent dead volume considerations, especially that associated with the injector valve and detector cell. The advances being made in miniaturization of measurement instrumentation make studies involving smaller volumes realistic possibilities. It should be possible using open tubular columns with voltammetric detection, such as those described by Knecht et al. (23), to work with volumes of less than a nanoliter. A major reason such analyses are possible is the ability of voltammetric detection to be scaled down without loss of sensitivity (30). The combination of microscale highperformance liquid chromatography and voltammetric detectors creates new opportunities for investigation of biological structures by electroanalytical chemistry.
ACKNOWLEDGMENT We thank Ivan Mefford for supplying several microbore columns. Registry No. DOPAC,102-32-9;5-HIAA, 54-16-0;dopamine, 51-61-6; homovanillic acid, 306-08-1. LITERATURE CITED Salamone, J. D.; Hamby, L. S.; Nelll, D. B.; Justice, J. B. fharmacol. Blochem. Behav. 1984, 2 0 , 609-612. Blakely, R. D.; Wages, S. A.; Justice, J. B.; Herndon, J. G.; Neill, D. B. Brain Res. 1984, 308, 1. Justice, J. B.; Wages, S. A.; Michael, A. C.; Blakely, R. D.; Neill, D. B. J . Liq. C h M t O g r . 1983, 6 , 1673-1896. Gaddum. J. H. J. physiol. 1961, 155, 1-2. Besson, J. J.; Cheramy, A.; Glowinski, J.; Gauchy, C. Naunyn-Schmbd e k g s Arch. Pharmacol. 1973, 278, 101-105. Wleraszko, A. Acta Neurobiol. 1980, 40, 687-707. Levine, J. E.; Ramlrez, V. C. Endocrinology 1980, 707, 1782-1790. Loulils, c. c.; Hingtgen, H. N.; Shea, P. A.; Arpison, N. H. fharmacol. Blochem. Behav. 1980, 72, 959-963. Bayon, A.; Shoemaker, W. J.; Lugo, L.; Azad, R.; Line, N.; DruckerColin, R. R.; Bloon, R. E. Neurosci. Len. 1981, 2 4 , 65-70. Tossman, U.; Ungerstedt. U. Neurosci. Len. Suppl. 1981, 7 , S479. Zetterstrom, T.; Sharp, T.; Marsden, C.; Ungerstedt, U. J. Neufochem. 1983, 41, 1769-1773. Zetterstrom. T.; Ungerstedt. U. Eur. J. Pharmacol. 1984, 9 7 , 29-36. Johnson. R. D.; Justice, J. B., Jr. Brain Res. Bull. 1983, 10, 567-571. Ungerstedt, U. I n Measurement of Neurotransitter Release in Vivo; Ed.; Marsden, C. A., Wiiey: New York, 1984; pp 81-105. Imperato. A.; DIChlara. G. J. Neurochem. 1984, 4 , 966-977. Justice. J. B.; Neill. D. B.; Wages, S. A.; Blakely, R. D. The 7983 LC€C Symposium, Abstract, pp 38-40. Scott,R. P. W.; Kucera, P. J. Chromtogr. 1976, 725, 251-263. Caliiuri. E. J.; Mefford, I.N. Brain Res. 1984, 296, 156-159. Myer, R. F.; Hartwlck, A., Jr. Anal. Chem. 1984, 5 6 , 2211-2214. Lin, P. Y. T.; Blank, C. L. Cur.Sep. 1983, 5 . 3-6. Myers, R. D. Methods Psychobiol. 1972, 2 , 169-21 1 . Rocca. J. C.; Hiaains. J. W.: Brownlee. R. J. J. Chromafmr. - Sci. 1985. 2 3 , 106-l-l?3. Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 5 6 , 470-482. Bard, . A G.: Faulkner, L. R. Elecfrochemicai Methods; Wlley: New York, 1980; p 153.
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(25) Nicholson, C. Dynamics of the Brain Cell Microenvironment, MIT: Boston, MA, 1980. (26) Bidlingmeyer, E. A.; Warren, F. V., Jr. Anal. Chem. 1984. 5 6 , 1582A-1596A. (27) Kirkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dilks, C. H. J . Chromat o g . Sci. 1977, 75, 303. (28) Weber, S.G.; Purdy, W. C. Anal. Chim. Acta 1978. 700. 531-544. (29) Michael, A. C.; Justice, J. E., Jr.; Neill, D. E. Neurosci. Lett. 1985,56,
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RECEIVED for review June 3, 1985. Resubmitted February 20, 1986. Accepted April 2,1986. This work was supported by NSF Grants BNS 8210773 and 8509576 and the Emory University Research Fund.
Determination of Fentanyl and Related Compounds by Capillary Gas Chromatography with Electron Capture Detection J a m e s M. Moore,* Andrew C. Allen,' Donald A. Cooper, a n d S u s a n M. C a r r
Special Testing and Research Laboratory, Drug Enforcement Administration, 7704 Old Springhouse Road, McLean, Virginia 22102-3494
A method has been developed that allows for the differentlation and quantltatlon of fentanyl and 25 analogues and homologues In ifflclt preparations. After isdatlon from the sample matrlx, the fentanyl compound is sublected to derlvatlratlon wlth heptafluorobutyrlc anhydride In the presence of 4(dhnethylamlno)pyrldie. Mosl fentanyls yielded two derlvatiratlon products, both vlnylogous amldes. Upon chromatographic analysis an addltlonal compound, belleved to be an injectlon port thermal elkninatkn product, was produced. By use of these three chrornatographlc peaks, the malority of fentanyls studied could be easily differentiated. Accurate quantitative results and good reproduciblllty were achieved at fentanyl levels of between 0.001 and 1% w/w. All fentanyls yielded heptafluorobutyryl derivatives that were easlly detected on-column at low picogram levels using a nonpolar fused dllca caplllary column In the splltless mode Interfaced wlth a 83Nielectron capture detector.
Fentanyl, N - [1-(2-phenethy1)-4-piperidyl]propionanilide, is a narcotic analgesic of the 4-anilidopiperidine series. Clinically, it has a potency approximately 100 times that of morphine ( I ) . In recent years, the appearance and subsequent abuse of compounds closely related to fentanyl have been of growing concern to law enforcement officials. Since the late 1970s, 10 homologues and 6 analogues of fentanyl, including a-methylfentanyl and 3-methylfentanyl, have appeared on the illicit market. Some have been implicated in overdose deaths, especially on the west coast of the United States. The analogues and homologues of fentanyl are often encountered on the illicit market in highly adulterated dosage forms at levels of between 0.1 and 1% w/w. Thus, there is an ever-increasing need for the development of methodology that will allow for the rapid screening and low-level quantitation of these compounds. Recent studies have primarily utilized high-performance liquid chromatography (HF'LC) and gas chromatography-mass spectrometry (GC-MS) to accomplish these analyses (2-9). We describe here methodology that is very sensitive and highly specific for the detection of fentanyl and related compounds. Furthermore, our studies indicate that accurate Present address: Western Regional Laboratory, Drug Enforcement Administration, 450 Golden Gate Ave., Box 36075, San Francisco, CA 94102.
quantitative results can be achieved between levels of 0.001 and 1%w/w (based upon 100-mg sample weight). The procedure involves isolation of the fentanyl from the sample matrix followed by derivatization with heptafluorobutyric anhydride (HFBA) in the presence of 4-(dimethylamino)pyridine (4-DMAP). The two major products resulting from this derivatization are both vinylogous amides, a structural feature that allows easy isolation and also accounts for good product stability in solution. Upon chromatographic injection in the splitless capillary mode, a chromatographic peak resulting from the thermal degradation of one of the vinylogous amide products is produced. The thermal degradation and vinylogous amide peaks exhibited good chromatography at low picogram levels when chromatographed on a nonpolar fused silica capillary column. When these peaks are used, the methodology has proven effective for the differentiation of fentanyl and 25 of its analogues and homologues. We have also applied this procedure for the sensitive detection of N-(2-phenethyl)-4-phenyl-4-acetoxypiperidine (PEPAP), a substance that is structurally similar to a compound that has been implicated in producing Parkinsoniantype symptoms in its users. Owing to the high sensitivity and specificity of this methodology, we believe it also has potential application in toxicological analyses. EXPERIMENTAL SECTION Instrumentation. Low-resolution mass spectra were acquired on a Finnigan MAT Model 4630 quadrupole mass spectrometer. The GC-MS was fitted with a 12-m X 0.25-mm-i.d. fused silica capillary column coated with DB-5 (J & W Scientific, Inc., Rancho Cordova, CA) at a film thickness of 0.25 pm. Sample injection was accomplished with an on-column injector (J & W Scientific) at a helium carrier gas velocity of 60 cm/s. Data were acquired at an ionization potential of 60 eV and sourve temperature of 120 "C. Both positive and negative ion (electron capture) chemical ionization utilized methane reagent gas at a filament potential of 100 eV. For positive ion chemical ionization, the source temperature was maintained at 140 "C and a source pressure of 0.35 torr (uncorrected) existed. Negative ion data were acquired at a source temperature of 80 "C and a pressure of 0.5 torr. High-resolution mass spectral data were obtained with a Finnigan MAT Model 8230 (San Jose, CA) double-focusing GC-MS operating at an ionization potential of 70 eV. Sample introduction was accomplished with split mode injection (Grob-type injector) into a 30-m x 0.25-mm fused silica capillary column coated with DB-1 (J & W Scientific) at a film thickness of 0.25 pm. Source temperature was approximately 150 "C and data were acquired at a resolution of 10000 (5% valley).
This article not subject to U S . Copyright. Published 1986 by the American Chemical Society
