Anal. Chem. l W 2 , 64, 2636-2641
2636
On-Line Coupling of in Vivo Microdialysis with Tandem Mass Spectrometry Leesa J. Deterding: Kelly Dixf Leo T. Burkaf and Kenneth B. Tomer'sl Laboratory of Molecular Biophysics and Experimental Toxicology Branch, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
The capablitty of Interfacing in VIVO microdlalyds with mass spectrometry has been de"trated. The goal of this research was to d e " t r a t e the feesiMmyof reai-thw anaiysk in Mdogical systems using mlcrodialysk In combination with tandem mass spectrometry(MS/MS). Microdialyek sampling was accompllshed by surgically Implanting a small mlcrodiaiysk probeinto a tissue or area of interest. Moleculesdiffuse through the membrane of the microdialysls probe due to concentrationdifferences. These molecules are collected In a sample loop and analyzed by tandem mass spectrometry. Sequentlei injections can be made in as ilttle as 2 min. Thls capaMiity is advantageous In the study of moleculeswith very rapideilmlnatbnrates. Trb( Il-chioroethyl)phosphate(TRCP) was used as a model compound in the development of thls analytical technique. As an example of an appilcation of the mkrodlatysWMS/MS technique, plasma concentration vs t h e curves were obtained and compared with the plasma concentration profiles obtalned using conventional studles. For the mlcrodialysk/MS/MS studies, the average slope from three animals was -0.086 mln-I. I n comparison, the average slope from four animals from the conventional studies was -0.035 mln-I.
In recent years, microdialysis has become an important technique for the in vivo sampling of the extracellular fluid in discrete compartments of living systems. This technique is being widely used in physiological, pharmacological, toxicological, and behavioral studies for the recovery of endogenous substances, such as neurotransmitters and their metabolites, or exogenous substances, such as drugs and toxicants, and has been recently reviewed.' Microdialysis is preferable to conventional sampling techniques becausethere is minimal damage to the sampling site. In addition, little alteration of the fluid balance results because no fluid is removed from or introduced into the system during the sampling process. Other advantages include the ability to introduce and collect substances simultaneously and the ability to continuously sample over a desired length of time. The microdialysis membrane excludes large molecules, and therefore, relatively "cleann samples can be collected. Microdialysis sampling is accomplished by implanting a small microdialysis probe intothe tissue of interest. Molecules with a molecular weight below the molecular weight cut-off of the dialysis membrane diffuse through the membrane due to concentration differences and are collected in the dialysate. Due to the low flow rates involved in microdialysis,collection times of about 5-30 min are frequently necessary in order to obtain sufficient sample and/or sample volumes (1-20 FL) for analysis. These small sample volumes place stringent requirements on sample handling and sample analysis tech+ Laboratory of Molecular Biophysics.
Experimental Toxicology Branch. (1)Lunte, C. E.;Scott, D. 0.;Kissinger, P. T. Anal. Chem. 1991,63, 773A-78OA. t
niques. A highly sensitive and selective detection method is necessary. One detection method which meets these requirements is mass spectrometry. For the analysis of biomolecules, fast atom bombardment mass spectrometry (FAB/MS) has receivedwidespread attention.* Several disadvantages of FAB/ MS include a high chemical background due to the matrix employed, often few structurally informative fragment ions are produced, the sample ion beam can be short-lived, and a suppression effect may be observed for some of the analytes. More recently, the technique of continuous-flow FAB (CFFAB)3t4was developed and has been found to alleviate some of these disadvantages of FAB. The continuous flow of aqueous solutions containing small amounts of matrix to the FAB probe tip results in a decrease in the chemical noise and, consequently, improved ~ensitivities.~It has also been observed that the use of CF-FAB significantly reduces the suppression effect because there is a constant flow of analyte to the FAB probe tip and a lower matrix concentration on the tip of the FAB probe.6 We have recently shown that coaxial CF-FAB is a very useful technique for the analysis of biomolecules.7-9 Due to the ability of the nanoscale capillary system to deliver a high concentration of analyte in a short period of time, the coaxial CF-FAB design allows for increased sensitivities over other CF-FAB interfaces. Detection limits of 500 am01 in the multichannel acquisition mode and in the scanningacquisition mode 1.8 fmol for a tripeptide and 10 fmol for C-peptide (Mr = 3617) have been obtained. These detection levels are in the concentration range of biochemicals in the body. The combination of microdialysis with CF-FAB allows the determination in real time of many biochemicals in the body. Recently, Justice and co-workers'o reported the thermospray mass spectrometry (TSP/MS) analysis of samples collected by off-line microdialysis. Caprioliet al. first coupled microdialysis with CF-FAB for the analysis of drug levels in blood.'' Using a 10-min collection period, the concentration of penicillin G in the blood of a rat was monitored on-line. Caprioli and co-workers have also examined the metabolism (2)Barber, M.; Bordoli, R. S.; Sedwick, R. D.; Tyler, A. N. J. Chem. SOC.,Chem. Commun. 1981,325-327. (3) Ito, Y.; Takeuchi, T.; Ishii, D.; Goto, M. J. Chromatogr. 1986,346, 161-166. (4)Caprioli, R.M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1986,58,29492954. (5)Caprioli, R. M.; Fan, T. Biochem. Biophys. Res. Commun. 1986, 141, 1058-1065. (6)Caprioli, R. M.; Moore, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987,1, 15-18. (7)de Wit, J. S. M.; Deterding, L. J.; Moseley, M. A.;Tomer, K. B.; Jorgenson, J. W. Rapid Commun. Mass Spectrom. 1988,2,100-104. (8)Moseley, M.A.;Deterding, L. J.; de Wit, J. S. M.; Tomer, K. B.; Kennedy, R. T.; Bragg, N.; Jorgenson, J. W. Anal. Chem. 1989,62,1577-
_.
1ARA
_-I
(9)Tomer, K. B.; Perkins, J. R.; Parker, C. E.; Deterding, L. J. Biol. Mass Spectrom. 1991,20,783-780. (10)Menacherry, S. D.; Justice, J. B., Jr. Anal. Chem. 1990,62,597601. (11)Caprioli, R. M.; Lin, S.-N. Proc. Natl. Acad. Sci. U.S.A. 1990, 240-243.
Thls article not subject to U.S. Copyrlght. Published 1992 by the Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
0
2637
Figure 1. Overall schematic of the in vivo microdialysis technique combined with mass spectrometry.
of substance P12 and valproic acidl3 in the rat brain by microdialysis/MS. The experimental parameters and operating conditions of in vivo microdialysis in combination with coaxial continuous-flow FAB used in our studies are described in the following sections. Tris(2-chloroethyl) phosphate (TRCP), an organophosphate flame retardant used in the plastic and synthetic fiber industries, was chosen as a model compound for the development of this technique. TRCP is toxic and carcinogenic to rats, yet there is little or no literature available on its plasma concentration profile with respect to time. This type of data is essential for the extrapolation of toxicity and carcinogenicity studies in rodents to predict possible health risks for humans. This paper demonstrates the first application of the on-line coupling of microdialysis with mass spectrometry for the study of the metabolism of xenobiotics. These combined techniques could play a major role in the research area of environmental toxicology. A comparison of the plasma concentration vs time profile obtained using microdialysis/mass spectrometry and conventional blood withdrawal followed by chromatographic analysis is made, and differences are discussed.
EXPERIMENTAL SECTION Chemicals. Tris(2-chloroethyl)phosphate was obtained from Radian Corporation (Austin, TX). A 20 mg/mL dosing solution was prepared in 8:l:l water/ethanol/emulphor. [1-14C]TRCP was obtained from Dupont New England Nuclear (5.9mCi/mmol). Animals. Male Fischer 344 rats were obtained from Charles River Breeding Laboratory (Raleigh, NC) at least 1week prior to treatment. Animals were housed in animal quarters with a 12-h light/dark cycle and received NIH-31 Rodent Chow and water ad libitum. Animal quarters were maintained at 21-22 "C and 50 f 10% relative humidity. Microdialysis System. The microdialysisprobes employed consisted of a 20-mm cannula with a 4-mm membrane (BioAnalytical Systems, West Lafayette, IN). The perfusion liquid is contained in a stainless steel reservoir under helium pressure. The flow rate through the dialysisprobe can be altered by varying the helium pressure. For our experiments, water is used as the perfusion liquid to minimize the potential for the formation of salt clusters on the FAB probe tip (either with the matrix or the (12) Johansson, P. E.; Caprioli,R. M. Proceedings of the 38th Annual ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, 1990, p 1327. (13) Lin, S.-N.;Slopis,J. M.; Johansson, P. E.; Chang, S.;Butler, I. J.; Caprioli, R. M. Proceedings of the 39th Annual ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991, p 583.
analyte). Formation of these clusters can increase the chemical background or decrease the abundance of the ion being analyzed, thus decreasing the sensitivity. The perfusate is pumped at a flow rate of either 2 or 0.8 pL/min. The flow rate through the microdialysis probe is determined using the FEP (fluorinated ethylenepropylene) tubing which is supplied with the microdialysis probes. The flow rate is calculated from the time required for the perfusate to fill the known dead volume of the tubing (1.2 pL/lOO mm). A sample loop of 7.5 or 1.1 pL is used to collect the dialysate at the injection system. The dialysate samples were injected by using a modification of the capillaryliquid chromatographysystem that was developed in this laboratory for use with open tubular liquid chromatogr a p h ~ The . ~ ~overallschematicfor an in vivo experiment is shown in Figure 1. A Valco injectionvalve is used to make the injections. The injection valve is connected with stainless steel tubing to a sample tee, which holds the fused silica capillary of the coaxial continuous-flow FAB probe. The other side of the sample tee is connected to a waste restrictor. This waste restrictor is selected so that the sampleloopcan be emptied during the desired injection interval. For our experiments, injections are made for 30 s (ca. 70 nL) at 5-min intervals or 20 s (ca. 30 nL) at 2-min intervals. Mass Spectrometry. All data were acquired on a VG ZAB4F mass spectrometer (VG Analytical, Altrincham, UK).15 This instrument is of B1E1-E2B2 design and is operated at 8 kV. An Ion Tech atom gun and a standard VG continuous-flow FAB source heated at 40-60 "C were used. The samples were bombarded with 8-keV xenon atoms. A coaxial continuous-flow FAB interface was used in this work and has been previously de~cribed.~J A fused silica capillary column from the injection system is inserted into a sheath capillary column. The dialysate flows through the inner column (typically 10-pm i.d., 150-pm 0.d.) while the matrix simultaneously flows through the outer column (160-pmi.d., 350-pm 0.d.). No mixing of the matrix with the dialysate occurs until it reaches the FAB probe tip. A microliter syringe pump (Isco, Inc., Lincoln, NE) was used to pump the matrix through the sheath column at a flow rate of 0.3 pL/min. The matrix composition was 25% glycerol in water (5 mM heptafluorobutyric acid). The flow rate of the inner column was ca. 95 nL/min which resulted in an elution time of ca. 1.67 min. This allowed for injections to be made at 2-min intervals. The MS/MS experiments were carried out by focusing the parent ion through MS-I. Daughter ion spectra were obtained by collisionalactivation at 8 keV. The parent ions were activated (14) de Wit, J. S. M.; Parker, C. E.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1987,59,2400-2404. (15) Ham, J. R.; Green, B. N.; Bateman, R. H.; Bott, P. A. Proceedings of the 32nd Annual ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 1984, p 380.
2txi8
ANALYTICAL
CHEMISTRY, VOL. 64, NO. 21, NOVEMBER I ,
by collision with helium gas (50% beam attenuation) in the collision cell located in the third field-free region. Collisionally activated decomposition spectra of the resulting daughter ions were obtained by a linear EzBz linked scan of MS-I1 (unit resolution). Selected reaction monitoring was performed by scanning MS-I1 over the desired mass range of the daughter ion. The data acquisition system used was a VG Analytical 11-250 data system. Data were acquired in the continuum mode. Additional data workup was performed using a Sun 3/60 work station operating Mach3 software (Kratos, Manchester, UK). Microdialysis/Mass Spectrometry. The initial microdialysis/MS experiments involved the acquisition of in vitro mass spectra of tris(2-chloroethyl) phosphate (TRCP) in water at a perfusion rate of 2 pL/min in order to optimize the conditions for the in vivo work. The mass spectrometer was set to scan over the region of the (M + H)+ ion, m/z 285 (m/z 293 to m/z 278 at 20 s/decade), of TRCP. Using a sample loop of 7.5 pL, injectiws of 30 s were made at 5-min intervals. Good S/N ratios were observed for TRCP at the 1 X M level. For the in vivo experiments, three male Fischer 344 rata (247260 g) were weighed prior to surgery and maintained under Metofane anesthesia throughout the experiment. A longitudinal incision was made over the right jugular vein, and the vein and pectoral muscle were exposed. The pectoral muscle was lifted and the intravenous (iv) guide (BioAnalytical Systems, West Lafayette, IN) for the microdialysis probe was implanted into the jugular vein through the muscle. Confirmation that the vein had been cannulated was achieved by removing the iv guide handle to observe blood flow back through the guide. The microdialysis probe was then inserted into the vein via the iv guide. After implantation of the microdialysisprobe, the incision was closed around the probe with wound clips. Immediately upon introduction of the microdialysis probe into the jugular vein, the biological system of the animal is perturbed. The microdialysisprobe, therefore, was allowed to equilibrate in the jugular vein for at least 30 min prior to dosing. Animals were dosed intravenously(20mg/kg)via the femoralvein using a TRCP dosing solution (20 mg of TRCP/mL in 81:1 water/ethanol/ emulphor). After TRCP administration, dialysate was collected and injections were made into the mass spectrometer. For the MS experiments, MS-I was scanned over the (M + H)+ ion of TRCP (m/z 285) using a narrow scan region of 15 amu. The mass spectrometer was scanned from m/z 293 to mlz 278 at 20 s/decade. For the selected reaction monitoring experiments, the (M + H)+ ion of TRCP was focused through MS-I while MS-I1 was scanned over a narrow daughter ion (m/z 223) region, m/z 225 to m/z220 at 20 s/decade. I n Vitro Calibration of Microdialysis Probes. In vitro experiments were performed to determine the linearity of the response of the combined techniques of microdialysis and mass spectrometry, as well as to establish a standard calibration curve for the in vivo analyses. TRCP was added to plasma to give a 2 mg/mL solution. Serial dilutions of the 2 mg/mL solution were performed to give standard TRCP solutions of 1.0,0.5,0.2,0.1, 0.02, 0.01, and 0.002 mg/mL in serum. Microdialysis was performed in vitro at room temperature on these vials with constant stirring using a stir bar. Triplicate injections for each concentration were made, and peak areas from the selected reaction monitoring experiments were averaged to generate a standard curve relating peak area to total TRCP concentration in plasma (Figure 2). Linearity over 4 orders of magnitude was observed (R2= 0.946). Plasma Protein Binding Studies. Blood was removed from a male F344 rat and centrifuged at approximately 2000g for 15 min to obtain plasma. Ten microliters of dosing solution containing [“CITRCP were added to 500-pL aliquota of plasma such that the total [“CITRCP concentrations in plasma were 4.9,24.5, and 49 pg/mL. Plasma samples (10-pL diquots) were counted in a liquid scintillation counter. Aliquots of 400 pL of each plasma sample were pipetted into Centricon-10 ultracentrifugation tubes and centrifuged at 4400g for ca. 5 min. Tenmicroliter aliquots of filtrate and sample reservoir were then counted. The unbound fraction of [14C]TRCPcfu,was calculated as (concentration in filtrate)/ (totalconcentration in plasma). The f u for TRCP is 0.42 (data not shown).
iesa
m
2
U X
0
a
““Vm-Y
, ~. G O
I
I
0 2
0 4
,
I
0 6
,
I
1 0
0 8
1 2
Total TRCP Concentration (mglmL)
Flguro 2. Standard concentration curve of in vitro monitoring of TRCP uslng microdialysis on-line with tandem mass spectrometry.
Conventional Sample Analysis. [WITRCP and unlabeled TRCP were mixed with an 81:1watedethanoVemulphor solution such that the final concentration of the solution was 20 mg/mL (50 pCi/mL). On the day prior to chemical treatment, animals were weighed and prepared for surgery. Four male (259-303 g) Fischer 344 rata were anesthetized by intraperitoneal (ip) injection of ketamine/xylazine (1-1.5 mL/kg of body weight, 100 mg of ketamine plus 3.2 mg of xylazine/mL). The right jugular vein was cannulated using the method of Harms and Ojeda.16 Followingsurgery,animals were housed in individual metabolism cages with free access to food and water for the remainder of the experiment. Animals were allowed to recover 18-24 h prior to chemicaltreatment. On the day of treatment, animalswere dosed intravenously(2Omg/kg)via the indwellingjugular cannula.Blood samples were drawn from the cannula at 2,4,6,8,10,15,20, and 30 min. Blood volume removed at each sample withdrawal (ca. 100rL) was replaced as heparinized saline (10 units of heparin/ mL of physiological saline). Blood samples were stored on ice in microfuge tubes for up to 20 min prior to centrifugation. Samples were centrifuged at 16000g for 5 min to obtain plasma. Fifty microliters of plasma were pipetted into a clean microfuge tube, and plasma proteins were precipitated with acetonitrile (100 pL). The samples were vortexed, then centrifuged for 10 min at 16000g to precipitate plasma proteins. This procedure resulted in 100% recovery of radioactivity over a concentration range of 4.9-49 pg/mL (data not shown). Samples were stored on ice or in the refrigerator until analyzed by reversed-phase liquid chromatography using an isocratic solvent system (65:35 A = 0.1 % trifluoracetic acid, B = acetonitrile) at a flow rate of 1.5mL/min withradiometricdetection (Flo-One/betaRadioactive Flow Detector, Radiomatic Instrument Company, Tampa, FL). Peak areaswere convertedtototdTRCPconcentration in plasma using a standard curve and free TRCP concentration in plasma was obtained by multiplying total concentration by fu.
RESULTS AND DISCUSSION Microdialysis/MaesSpectrometry. In our initial in vivo microdialyeia/MS experiments, TRCP was detected at MS-I. Background interferences from the FAB matrix occurred, however (datanot shown). To verifythat TRCP was actually being observed in vivo from the rat, an MS/MS experiment of the (M + HI+ ion of TRCP was performed. The resulting spectrum is shown in Figure 3A. As a comparison, the MS/ MS spectrum of standard TRCP is shown in Figure 3B. Upon collisional activation,the most abundant daughter ion formed is due to the loss of ClCH==CHZ from the (M H)+ion (mlz 285) of TRCP. Because mass spectrometry is a separation
+
(16)Harms,P. G.;Ojeda, S.R.J. Appl. Physiol. 1974,36 (3), 391-392.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 0 II
2839
'"1
CICH2CH20-P-OCH2CH2CI
I
(M+H)'
OCH2CH2CI
n
CICH-CHZ
I
p3
1
83;
g
,a;
m
'D 63;
:
2 j31
-HCI
4
: : - &&t
c
. m , 5 33:
)I, !,b 1
161
IH
ma
221
240
?E1
6
1.4 \
O i
,L,L,
L,
3 9
mlz
Flgure 3. MS/MS spectrum of the (M + H)+ ion of mlr 285 of TRCP acquired (A) in vivo from microdialysls/MS and (e)from standard TRCP.
-.
-
I
0
{'
Rat2 Rat3
technique based on mass, tandem mass spectrometry (MS/ MS) adds a second dimension of separation in that only ions of the selected mass of the compound of interest are transmitted. As a result, interference from background is typically reduced in MS/MS data. Chemical interferences can be further reduced using selected reaction monitoring (SRM). In these experiments, the detection is restricted to only those compounds undergoing a specific loss from the focused ion. MS/MS and SRM were, therefore, integrated into the protocol to remove as much background interferences as possible. The fragment ion of m/z 223 (lossof C1CH=CH2) was chosen to be monitored during all subsequent on-line microdialysis analyses. In vivo microdialysia/MS/MS experiments (SRM) in the jugular vein of rats were then performed (data not shown). The results, however, indicated very low levels of TRCP, and the results were inconsistent with disposition studies by Herr et al.17 In those studies, plasma concentration data following oral administration of TRCP (175 mg/kg) demonstrate an elimination half-life of TRCP in plasma on the order of 2 h. We, therefore, expected to detect TRCP in the dialysate from the microdialysis/mass spectrometry experiments for at least 2 h. To improve the sensitivity and consistency of the microdialysis/mass spectrometry technique, the perfusion rate was decreased from 2 to 0.8 ctL/min. This was achieved by simply lowering the helium pressure of the stainless steel reservoir. At this time, preliminary results from a study of the iv administration of TRCP indicated that the half-life was significantly lese than 2 h. The sample loop, therefore, was decreased from 7.5 to 1.1r L so that injections could be made a t 2-min intervals vs 5-min intervals. A typical example of the mass chromatograms obtained from in vivo microdialysis in the jugular vein of a rat in combination with selected reaction monitoring is shown in Figure 4. The experiment was repeated with two additional animals. One important factor in the microdialysis technique is the extent to which the compound of interest is bound to proteins in the blood. The protein binding will interfere with recovery because microdialysis only samples free analyte in plasma. A plasma protein binding study was, therefore, performed using [WITRCP. The unbound fraction of [WITRCP was calculated and found to be 0.42. Using the standard calibration curve and the results of the protein binding study, peak areas obtained from the microdialysis/MS/MS experiments for each animal were converted t o free TRCP
concentration in plasma. Figure 5 shows the free TRCP concentration vs time profiles following intravenous administration of TRCP (20 mg/kg). The symbols represent data from three individual animals. Data from each animal were analyzed by nonlinear regression to determine the best-fit slope of the log concentration versus time graph over the first 30 min. Only the data from the first 30 min was considered, because after this time, TRCP could no longer be detected in the HPLC analyses from the conventional studies. The average slope from the three animals was 4.086 f 0.015 min-1. The accuracy and utility of any new analytical technique is not known unless it is compared with a standard method. We therefore performed conventional studies to generate free TRCP concentration in plasma versus time profiles following intravenous administration. Herr e t al." and Burka and coworkers's have studied the metabolism, distribution, and toxicity of orally-administeredTRCP, yet we found no reports in the literature regarding concentration/time profiles following intravenous administration of TRCP. Conventional Studies. In conventional plasma analysis studies, serial blood samples are withdrawn after chemical administration with subsequent extraction and analysis by chromatographicmethods. In our studies, radiolabeledTRCP was administered to F344 rats, and plasma samples were analyzed by HPLC. Free TRCP concentration in plasma versus time data following intravenous administration (20 mg/kg) is shown in Figure 6. The symbols represent data
(17) Herr, D. W.; Sanders,J. M.;Matthews, H. B. Drug Metab. Dispos. 1991, 19 (2), 436-442.
(18) Burka, L. T.; Sanders, J. M.; Herr, D. W.; Matthews, H. B. Drug Metab. Dispos. 1991, 19 (2), 43-47.
vu)
330
lo
-c
0
e ~
I
c
c
u n
200
0
U I-
a
1co
1
A
A
A
I
a .
-
] .0..
2
U.
0
10
20
30
40
50
Time (min.)
Flgure 5. Free concentration of TRCP In plasma vs time as observed in three individual animals In the mlcrod&lysls/MS/MSexperlments.
2640
ANALYTICAL CHEMISTRY. VOL. 64, NO. 21, NOVEMBER 1, 1992
. .-e
Rail
4
Rat2 Rat3
A
Rat4
Q
0
I
L
C 0
A
E
A
0
n
u a t-
13
-
I
0
E
Y
0
p : . O
X
A
A
P
o
. 0
4
. A A
- , 10
20
30
43
Time (min.)
Figure 6. Free concentration of TRCP in plasma vs time as observed in four IndlvMuai animals in the conventional pharmacokinetic studies.
200
100
0
Time (min.)
Figure 7. Free concentration of TRCP in plasma vs time as observed in one anlmal in the conventionalstudies (open circles) and one animal in the mlcrodlalysis/MS/MS studies (closed squares).
from four individual animals. As in the microdialysis experiments, plasma concentration versus time data for each animal were fit using nonlinear regression analysis. The slopes of log concentration vs time graphs obtained for each animal were averaged and the result was found to be -0.035 f 0.002 mi+. Microdialysis/MS/MSvs Conventional Methods. Figure 7 shows the data obtained from one animal of each of the two techniques (only one animal from each is plotted for simplification of the graph). The data from one animal in the MD/MS/MS studies (solid squares) is plotted on the same graph as data from one animal in the conventional studies (open circles). The left vertical axis corresponds to the free TRCP concentration (0-300 mg/L) from the MD/MS/MS studies while the right vertical axis corresponds to the free TRCP concentration (0-20 mg/L) from the conventional studies. The slopes of the log concentration vs time graphs obtained using the average of three animals by microdialysisl mass spectrometry (slope = -0.086 f 0.015 min-l) and the average of four animals by conventional methods (slope = -0.035 f 0.002 mi+) are significantly different at the p = 0,011level (student's t-test). These statistical differences in slope may result from several factors.
First, the conventional method uses awake, freely moving animals whereas the microdialysis/MS/MS method uses anesthetized animals. Metofane, the anesthetic used in the MD/MS/MS studies, is known to alter the normal physiology of the animal in several ways. A major effect is altered blood flow, which in turn may change the disposition and, consequently, the metabolism of xenobiotics in solution in plasma. In addition, Metofane may also alter TRCP plasma protein binding. Plasma saturated with Metofane may bind TRCP to a lesser or greater extent than control plasma. Because Metofane was administered on an as-needed basis rather than continuously, plasma levels of anesthetic were constantly changing. Second, the process of TRCP elimination from plasma is a very dynamic process as demonstrated by the slopes of the log concentration vs time curves. To adequately define the shape of the curve, a sufficient number of data points are required. The MD/MS/MS system allows sampling at 2 minute intervals. If samples were taken every 2 min by conventionalmethods, the fluid balance would be significantly altered in the animals. In turn, the disposition, metabolism, and elimination of compounds in plasma would also be changed. Therefore, samples are taken less frequently in conventional studies. For the conventional studies of TRCP, samples were taken a t 2,4,6,8,10,15,20, and 30 min. Beyond 10 minutes, more scatter is observed in the data points. Furthermore, the limits of detection of the HPLC are approached for the 30-min sample, and [WITRCP was not consistently detected in plasma samples taken after 30 min. On the other hand, TRCP was consistently detected in the dialysate from the microdialysis probe beyond 40 min after dosing. Not only does the increased number of data points in the MD/MS/MS studies (ca. 20 versus 7) better define the profile of TRCP in plasma, but the MD/MS/MS method is able to detect TRCP for a longer period of time, therefore, better defining the curve a t later time points. Third, it has been reported that some limitations may exist in the in vitro calibration of the microdialysis probes.lS21 In vitrorecovery may not be a reliable estimate of in vivo recovery because of differences between the in vitro and the in vivo environments (e.g. blood flows,temperatures, eta.). Although these reports have primarily addressed microdialysisin tissue rather microdialysis in blood, it has been reported that the relative efficiency of the microdialysis probes implanted in the jugular vein may vary during the course of an experiment.22 Fourth, the abundance of the protonated molecular ion may be affected by the presence of some Components found in biological fluids. In particular, salts have a tendency to form adducts with the components of interest. As a result, (M + Na)+ions are often observed. Because the microdialysis mass spectrometry experiments employ selected reaction monitoring, we are unable to determine if this adduct formation is actually occurring. If these adducts are formed, a decrease in the abundance of the (M + H)+ ion of TRCP would be observed. After submission of this article, Telting-Dim et al.23 published a paper showing differences in pharmacokinetic results obtained from conventional and microdialysis (UV detection) techniques that are similar to the results we have observed. These researchers observed that the pharmaco(19) Menacherry, S.;Hubert, W.; Justice, J. B., Jr. Anal. Chem. 1992, 64, 577-583. (20) Microdialysis User's Manual, Carnegie Medicine, 3rd ed.; Carnegie Medicine: Stockholm,Sweden, 1987; Section 4. (21)Alexander, G. M.; Grothusen,. J. R.:. Schwartzman, R. J. Life Sci. 1988,43, 595-601. (22) Sjoberg, P.; Olofsson, I. M.; Lundqvist, T. Curr. Sep. 1991,10,88 (Abstract No. 69). (23) Telting-Dim, M.; Scott, D. 0.; Lunte, C. E. Anal. Chem. 1992,64, 806-810.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21. NOVEMBER 1, 1992
kinetics of theophylline varied considerably between awake and anesthetized animals and whether or not blood samples were drawn (as in conventional studies) simultaneously with microdialysis sampling. We are currently investigating the possibility of some of the differencesbetween the conventional studies and the microdialysis/MS/MS studies. Conventional studies on anesthetized animals are being performed. In addition, the possibility of the in vivo calibration of microdialysis probes and microdialysis/MS/MSexperiments on awake animals is being investigated. The combined technique of microdialysis with mass spectrometry offers several advantages over conventional sampling methods. With the microdialysisIMS technique, the fluid balance in the animal is maintained even though the blood of the rat is being sampled every 2 min for up to 40 min postdosing. As a result, the plasma concentration vs time curve is well-defined. In conventional studies, blood volume loss is a concern when multiple samples are withdrawn over a short period of time. Consequently, fewer time points are available for defining the plasma concentration profiles. Conventional methods, however, are appropriate for compounds which are not rapidly eliminated from plasma by the processes of distribution and metabolism or which cannot pass through the microdialysis membrane. For compounds with slower elimination rates, fewer blood samples are drawn over a longer period of time. Consequently, maintaining the fluid balance is not an issue. An additional advantage of the microdialysisIMS technique is the time spent on sample analysis is significantly reduced in comparison with conventional methods which utilize HPLC or GC. Sample workup and replicate chromatographic analyses per sample (characteristics of conventionalstudies) are not required. An added dimension of the mass spectrometric technique is that the molecular identity of the analyte of interest is confirmed, unlike conventional studies employing HPLC or GC where compounds are identified solely on the basis of chromato-
2641
graphic retention time. One further advantage of the combined microdialysis/maasspectrotnetric technique ia that mass spectrometry detection does not require radiolabeled compounds. In conventionalstudies,radiolabeled compounds are often necessary for detection.
CONCLUSIONS The use of microdialysis in conjunction with coaxial continuous-flowFAB and tandem mass spectrometry has been shown to be an effective technique for the real-time analysis of molecules in biological systems. This combined technique can be used for the on-line mass spectrometric determination of compounds sampled in vivo. The plasma concentration profile of TRCP in rats administered intravenously has been determined. In addition, this technique has potential to be used for on-line pharmacokinetic analyses. This on-line capability should provide further insight into the metabolism and kinetics of biological processes.
ACKNOWLEDGMENT The authors would like to thank Arthur Moseley for valuable discussions in the early development of the microdialysis/MS system, Bob Hall, Chief of Instrumentation a t NIEHS, for his assistance in the design and fabrication of the coaxial CF-FAB probe and the microdialysis injection system used in this work, Kathleen Thomas for her help in preparation of the figures, Adrian Phillips for her assistance with HPLC analyses, and Karen Demby for her critical review of the manuscript.
RECEIVED for review April 7, 1992. Accepted July 28, 1992. Registry No. TRCP, 115-96-8.
