006
Anal. Chem. 1992, 6 4 , 806-810
REFERENCES
(9) Ndiomu, D. P.; Slmpson, C. F. Anel. Chlm. Act8 1988, 273,237-243. (10) Nam, K. s.;K a P h S.;Yanders, A. F.; Purl, R. K. c h e m s p b r e 1990, 20,873-880. (11) Nlessen, W. M. A.; Bergers, P. J. M.; Tjaden, U. R.; Van Der Greef, J. J . Chromatogr. 1988, 454, 243-251. (12) Stadallus, M. A.; Berus, J. S.; Snyder, L. R. LC-GC 1988, 6, 494-500.
( 1 ) Yacobl, A.; Skelly, J. P.; Batra, V. K. Tox/c&inetics and New Drug Development; Pergamon Press: New York, 1989. (2) Lim, C. K. Trends And. Chem. 1988, 7, 340-345. (3) Poole, S. K.; Dean, T. A.; Oudsema, J. W.; Poole, C. F. Anal. Chim Act8 1990, 236,3-42. (4) Hawthorne, S. B. Anal. Chem. 1990, 62,633A-642A. (5) Klng, J. W. J . Chromatogr. Sci. 1989, 27,355-364. (8) Hedrick, J. L.; Taylor, L. T. J . High Resolut. Chromatogr. 1990, 73, 312-316 - .- . . (7) Hedrlck, J. L.; Taylor, L. T. Anal. Chem. 1989, 67,1986-1988. (8) Ong, C . P.; Ong, H. M.; Li, S. F. Y.; Lee, H. K. J . Microcolumn Sep. 1990, 2,69-73.
- -
RECEIVED for review October 3, 1991. Accepted January 2, 1992.
Intravenous Microdialysis Sampling in Awake, Freely-Moving Rats Martin Telting-Diaz, Dennis 0. Scott, and Craig E. Lunte* Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045
Intravenous mlcrodlalysls sampllng In the awake, freelymovlng rat for the determlnatlon of free drug concentratlons In blood Is descrlbed. Intravenous mlcrodlalysls was performed wlth a nonmetallic, flexlble dlalysls probe. The pharmacoklnetlcsof theophylline were determlned uslng both mlcrodlalysls sampling and cdlectlon of whde blood folbwlng an Iv dose. There was no difference In the half-IHe of ellmlnatlon of theophylline determlned by mlcrodlalysls, 4.4 f 0.4 h, and whole blood sampllng, 4.5 f 0.7 h. The kinetics of ellmlnatlon were affected by removing Mood samples and by uslng anesthesla. The half-life of ellmlnatlon was 4.4 f 0.4 h when uslng slmultaneous mlcrodlalysls and whole-blood sampHng and only 3.0 f 0.4 h uslng mlcrodlalyslsabne. The half-llfe of ellmlnatlon was 17.0 f 7.1 h In chloral hydrate anestheslzed rats. Mlcrodlalysls samples were contlnuously collected for over 7 h wlthout fluld loss uslng a single experimental animal. Mlcrodlalysls sampllng directly assesses the free drug concentration In blood. The extent of theophylline blndlng to blood proteins was determined In vltro In rat plasma and rat whole blood uslng both ultraflltratlon and mlcrodlalysls. Theophylllne was (47.3 f 1.3)% bound In rat plasma and (52.2 f 1.6)% bound In rat whole blood. MIcrodlalysls sampling Is a powerful tool for pharmacoklnetlc studies, provldlng accurate and preclse phamacoklnetk data.
INTRODUCTION In vivo microdialysis has been demonstrated to be a powerful tool to continuously sample the blood and tissue of animals for metabolic and pharmacokinetic experiments.'* Experiments outside the central nervous system have been limited to anesthetized animals typically because of the rigid design of the microdialysis probes commonly employed. For neurochemical experiments the rigid probe can be used in awake, freely-moving animals because it can be secured to the In other tissues, the integrity of the microdialysis probe system is often compromised when the animal moves. Alternative designs of microdialysis probes that are flexible have been described.'O-13 The use of a flexible microdialysis probe overcomes the limitations of rigid probes and permits
experiments to be performed in all parts of a freely-moving animal. This report describes the application of in vivo microdialysis perfusion to the determination of the pharmacokinetics of theophylline in awake, freely-moving rats. It is well established that anesthetics can have a pronounced effect on the observed pharmacokinetics of drugs.14 The ability to use microdialysis sampling for pharmacokinetic determinations in awake, freely-moving rats greatly improves the relevance of the data obtained. In addition, microdialysis sampling directly provides the concentration of unbound drug in the blood relative to the total concentration determined from whole blood samples. It is generally considered that the unbound concentration is the pharmacologically more re1e~ant.l~ When binding is accounted for microdialysis sampling gives equivalent results to whole blood sampling.
EXPERIMENTAL SECTION Chemicals. Theophylline was purchased from Sigma Chemical Company (St. Louis, MO). HPLC-grade acetonitrile wm obtained from Fisher Scientific (FairLawn, NJ). All other chemicals were reagent grade or better and were used as received. Apparatus. Dialysis System. Microdialysis sampling was performed using a CMA/100 microinjection pump from Bioanalytical Systems, Inc./Ch4A (West Lafayette, IN) coupled to a microdialysis probe inserted into the jugular vein of the experimental animal. The perfusion medium was pumped through the probes at a flow rate of 1 pL/min for all experiments. Microdialysis samples were collected by a CMA/200 refrigerated fraction collector. Chromatographic System. The liquid chromatographic system consisted of a BAS PM-60 pump, an SPD-6AV variable wavelength UV-vis absorbance detector with a microbore cell (Shimadzu Scientific Instruments, Inc., Columbia, MD), and a Rheodyne 7125 injection valve with a 5-pL sample loop. Separation was achieved using a Brownlee 5 pm ODS (1-mm X 15-cm) column and a flow rate of 50 pL/min. For all experiments the UV detector was operated at 270 nm. Microdialysis Probe. The flexible microdialysis probe was similar to those described p r e ~ i o u s l y .The ~ ~ probe ~ ~ was constructed as shown in Figure 1by inserting two pieces of fused silica tubing, 75-pm i.d. and 147-pm 0.d. (Polymicro Technologies Inc., Phoenix, AZ),into a 5-mm length of polyethylene tubing, 0.2%" i.d. and 0.61" 0.d. One of the pieces of fused silica was inserted into but not through the polyethylene tubing. The other piece
0003-2700/92/0364-0806$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992
Fused Silica Tubing 0.075 mm LD 0.14 mm O.D.
I/ i
0.61 mm O D
PolyethyleneTubing 0.28 mm I.D. 0.61 mm O.D.
EPOXY
1 11- :rD~ I
;;;ec
0.250 mm O.D.
Flguro 1. Schematic diagram of the flexible micrcdlalysis probe and parallel iv cannula.
of fused silica was inserted through the polyethylene tubing such that 5 mm was exposed. The exposed piece of fused silica tubing was covered with the dialysis fiber which was then attached to the polyethylene tubing with epoxy (Devcon, Danvers, MA). The other end of the dialysis fiber was sealed with the epoxy. Regenerated cellulose dialysis fibers with a 232-pm i.d. and 250-pm 0.d. and a molecular weight cut-off of 5000 were used (Dow Chemical Corporation, Midland, MI). Microdialysis Probe Characterization. To determine the in vivo concentration of theophylline giving rise to the concentration detected in the perfusion medium, it is necessary to know the recovery of the dialysis probe. The recovery is defined as the ratio of the analyte concentration determined in the dialysate to the actual concentration of the analyte in the sample. Recoveries were determined in rat whole blood, rat plasma, and pH 7.4 Ringer's solution consisting of 155 mM NaC1,5.5 mM KC1, and 2.3 mM CaC12 All experiments were performed in a thermostated shaker bath to both maintain the temperature at 37 "C and provide the hydrodynamic system to model flowing blood. The free drug concentration in the whole-blood and plasma samples was determined by ultrafiltration. There was no difference in the recoveries from the different matrices when binding of theophylline to blood proteins was taken into acount and was consistent with previous reports.1° The average recovery of theophylline for all probes used in these experiments was (24.2 f 1.8)%. The recovery of each dialysis probe was determined both before and after implantation. The recovery determined after the implantation was used to calculate in vivo concentrations although the two recoveries never differed by more than 5% relative. Protein Binding. Because microdialysis only samples the free drug in blood, the extent of binding of the drug to blood proteins must be known to compare results from microdialysis to those from whole b1ood.l6 The extent of binding of theophylline to rat blood proteins was determined by ultrafiltration using both whole blood and plasma. Ultrafiltration was performed with an MPS-1 micropartition system with a YMT-membrane filter (Amicon, Lexington, MA). Samples of both EDTA-treated rat whole blood and rat plasma were spiked with known concentrations of theophylline and equilibrated for 1h at 37 "C. The free fraction was then separated by ultrafiltration and analyzed by liquid chromatography. In Vivo Pharmacokinetic Experiments. Four to five month old SpragueDawley rats weighing approximately 400 g were used. Rats were anesthetized with the inhalation anesthetic isoflurane. Isoflurane was chosen because it has a very short duration of action. The rat is typically fully recovered in less than 30 min after the administration of isoflurane is stopped. For experiments in anesthetized animals, the rat was initially anesthetized with isoflurane but then given a 400 mg/kg ip dose of chloral hydrate. After 2 h, maintenance doses of 200 mg/kg chloral hydrate were given every hour by iv administration.
807
A small incision through the skin was made at the back of the neck and on the right shoulder. The jugular vein was exposed and a small nick made into the vein. The microdialysis probe and a cannula were inserted through this nick, and the probe and cannula were threaded through the vein to a region near the heart (ca. 2.5 cm). The cannula was backed-off 5 mm from the dialysis membrane. The jugular vein was then ligated. The inlet and outlet tubing of the dialysis probe and the cannula tubing were then threaded under the skin and out the incision on the back of the neck. Both incisions were closed with surgical staples and the rat was placed in a flexible jacket that covered both incisions and supported the placement of the dialysis probe. The rat was then attached to the awake animal system (CMA/BAS) with a stiff wire attached to a collar on the rat and to a counterbalanced arm attached to the animal container. The counterbalanced arm also had a two-channel liquid swivel through which the dialysis probe was attached to the perfusion pump and fraction collector. The liquid swivel allows the animal fullmotion without the tubing becoming entwined and kinked. To validate the microdialysis sampling technique, whole-blood samples were simultaneously collected. This was done through a cannula implanted along with the dialysis probe. A 100-pL sample of whole blood was collected every 30 min for the first hour then once every hour. The whole-blood sample was centrifuged for 10 min; 40 pL of the resulting plasma was treated with an equal volume of 0.7 M perchloric acid to precipitate proteins and centrifuged for 10 min. The supernatant was injected into the chromatographic system. Pharmacokinetic experiments were performed by perfusing the implanted probe with a Ringer's solution a t a perfusion rate of 1 pL/min. Samples were continuously collected over 10-min intervals. Dialysis samples were diluted with Ringer's solution as needed to keep the concentration in the range of calibration. Blanks were collected for at least 1h following insertion of the microdialysis probes. No chromatographic interferences were observed in the blanks. The animal was then dosed with theophylline (15 mg/kg) in 0.5 mL of Ringer's solution iv at 37 "C. Dialysis samples were collected for 6-7 h after dosing. The in vivo concentration of theophylline was calculated by determining the concentration in the dialysate from a standard curve and then accounting for the recovery of the microdialysis probe. Microdialysis is a continuous sampling technique, therefore each sample represents the average concentration of analyte in the blood during the sampling interval. This is compared to taking discrete blood samples that represent the concentration in the blood only at the time of sampling. Because of the continuous sampling, microdialysis is an integrating technique that is less prone to fluctuations than an instantaneous sampling technique. Pharmacokinetic parameter calculationsare also simplified. For example, area-under-the-curve (AUC) calculations are performed by summing the product of the concentration (jg/mL), perfusion rate (mL/min), and sample interval (min) for all samples instead of interpolating between points with the trapezoidal rule. Pharmacokinetic Calculations. Terminal half-lives of free theophylline were estimated by fitting an equation of the form C ( t ) = A exp(-crt) + B exp(-ot) (1) using a nonlinear least-squares fit. The first term in eq 1describes
the distribution phase following an iv bolus injection of theophylline where a is the distribution rate constant. The second term describes the elimination phase were 6 is the elimination rate constant. The half-life of elimination can be determined from the elimination rate constant (PI. The concentration in the blood at the time of injection (Co)can be calcuIated by adding the terms A and B. The AUC values for blood sampling were estimated using the linear trapezoidal rule and were extrapolated to infiity by adding C,/& where C , is the concentration of the last measurement. For the microdialysis data, the AUC was estimated by summing the products of the measured concentrations and the collection time interval with the addition of C,/O (eq 2). Noncompartmental
-
AUC = E C t A t 0
ct +P
analysis was used to obtain the total body clearance of theophylline
808
ANALYTICAL CHEMISTRY, VOL. 64,
NO. 7, APRIL 1, 1992
Table I. Binding of Theophylline to Blood Protein"
theophylline concentration 10 pg/mL 20 pg/mL
1 pg/mL
plasma microdialysis ultrafiltration whole blood microdialysis ultrafiltration
47.7 f 2.7 47.5 f 2.6
48.4 f 3.6 50.1 f 2.9
45.9 f 4.5 52.2 f 3.3
53.8 f 4.6 49.9 f 2.8
52.1 f 3.0 53.0 f 7.0
50.7 f 6.7 54.6 f 5.0
n n = 3. Table 11. Recovery of Theophylline by Microdialysis"
theophylline concentration 1 pg/mL 10 pg/mL 20 pg/mL Ringer's plasma whole blood O n
24.4 f 1.9 26.6 f 3.0 22.4 f 3.2
24.9 f 2.2 25.6 f 5.1 22.4 f 4.5
21.7 f 1.7 22.6 f 4.7 21.1 f 6.3
I
,
,
,
,
0
2
4
6
8
,
1
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l
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3
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4
6
8
i
d
1012
- n e lm1"JteS)
Figure 2. Typical chromatograms of iv microdlalysis samples: (A) blank prior to dosing, (B) 30 min after a 15 mg/kg iv dose of theophylline, (C) 300 min after a 15 mg/kg iv dose.
= 3.
(C, = dose/AUC) and the volume of distribution (Vd = C,/p). The mean residence time (MRT) was calculated by dividing the AUMC (first moment of the area under the curve) by the AUC. All results are expressed as the mean f standard deviation.
RESULTS AND DISCUSSION Probe Calibration and Protein Binding. The extent of theophylline binding to blood proteins is listed in Tible I. The percent bound is independent of the concentration of theophylline in the range studied. Theophylline was found to be (52.2 f 1.6)% bound in rat whole blood and (47.7 f 0.8) % bound in rat plasma. This is in good agreement with previous results using human plasma." The whole blood value was used in subsequent comparisons of microdialysis samples to whole-blood samples. The recovery of the microdialysis probe was determined in whole blood, plasma, and Ringer's solution over the concentration range needed for the pharmacokinetic experiments. The recovery results are tabulated in Table 11. The recovery is independent of the concentration and the matrix for these experiments. Based on these results, subsequent probes were only calibrated using the Ringer's solution. Intravenous Microdialysis Sampling. Typical chromatograms of blood dialysate obtained by in vivo sampling are shown in Figure 2. In vivo dialysis samples were directly injected into the chromatograph. The blank sample was run at the most sensitive detector gain setting used to determine if any endogenous interferences occur a t high gain. As can be seen (Figure 2A), no interferences occur in the blanks obtained prior to dosing of the animal with theophylline. The whole-blood samples were more difficult to analyze, requiring protein precipitation and removal before injection into the chromatographic system. These samples exhibited more potential interferences in the blanks relative to the dialysis samples (Figure 3). Fortunately, none of these endogenous compounds interfered with the determination of theophylline with this separation. For comparison, the whole-blood samples of Figure 3 correspond in time to the dialysis samples of Figure 2. Pharmacokinetic Comparison to Whole-Blood Samples. Microdialysis sampling was validated by simultaneously collecting blood samples. Pharmacokinetic curves obtained by the two methods were identical (Figure 4) when the samples were taken simultaneously and binding of theophylline to blood proteins is taken into account. Also shown in Figure 5 is a semi-log plot for the microdialysis sampling data clearly showing the fast distribution phase followed by the slower
L
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1
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2
4
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,
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Flgure 3. Typical chromatograms of iv whole-blood samples: (A) blank prior to dosing, (B)30 min after a 15 mg/kg iv dose of theophylline, (C) 300 min after a 15 mg/kg iv dose. 'O
1
60
3
50
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100
150
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250
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Flgure 4. Concentration-versus-tlme profile for a 15 mg/kg Iv dose of theophylline In the awake, freely-movlng rat. The bars represent the microdialysis samples and the circles represent the whole-blood samples. The concentration of theophylline in whole blood is corrected for protein binding. Shown in the inset is a plot of log concentrationversus-time using the microdialysis sampling data.
elimination phase. The pharmacokinetic parameters extracted from the data are listed in Table III. The results for the whole blood are in good agreement with previously reported values.18 The values for whole blood sampling and simultaneous microdialysis sampling show no statistical difference. The half-life of elimination determined from microdialysis samples
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7,APRIL 1, 1992
809
1 Table IV. Pharmacokinetics of Theophylline (15 mg/kg iv) in Awake and Anesthetized Rats"
100
3
[\ i
3 .-c5 e
tl,z distribution, min t , elimination, h dRT, h
AUC,pg min/L vd, L
1
C,, mL/min
0
awake
anesthetized
3.6 f 2.2 3.0 f 0.4 2.5 0.6 3523 f 1414 1.2 f 0.4 4.7 f 1.9
2.4 f 0.6 17.0 f 7.1 24.6 f 1.1 32626 f 8084 0.15 f 0.03 0.10 f 0.02
*
C
8
n n = 3.
10
0
I
I
50
100
150
200
Time (minutes)
Flgurr 5. Semi-log presentation of the concentratlon-versus-tlme profile for 15 mg/kg iv doses of theophylline In rats determined using
mlcrodlalysls sampllng. Awake animal without blood sampling (m): awake animal wlth simultaneous blood sampling (0):anesthetized animal (+I. Table 111. Pharmacokinetic Parameters for Theophylline-Dosed iv at 15 mg/kg" microdialysis sampling w/o blood sampling
tlI2 distribution, min h
o n , itn a& J$ i
AUC,bg min/mL v,, L C,, mL/min
w / blood sampling
blood samplingb
3.6 f 2.2 5.2 f 1.3 7.8 f 4.1 3.0 f 0.4 4.4 f 0.7 4.4 f 0.4 2.5 0.6 2.8 f 0.8 2.9 f 0.8 3525 f 1414 5616 f 1614 6135 f 1267 1.2 f 0.4 1.1 f 0.3 1.1 f 0.3 4.7 f 1.9 2.9 f 0.7 2.5 f 0.5
" n = 3. D a t a corrected for protein binding; values reflect unbound theoDhvllin concentration. was 4.4 f 0.4 h (n = 3) and from whole-blood samples was 4.5 f 0.7 h (n = 3). However, when microdialysis was performed alone a half-life of only 3.0 f 0.4 h (n = 3) was found. This half-life is statistically different from the half-life with blood sampling, p < 0.004. In addition, when only microdialysis sampling is performed the AUC and clearance are significantly different while the MRT and V , are the same relative to simultaneous blood sampling. It has been reported previously that the pharmacokinetics can be affected by blood sampling because of resulting changea in blood volume. These data indicate that microdialysis sampling eliminates such perturbations because no net fluid is removed even with continuous sampling for several hours. Effect of Anesthesia. The pharmacokinetics of theophylline are also greatly affected by the use of anesthesia during the pharmacokinetic experiment. This is shown in Table IV where the pharmacokinetic parameters obtained by microdialysis sampling in awake, freely-moving rata are compared to those in anesthesized rata. All parameters affected by the rate of metabolism are dramatically different in the anesthetized rat whereas the rate of distribution, which depends predominantly on blood flow, is unchanged. That the half-life of elimination more than quadruples is an indication that the anesthetic is inhibiting the metabolic enzymes of the liver. The effect of experimental conditions on the pharmacokinetics of theophylline in rats as determined by microdialysis sampling is shown graphically in Figure 5. Using the semi-log presentation of the concentration-versus-time curve, the slope of the lines is proportional to the rate of that phase. As can be seen, the initial rapid distribution phase is the same
in all cases. However, the elimination phase varies considerably depending upon both whether the animal is anesthetized and whether blood samples are drawn simultaneously with the microdialysis.
CONCLUSIONS Microdialysis offers several advantages for pharmacokinetic studies. The temporal resolution is much higher than for other methods. While 10-min intervals were used for these experiments, shorter times are easily achieved if needed. Since no blood is drawn, a large number of samples can be collected from a single animal without loss of fluid volume. Simultaneous sampling can be achieved using multiple dialysis probes. This provides the ability to monitor pharmacokinetics at multiple sites in a single animal. Because complete pharmacokinetic curves can be obtained for several organs using a single experimental animal, overall fewer animals will be necessary to obtain data on a given drug. Microdialysis samples only the free fraction of the drug in blood. In addition, microdialysis coupled to collection of whole blood samples provides a technique to determine the degree of drug binding in vivo during a pharmacokinetic experiment. The major limitation of microdialysis sampling is the small sample volumes obtained. The sample volume, perfusion rate, recovery, and sampling interval are all interrelated. Slow perfusion rates provide higher recoveries but smaller samples per unit time. Therefore the detection method must be sufficient to directly determine the lowest drug concentration necessary. ACKNOWLEDGMENT We wish to acknowledge the Center for Bioanalytical Research at The University of Kansas, Bioanalytical Systems, Inc., and Merck, Sharpe & Dohme for partial financial support of this work. D.O.S.acknowledges the financial assistance of the Procter & Gamble Co. through the P&G Bioanalytical Fellowship a t The University of Kansas. REFERENCES (1)
Scott. D. 0.;Sorenson. L. R.; Lunte, C. E. J . Chromefogr. 1990, 506,
461-469. (2) Scott, D. 0.;Sorenson, L. R.; Steeie, K. L.; Puckett, D.L.; Lunte, C. E. Pharm. Res. 1991, 8 . 389-392. (3) Steele, K. L.; Scott, D. 0.;Lunte, C. E. Anal. Chlm. Acta 1991, 246, 18 1-186. (4) Lunte, C. E.; Scott, D. 0.; Kisslnger, P. T. Anal. Chem. 1991, 63, 773A-780A. ( 5 ) Caprioli, R. M.; Lin, S. Roc. Nafl. Aced. Scl. U . S . A . 1990, 87, 240-243. (6) Menacherry, S. D.; Justice, J. B. Anal. Chem. 1990, 62, 597-601. (7) Ungerstedt. U. Usasuremnf of Newotransm/tter Rebase In Vivo; Marsden, C. A., Ed.; Wiley-Interscience: Chlchester, 1984 pp 81-105. (8) Ungerstedt, U.; Forster, C.; Herrera-Marschitz, M.; Hoffman, I.; Jungnelius, U.; Tossman, U.; Zetterstrom, T. Neuroscl. Left. (Suppl.)1982, 10,493. (9) Sandberg, M.; Lindstrom, S. J . Neuroscl. Methods 1983, 9 , 65-74. (10) Wages, S. A.; Church, W. H.; Justice, J. B., Jr. Anal. Chem. 1986, 58, 1649-1656. (11) Church, W. H.; Justlce, J. B., Jr. Anal. Chem. 1987, 59, 712-716. (12) Pettit, H. 0.; Pan, H. T.; Parsons, L. H.; Justice, J. E., Jr. J . Nevochem. 1990, 55, 798-804. (13) Pan, H. T.; Menacherry, S.; Justice, J. B., Jr. J . Neurochem. 1991, 56, 1299-1306.
Anal. Chem. 1992, 64, 810-814
810
(14) Mather, L. E.; Runciman, W. B.; Ilsley, A. H. Reg. Anaestb. 1982, 7 (SUPPI 4), S24433. (15) Benet, L. 2.; Mitchell, J. R.; Sheiner, L. E. The Pharmacological Basis
Theramutics. 8th ed.: Gilman. A. G.. Rall. T. W.. Nies. A. S.. Tavlor. P.. Eds.;'Pergamon Press: New York, t991;p 12. (16) Herrera, A. M.; scott, D. 0.;Lunte, G. E. /'harm. Res, 1990, 7,
(17) Ogilvie, R . I. Clin. Pharm. 1978, 3,267-293. (18) Fruncillo, R . J.; Digregorio, G. J. J . Pharm. Sci. 1984, 73, 1117-1 121.
Of
1077-10Sl.
RECEIVED for review October 14, 1991. Accepted January 2, 1992.
Parts-per-Trillion Determination of Trihalomethanes in Water by Purge-and-Trap Gas Chromatography with Electron Capture Detection Louis LBpine* and Jean-Franqois Archambault Znstitut de recherche d'Hydro-QuCbec, 1800 mont6e Sainte- Julie, Varennes, QuCbec, Canada J3X 1Sl
A gas chromatographlc method uslng a purge-and-trap concentrator and electron capture detectlon to monltor trlhalomethanes (THMs) at the low parts-per-bllllon (ppb) and the parts-per-trllllon (ppt) levels Is described In thls paper. The method shows good Ilneartty over 4 orders of magnltude wlth a detectlon llmlt between 0.01 and 0.05 ppt for each THM normally found after chlorlnatlon: chloroform, bromodlchloromethane, dlbromochloromethane, and bromoform. Speclal procedures, for sample handllng and the preparatlon of blank water and analytlcal standards, requlred at such a low Concentration, are a h dtscussed. The anatytlcat method has been successfully applled In the analysls of samples taken at dlfferent polnts of the water treatment plant of HydroQuibec's Gentllly 2 nuclear power statlon. Analysls of samples taken after chlorlnatlon and demlnerallzatlonrevealed the formatlon of all four THMs and other unldenttfled halogenated volatlle compounds. Chlorotorm and bromodlchloromethane were found at the low ppb level, whlle dlbromochloromethane and bromoform were present at the ppt level.
INTRODUCTION Nuclear and fossil-fueled power plants generally have their own water treatment unit, similar to those used to produce municipal drinking water, which includes a chlorine-disinfection step that is known to produce trihalomethanes (THMs). The far most common THM in chlorinated water is chloroform (CHCl,), although lower concentrations of brominated THMs such as bromodichloromethane (CHEW&), dibromochloromethane (CHBr,Cl), and bromoform (CHBr,) are often The iodine-containing compounds reported by a few workers4,j are not usually found. These molecules are not likely to be removed by the demineralization system and are therefore introduced into the steam-generation cycle where the water is heated at temperatures reaching 260 OC or more. A t Hydro-QuBbec's Gentilly 2 nuclear power plant, a 685 MW CANDU-PHW (CANadian Deuterium Uranium-Pressurized Heavy Water), abnormally high chloride concentrations have been observed in steam-generator blowdown and found to be related to the THM level in the makeup water.6 A recent study conducted in our laboratories on the thermal stability of chloroform confirmed the latter's rapid degradation to produce three C1- ions? It is believed that other
THMs will follow the same degradation mechanism to yield the corresponding acid (HCl, HBr) and subsequently increase the bromide and chloride level.8 This is of great concern to the nuclear industry because of the known corrosive effect of chloride on structural components. Owing to the concentrating factor involved in steam generators for nonvolatile species such as chloride, even a small amount of THMs in the makeup water can result in chloride concentrations exceeding the limit for safe system operation. The vast majority of methods used for THM analysis are designed for quality control of drinking water. Their analytical range is limited to parts per billion (ppb), in compliance with the 100 ppb total THM maximum contaminant level prescribed in the USEPA Safe Drinking Water Act. The particular application to the nuclear plant operation called for a more sensitive technique to analyze samples with individual THM concentrations in the low-ppb or even in the partsper-trillion (ppt) range and, in our study on thermal degradationP7to monitor their residual concentration in the low-ppt range. Gas chromatography with electron capture detection (ECD) is an attractive method for THM analysis since it offers a wide linear dynamic range and is the most sensitive method available for halogenated organic compounds. To a certain extent, it is specific to organohalides, which means the latter can be discriminated from a complex background. However, this detector cannot tolerate a large quantity of water and, although direct aqueous injection with solvent bypass has been r e p ~ r t e da, ~preextraction step is generally performed. Three different approaches have been considered: liquid-liquid extraction,1°'12headspace sampling,12-14and purge-and-trap.'j Comparison studies12J6have shown that even with ECD detection, liquid-liquid extraction and headspace sampling are limited to the ppb range; these methods are also subjected to airborne or solvent contamination, often giving high blank values. The purge-and-trap technique is free from these problems because it uses an inert gas to extract THMs from water and the sample, once introduced, is never in contact with the atmosphere. The concentrating factor obtained by trapping the analytes selectively on an adsorbent material allows analysis of very low-concentration samples. This paper describes the analytical method developed for ultratrace analysis of the four THMs of interest in water samples, using a purge-and-trap concentrator coupled to a gas chromatograph
0003-2700/92/0364-0810$03.00/0 0 1992 American Chemical Society
Anal. Chem. 1992, 6 4 , 806-810
REFERENCES
(9) Ndiomu, D. P.; Slmpson, C. F. Anel. Chlm. Act8 1988, 273,237-243. (10) Nam, K. s.;K a P h S.;Yanders, A. F.; Purl, R. K. c h e m s p b r e 1990, 20,873-880. (11) Nlessen, W. M. A.; Bergers, P. J. M.; Tjaden, U. R.; Van Der Greef, J. J . Chromatogr. 1988, 454, 243-251. (12) Stadallus, M. A.; Berus, J. S.; Snyder, L. R. LC-GC 1988, 6, 494-500.
( 1 ) Yacobl, A.; Skelly, J. P.; Batra, V. K. Tox/c&inetics and New Drug Development; Pergamon Press: New York, 1989. (2) Lim, C. K. Trends And. Chem. 1988, 7, 340-345. (3) Poole, S. K.; Dean, T. A.; Oudsema, J. W.; Poole, C. F. Anal. Chim Act8 1990, 236,3-42. (4) Hawthorne, S. B. Anal. Chem. 1990, 62,633A-642A. (5) Klng, J. W. J . Chromatogr. Sci. 1989, 27,355-364. (8) Hedrick, J. L.; Taylor, L. T. J . High Resolut. Chromatogr. 1990, 73, 312-316 - .- . . (7) Hedrlck, J. L.; Taylor, L. T. Anal. Chem. 1989, 67,1986-1988. (8) Ong, C . P.; Ong, H. M.; Li, S. F. Y.; Lee, H. K. J . Microcolumn Sep. 1990, 2,69-73.
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RECEIVED for review October 3, 1991. Accepted January 2, 1992.
Intravenous Microdialysis Sampling in Awake, Freely-Moving Rats Martin Telting-Diaz, Dennis 0. Scott, and Craig E. Lunte* Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045
Intravenous mlcrodlalysls sampllng In the awake, freelymovlng rat for the determlnatlon of free drug concentratlons In blood Is descrlbed. Intravenous mlcrodlalysls was performed wlth a nonmetallic, flexlble dlalysls probe. The pharmacoklnetlcsof theophylline were determlned uslng both mlcrodlalysls sampling and cdlectlon of whde blood folbwlng an Iv dose. There was no difference In the half-IHe of ellmlnatlon of theophylline determlned by mlcrodlalysls, 4.4 f 0.4 h, and whole blood sampllng, 4.5 f 0.7 h. The kinetics of ellmlnatlon were affected by removing Mood samples and by uslng anesthesla. The half-life of ellmlnatlon was 4.4 f 0.4 h when uslng slmultaneous mlcrodlalysls and whole-blood sampHng and only 3.0 f 0.4 h uslng mlcrodlalyslsabne. The half-llfe of ellmlnatlon was 17.0 f 7.1 h In chloral hydrate anestheslzed rats. Mlcrodlalysls samples were contlnuously collected for over 7 h wlthout fluld loss uslng a single experimental animal. Mlcrodlalysls sampllng directly assesses the free drug concentration In blood. The extent of theophylline blndlng to blood proteins was determined In vltro In rat plasma and rat whole blood uslng both ultraflltratlon and mlcrodlalysls. Theophylllne was (47.3 f 1.3)% bound In rat plasma and (52.2 f 1.6)% bound In rat whole blood. MIcrodlalysls sampling Is a powerful tool for pharmacoklnetlc studies, provldlng accurate and preclse phamacoklnetk data.
INTRODUCTION In vivo microdialysis has been demonstrated to be a powerful tool to continuously sample the blood and tissue of animals for metabolic and pharmacokinetic experiments.'* Experiments outside the central nervous system have been limited to anesthetized animals typically because of the rigid design of the microdialysis probes commonly employed. For neurochemical experiments the rigid probe can be used in awake, freely-moving animals because it can be secured to the In other tissues, the integrity of the microdialysis probe system is often compromised when the animal moves. Alternative designs of microdialysis probes that are flexible have been described.'O-13 The use of a flexible microdialysis probe overcomes the limitations of rigid probes and permits
experiments to be performed in all parts of a freely-moving animal. This report describes the application of in vivo microdialysis perfusion to the determination of the pharmacokinetics of theophylline in awake, freely-moving rats. It is well established that anesthetics can have a pronounced effect on the observed pharmacokinetics of drugs.14 The ability to use microdialysis sampling for pharmacokinetic determinations in awake, freely-moving rats greatly improves the relevance of the data obtained. In addition, microdialysis sampling directly provides the concentration of unbound drug in the blood relative to the total concentration determined from whole blood samples. It is generally considered that the unbound concentration is the pharmacologically more re1e~ant.l~ When binding is accounted for microdialysis sampling gives equivalent results to whole blood sampling.
EXPERIMENTAL SECTION Chemicals. Theophylline was purchased from Sigma Chemical Company (St. Louis, MO). HPLC-grade acetonitrile wm obtained from Fisher Scientific (FairLawn, NJ). All other chemicals were reagent grade or better and were used as received. Apparatus. Dialysis System. Microdialysis sampling was performed using a CMA/100 microinjection pump from Bioanalytical Systems, Inc./Ch4A (West Lafayette, IN) coupled to a microdialysis probe inserted into the jugular vein of the experimental animal. The perfusion medium was pumped through the probes at a flow rate of 1 pL/min for all experiments. Microdialysis samples were collected by a CMA/200 refrigerated fraction collector. Chromatographic System. The liquid chromatographic system consisted of a BAS PM-60 pump, an SPD-6AV variable wavelength UV-vis absorbance detector with a microbore cell (Shimadzu Scientific Instruments, Inc., Columbia, MD), and a Rheodyne 7125 injection valve with a 5-pL sample loop. Separation was achieved using a Brownlee 5 pm ODS (1-mm X 15-cm) column and a flow rate of 50 pL/min. For all experiments the UV detector was operated at 270 nm. Microdialysis Probe. The flexible microdialysis probe was similar to those described p r e ~ i o u s l y .The ~ ~ probe ~ ~ was constructed as shown in Figure 1by inserting two pieces of fused silica tubing, 75-pm i.d. and 147-pm 0.d. (Polymicro Technologies Inc., Phoenix, AZ),into a 5-mm length of polyethylene tubing, 0.2%" i.d. and 0.61" 0.d. One of the pieces of fused silica was inserted into but not through the polyethylene tubing. The other piece
0003-2700/92/0364-0806$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992
Fused Silica Tubing 0.075 mm LD 0.14 mm O.D.
I/ i
0.61 mm O D
PolyethyleneTubing 0.28 mm I.D. 0.61 mm O.D.
EPOXY
1 11- :rD~ I
;;;ec
0.250 mm O.D.
Flguro 1. Schematic diagram of the flexible micrcdlalysis probe and parallel iv cannula.
of fused silica was inserted through the polyethylene tubing such that 5 mm was exposed. The exposed piece of fused silica tubing was covered with the dialysis fiber which was then attached to the polyethylene tubing with epoxy (Devcon, Danvers, MA). The other end of the dialysis fiber was sealed with the epoxy. Regenerated cellulose dialysis fibers with a 232-pm i.d. and 250-pm 0.d. and a molecular weight cut-off of 5000 were used (Dow Chemical Corporation, Midland, MI). Microdialysis Probe Characterization. To determine the in vivo concentration of theophylline giving rise to the concentration detected in the perfusion medium, it is necessary to know the recovery of the dialysis probe. The recovery is defined as the ratio of the analyte concentration determined in the dialysate to the actual concentration of the analyte in the sample. Recoveries were determined in rat whole blood, rat plasma, and pH 7.4 Ringer's solution consisting of 155 mM NaC1,5.5 mM KC1, and 2.3 mM CaC12 All experiments were performed in a thermostated shaker bath to both maintain the temperature at 37 "C and provide the hydrodynamic system to model flowing blood. The free drug concentration in the whole-blood and plasma samples was determined by ultrafiltration. There was no difference in the recoveries from the different matrices when binding of theophylline to blood proteins was taken into acount and was consistent with previous reports.1° The average recovery of theophylline for all probes used in these experiments was (24.2 f 1.8)%. The recovery of each dialysis probe was determined both before and after implantation. The recovery determined after the implantation was used to calculate in vivo concentrations although the two recoveries never differed by more than 5% relative. Protein Binding. Because microdialysis only samples the free drug in blood, the extent of binding of the drug to blood proteins must be known to compare results from microdialysis to those from whole b1ood.l6 The extent of binding of theophylline to rat blood proteins was determined by ultrafiltration using both whole blood and plasma. Ultrafiltration was performed with an MPS-1 micropartition system with a YMT-membrane filter (Amicon, Lexington, MA). Samples of both EDTA-treated rat whole blood and rat plasma were spiked with known concentrations of theophylline and equilibrated for 1h at 37 "C. The free fraction was then separated by ultrafiltration and analyzed by liquid chromatography. In Vivo Pharmacokinetic Experiments. Four to five month old SpragueDawley rats weighing approximately 400 g were used. Rats were anesthetized with the inhalation anesthetic isoflurane. Isoflurane was chosen because it has a very short duration of action. The rat is typically fully recovered in less than 30 min after the administration of isoflurane is stopped. For experiments in anesthetized animals, the rat was initially anesthetized with isoflurane but then given a 400 mg/kg ip dose of chloral hydrate. After 2 h, maintenance doses of 200 mg/kg chloral hydrate were given every hour by iv administration.
807
A small incision through the skin was made at the back of the neck and on the right shoulder. The jugular vein was exposed and a small nick made into the vein. The microdialysis probe and a cannula were inserted through this nick, and the probe and cannula were threaded through the vein to a region near the heart (ca. 2.5 cm). The cannula was backed-off 5 mm from the dialysis membrane. The jugular vein was then ligated. The inlet and outlet tubing of the dialysis probe and the cannula tubing were then threaded under the skin and out the incision on the back of the neck. Both incisions were closed with surgical staples and the rat was placed in a flexible jacket that covered both incisions and supported the placement of the dialysis probe. The rat was then attached to the awake animal system (CMA/BAS) with a stiff wire attached to a collar on the rat and to a counterbalanced arm attached to the animal container. The counterbalanced arm also had a two-channel liquid swivel through which the dialysis probe was attached to the perfusion pump and fraction collector. The liquid swivel allows the animal fullmotion without the tubing becoming entwined and kinked. To validate the microdialysis sampling technique, whole-blood samples were simultaneously collected. This was done through a cannula implanted along with the dialysis probe. A 100-pL sample of whole blood was collected every 30 min for the first hour then once every hour. The whole-blood sample was centrifuged for 10 min; 40 pL of the resulting plasma was treated with an equal volume of 0.7 M perchloric acid to precipitate proteins and centrifuged for 10 min. The supernatant was injected into the chromatographic system. Pharmacokinetic experiments were performed by perfusing the implanted probe with a Ringer's solution a t a perfusion rate of 1 pL/min. Samples were continuously collected over 10-min intervals. Dialysis samples were diluted with Ringer's solution as needed to keep the concentration in the range of calibration. Blanks were collected for at least 1h following insertion of the microdialysis probes. No chromatographic interferences were observed in the blanks. The animal was then dosed with theophylline (15 mg/kg) in 0.5 mL of Ringer's solution iv at 37 "C. Dialysis samples were collected for 6-7 h after dosing. The in vivo concentration of theophylline was calculated by determining the concentration in the dialysate from a standard curve and then accounting for the recovery of the microdialysis probe. Microdialysis is a continuous sampling technique, therefore each sample represents the average concentration of analyte in the blood during the sampling interval. This is compared to taking discrete blood samples that represent the concentration in the blood only at the time of sampling. Because of the continuous sampling, microdialysis is an integrating technique that is less prone to fluctuations than an instantaneous sampling technique. Pharmacokinetic parameter calculationsare also simplified. For example, area-under-the-curve (AUC) calculations are performed by summing the product of the concentration (jg/mL), perfusion rate (mL/min), and sample interval (min) for all samples instead of interpolating between points with the trapezoidal rule. Pharmacokinetic Calculations. Terminal half-lives of free theophylline were estimated by fitting an equation of the form C ( t ) = A exp(-crt) + B exp(-ot) (1) using a nonlinear least-squares fit. The first term in eq 1describes
the distribution phase following an iv bolus injection of theophylline where a is the distribution rate constant. The second term describes the elimination phase were 6 is the elimination rate constant. The half-life of elimination can be determined from the elimination rate constant (PI. The concentration in the blood at the time of injection (Co)can be calcuIated by adding the terms A and B. The AUC values for blood sampling were estimated using the linear trapezoidal rule and were extrapolated to infiity by adding C,/& where C , is the concentration of the last measurement. For the microdialysis data, the AUC was estimated by summing the products of the measured concentrations and the collection time interval with the addition of C,/O (eq 2). Noncompartmental
-
AUC = E C t A t 0
ct +P
analysis was used to obtain the total body clearance of theophylline
808
ANALYTICAL CHEMISTRY, VOL. 64,
NO. 7, APRIL 1, 1992
Table I. Binding of Theophylline to Blood Protein"
theophylline concentration 10 pg/mL 20 pg/mL
1 pg/mL
plasma microdialysis ultrafiltration whole blood microdialysis ultrafiltration
47.7 f 2.7 47.5 f 2.6
48.4 f 3.6 50.1 f 2.9
45.9 f 4.5 52.2 f 3.3
53.8 f 4.6 49.9 f 2.8
52.1 f 3.0 53.0 f 7.0
50.7 f 6.7 54.6 f 5.0
n n = 3. Table 11. Recovery of Theophylline by Microdialysis"
theophylline concentration 1 pg/mL 10 pg/mL 20 pg/mL Ringer's plasma whole blood O n
24.4 f 1.9 26.6 f 3.0 22.4 f 3.2
24.9 f 2.2 25.6 f 5.1 22.4 f 4.5
21.7 f 1.7 22.6 f 4.7 21.1 f 6.3
I
,
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8
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Figure 2. Typical chromatograms of iv microdlalysis samples: (A) blank prior to dosing, (B) 30 min after a 15 mg/kg iv dose of theophylline, (C) 300 min after a 15 mg/kg iv dose.
= 3.
(C, = dose/AUC) and the volume of distribution (Vd = C,/p). The mean residence time (MRT) was calculated by dividing the AUMC (first moment of the area under the curve) by the AUC. All results are expressed as the mean f standard deviation.
RESULTS AND DISCUSSION Probe Calibration and Protein Binding. The extent of theophylline binding to blood proteins is listed in Tible I. The percent bound is independent of the concentration of theophylline in the range studied. Theophylline was found to be (52.2 f 1.6)% bound in rat whole blood and (47.7 f 0.8) % bound in rat plasma. This is in good agreement with previous results using human plasma." The whole blood value was used in subsequent comparisons of microdialysis samples to whole-blood samples. The recovery of the microdialysis probe was determined in whole blood, plasma, and Ringer's solution over the concentration range needed for the pharmacokinetic experiments. The recovery results are tabulated in Table 11. The recovery is independent of the concentration and the matrix for these experiments. Based on these results, subsequent probes were only calibrated using the Ringer's solution. Intravenous Microdialysis Sampling. Typical chromatograms of blood dialysate obtained by in vivo sampling are shown in Figure 2. In vivo dialysis samples were directly injected into the chromatograph. The blank sample was run at the most sensitive detector gain setting used to determine if any endogenous interferences occur a t high gain. As can be seen (Figure 2A), no interferences occur in the blanks obtained prior to dosing of the animal with theophylline. The whole-blood samples were more difficult to analyze, requiring protein precipitation and removal before injection into the chromatographic system. These samples exhibited more potential interferences in the blanks relative to the dialysis samples (Figure 3). Fortunately, none of these endogenous compounds interfered with the determination of theophylline with this separation. For comparison, the whole-blood samples of Figure 3 correspond in time to the dialysis samples of Figure 2. Pharmacokinetic Comparison to Whole-Blood Samples. Microdialysis sampling was validated by simultaneously collecting blood samples. Pharmacokinetic curves obtained by the two methods were identical (Figure 4) when the samples were taken simultaneously and binding of theophylline to blood proteins is taken into account. Also shown in Figure 5 is a semi-log plot for the microdialysis sampling data clearly showing the fast distribution phase followed by the slower
L
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Flgure 3. Typical chromatograms of iv whole-blood samples: (A) blank prior to dosing, (B)30 min after a 15 mg/kg iv dose of theophylline, (C) 300 min after a 15 mg/kg iv dose. 'O
1
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50
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100
150
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250
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Flgure 4. Concentration-versus-tlme profile for a 15 mg/kg Iv dose of theophylline In the awake, freely-movlng rat. The bars represent the microdialysis samples and the circles represent the whole-blood samples. The concentration of theophylline in whole blood is corrected for protein binding. Shown in the inset is a plot of log concentrationversus-time using the microdialysis sampling data.
elimination phase. The pharmacokinetic parameters extracted from the data are listed in Table III. The results for the whole blood are in good agreement with previously reported values.18 The values for whole blood sampling and simultaneous microdialysis sampling show no statistical difference. The half-life of elimination determined from microdialysis samples
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7,APRIL 1, 1992
809
1 Table IV. Pharmacokinetics of Theophylline (15 mg/kg iv) in Awake and Anesthetized Rats"
100
3
[\ i
3 .-c5 e
tl,z distribution, min t , elimination, h dRT, h
AUC,pg min/L vd, L
1
C,, mL/min
0
awake
anesthetized
3.6 f 2.2 3.0 f 0.4 2.5 0.6 3523 f 1414 1.2 f 0.4 4.7 f 1.9
2.4 f 0.6 17.0 f 7.1 24.6 f 1.1 32626 f 8084 0.15 f 0.03 0.10 f 0.02
*
C
8
n n = 3.
10
0
I
I
50
100
150
200
Time (minutes)
Flgurr 5. Semi-log presentation of the concentratlon-versus-tlme profile for 15 mg/kg iv doses of theophylline In rats determined using
mlcrodlalysls sampllng. Awake animal without blood sampling (m): awake animal wlth simultaneous blood sampling (0):anesthetized animal (+I. Table 111. Pharmacokinetic Parameters for Theophylline-Dosed iv at 15 mg/kg" microdialysis sampling w/o blood sampling
tlI2 distribution, min h
o n , itn a& J$ i
AUC,bg min/mL v,, L C,, mL/min
w / blood sampling
blood samplingb
3.6 f 2.2 5.2 f 1.3 7.8 f 4.1 3.0 f 0.4 4.4 f 0.7 4.4 f 0.4 2.5 0.6 2.8 f 0.8 2.9 f 0.8 3525 f 1414 5616 f 1614 6135 f 1267 1.2 f 0.4 1.1 f 0.3 1.1 f 0.3 4.7 f 1.9 2.9 f 0.7 2.5 f 0.5
" n = 3. D a t a corrected for protein binding; values reflect unbound theoDhvllin concentration. was 4.4 f 0.4 h (n = 3) and from whole-blood samples was 4.5 f 0.7 h (n = 3). However, when microdialysis was performed alone a half-life of only 3.0 f 0.4 h (n = 3) was found. This half-life is statistically different from the half-life with blood sampling, p < 0.004. In addition, when only microdialysis sampling is performed the AUC and clearance are significantly different while the MRT and V , are the same relative to simultaneous blood sampling. It has been reported previously that the pharmacokinetics can be affected by blood sampling because of resulting changea in blood volume. These data indicate that microdialysis sampling eliminates such perturbations because no net fluid is removed even with continuous sampling for several hours. Effect of Anesthesia. The pharmacokinetics of theophylline are also greatly affected by the use of anesthesia during the pharmacokinetic experiment. This is shown in Table IV where the pharmacokinetic parameters obtained by microdialysis sampling in awake, freely-moving rata are compared to those in anesthesized rata. All parameters affected by the rate of metabolism are dramatically different in the anesthetized rat whereas the rate of distribution, which depends predominantly on blood flow, is unchanged. That the half-life of elimination more than quadruples is an indication that the anesthetic is inhibiting the metabolic enzymes of the liver. The effect of experimental conditions on the pharmacokinetics of theophylline in rats as determined by microdialysis sampling is shown graphically in Figure 5. Using the semi-log presentation of the concentration-versus-time curve, the slope of the lines is proportional to the rate of that phase. As can be seen, the initial rapid distribution phase is the same
in all cases. However, the elimination phase varies considerably depending upon both whether the animal is anesthetized and whether blood samples are drawn simultaneously with the microdialysis.
CONCLUSIONS Microdialysis offers several advantages for pharmacokinetic studies. The temporal resolution is much higher than for other methods. While 10-min intervals were used for these experiments, shorter times are easily achieved if needed. Since no blood is drawn, a large number of samples can be collected from a single animal without loss of fluid volume. Simultaneous sampling can be achieved using multiple dialysis probes. This provides the ability to monitor pharmacokinetics at multiple sites in a single animal. Because complete pharmacokinetic curves can be obtained for several organs using a single experimental animal, overall fewer animals will be necessary to obtain data on a given drug. Microdialysis samples only the free fraction of the drug in blood. In addition, microdialysis coupled to collection of whole blood samples provides a technique to determine the degree of drug binding in vivo during a pharmacokinetic experiment. The major limitation of microdialysis sampling is the small sample volumes obtained. The sample volume, perfusion rate, recovery, and sampling interval are all interrelated. Slow perfusion rates provide higher recoveries but smaller samples per unit time. Therefore the detection method must be sufficient to directly determine the lowest drug concentration necessary. ACKNOWLEDGMENT We wish to acknowledge the Center for Bioanalytical Research at The University of Kansas, Bioanalytical Systems, Inc., and Merck, Sharpe & Dohme for partial financial support of this work. D.O.S.acknowledges the financial assistance of the Procter & Gamble Co. through the P&G Bioanalytical Fellowship a t The University of Kansas. REFERENCES (1)
Scott. D. 0.;Sorenson. L. R.; Lunte, C. E. J . Chromefogr. 1990, 506,
461-469. (2) Scott, D. 0.;Sorenson, L. R.; Steeie, K. L.; Puckett, D.L.; Lunte, C. E. Pharm. Res. 1991, 8 . 389-392. (3) Steele, K. L.; Scott, D. 0.;Lunte, C. E. Anal. Chlm. Acta 1991, 246, 18 1-186. (4) Lunte, C. E.; Scott, D. 0.; Kisslnger, P. T. Anal. Chem. 1991, 63, 773A-780A. ( 5 ) Caprioli, R. M.; Lin, S. Roc. Nafl. Aced. Scl. U . S . A . 1990, 87, 240-243. (6) Menacherry, S. D.; Justice, J. B. Anal. Chem. 1990, 62, 597-601. (7) Ungerstedt. U. Usasuremnf of Newotransm/tter Rebase In Vivo; Marsden, C. A., Ed.; Wiley-Interscience: Chlchester, 1984 pp 81-105. (8) Ungerstedt, U.; Forster, C.; Herrera-Marschitz, M.; Hoffman, I.; Jungnelius, U.; Tossman, U.; Zetterstrom, T. Neuroscl. Left. (Suppl.)1982, 10,493. (9) Sandberg, M.; Lindstrom, S. J . Neuroscl. Methods 1983, 9 , 65-74. (10) Wages, S. A.; Church, W. H.; Justice, J. B., Jr. Anal. Chem. 1986, 58, 1649-1656. (11) Church, W. H.; Justlce, J. B., Jr. Anal. Chem. 1987, 59, 712-716. (12) Pettit, H. 0.; Pan, H. T.; Parsons, L. H.; Justice, J. E., Jr. J . Nevochem. 1990, 55, 798-804. (13) Pan, H. T.; Menacherry, S.; Justice, J. B., Jr. J . Neurochem. 1991, 56, 1299-1306.
Anal. Chem. 1992, 64, 810-814
810
(14) Mather, L. E.; Runciman, W. B.; Ilsley, A. H. Reg. Anaestb. 1982, 7 (SUPPI 4), S24433. (15) Benet, L. 2.; Mitchell, J. R.; Sheiner, L. E. The Pharmacological Basis
Theramutics. 8th ed.: Gilman. A. G.. Rall. T. W.. Nies. A. S.. Tavlor. P.. Eds.;'Pergamon Press: New York, t991;p 12. (16) Herrera, A. M.; scott, D. 0.;Lunte, G. E. /'harm. Res, 1990, 7,
(17) Ogilvie, R . I. Clin. Pharm. 1978, 3,267-293. (18) Fruncillo, R . J.; Digregorio, G. J. J . Pharm. Sci. 1984, 73, 1117-1 121.
Of
1077-10Sl.
RECEIVED for review October 14, 1991. Accepted January 2, 1992.
Parts-per-Trillion Determination of Trihalomethanes in Water by Purge-and-Trap Gas Chromatography with Electron Capture Detection Louis LBpine* and Jean-Franqois Archambault Znstitut de recherche d'Hydro-QuCbec, 1800 mont6e Sainte- Julie, Varennes, QuCbec, Canada J3X 1Sl
A gas chromatographlc method uslng a purge-and-trap concentrator and electron capture detectlon to monltor trlhalomethanes (THMs) at the low parts-per-bllllon (ppb) and the parts-per-trllllon (ppt) levels Is described In thls paper. The method shows good Ilneartty over 4 orders of magnltude wlth a detectlon llmlt between 0.01 and 0.05 ppt for each THM normally found after chlorlnatlon: chloroform, bromodlchloromethane, dlbromochloromethane, and bromoform. Speclal procedures, for sample handllng and the preparatlon of blank water and analytlcal standards, requlred at such a low Concentration, are a h dtscussed. The anatytlcat method has been successfully applled In the analysls of samples taken at dlfferent polnts of the water treatment plant of HydroQuibec's Gentllly 2 nuclear power statlon. Analysls of samples taken after chlorlnatlon and demlnerallzatlonrevealed the formatlon of all four THMs and other unldenttfled halogenated volatlle compounds. Chlorotorm and bromodlchloromethane were found at the low ppb level, whlle dlbromochloromethane and bromoform were present at the ppt level.
INTRODUCTION Nuclear and fossil-fueled power plants generally have their own water treatment unit, similar to those used to produce municipal drinking water, which includes a chlorine-disinfection step that is known to produce trihalomethanes (THMs). The far most common THM in chlorinated water is chloroform (CHCl,), although lower concentrations of brominated THMs such as bromodichloromethane (CHEW&), dibromochloromethane (CHBr,Cl), and bromoform (CHBr,) are often The iodine-containing compounds reported by a few workers4,j are not usually found. These molecules are not likely to be removed by the demineralization system and are therefore introduced into the steam-generation cycle where the water is heated at temperatures reaching 260 OC or more. A t Hydro-QuBbec's Gentilly 2 nuclear power plant, a 685 MW CANDU-PHW (CANadian Deuterium Uranium-Pressurized Heavy Water), abnormally high chloride concentrations have been observed in steam-generator blowdown and found to be related to the THM level in the makeup water.6 A recent study conducted in our laboratories on the thermal stability of chloroform confirmed the latter's rapid degradation to produce three C1- ions? It is believed that other
THMs will follow the same degradation mechanism to yield the corresponding acid (HCl, HBr) and subsequently increase the bromide and chloride level.8 This is of great concern to the nuclear industry because of the known corrosive effect of chloride on structural components. Owing to the concentrating factor involved in steam generators for nonvolatile species such as chloride, even a small amount of THMs in the makeup water can result in chloride concentrations exceeding the limit for safe system operation. The vast majority of methods used for THM analysis are designed for quality control of drinking water. Their analytical range is limited to parts per billion (ppb), in compliance with the 100 ppb total THM maximum contaminant level prescribed in the USEPA Safe Drinking Water Act. The particular application to the nuclear plant operation called for a more sensitive technique to analyze samples with individual THM concentrations in the low-ppb or even in the partsper-trillion (ppt) range and, in our study on thermal degradationP7to monitor their residual concentration in the low-ppt range. Gas chromatography with electron capture detection (ECD) is an attractive method for THM analysis since it offers a wide linear dynamic range and is the most sensitive method available for halogenated organic compounds. To a certain extent, it is specific to organohalides, which means the latter can be discriminated from a complex background. However, this detector cannot tolerate a large quantity of water and, although direct aqueous injection with solvent bypass has been r e p ~ r t e da, ~preextraction step is generally performed. Three different approaches have been considered: liquid-liquid extraction,1°'12headspace sampling,12-14and purge-and-trap.'j Comparison studies12J6have shown that even with ECD detection, liquid-liquid extraction and headspace sampling are limited to the ppb range; these methods are also subjected to airborne or solvent contamination, often giving high blank values. The purge-and-trap technique is free from these problems because it uses an inert gas to extract THMs from water and the sample, once introduced, is never in contact with the atmosphere. The concentrating factor obtained by trapping the analytes selectively on an adsorbent material allows analysis of very low-concentration samples. This paper describes the analytical method developed for ultratrace analysis of the four THMs of interest in water samples, using a purge-and-trap concentrator coupled to a gas chromatograph
0003-2700/92/0364-0810$03.00/0 0 1992 American Chemical Society
