802
Anal. Chsm. 1992, 64, 802-806
Combined Supercritical Fluid Extraction/Solid-Phase Extraction with Octadecylsilane Cartridges as a Sample Preparation Technique for the Ultratrace Analysis of a Drug Metabolite in Plasma Hanjiu Liu, Linda M. Cooper, Douglas E. Raynie, J. David Pinkston, a n d Kenneth R. Wehmeyer* The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio 45239-8707
Supercrltlcal fluld extraction was coupled wlth solld-phase extraction uslng octadecylsllane cartrldges for the selectlve lsolatlon of ultratrace levels of a drug metabollte, mebeverlne alcohol, from plasma. Plasma was directly applied to the extraction cartrldge, the cartridge was washed to remove pcoteln and then extracted under supercrklcal condltlons uslng Cod5 % methanol. The effluent from the extraction cell was bubbled through a small volume of 2-propanol to trap the extracted mebeverlne alcohol. The effects of extractlon pressure and temperature on analyte recovery were examIned. The absolute recovery, selectlvlty, preclslon, and accuracy of the comblned supercrltlcal fluld extractlon/solldphase extractlon approach were compared to those of conventlonal solid-phase extractlon uslng gas chromatography/ mass spectrometry In the selected-Ion monltorlng mode. Mebeverlne alcohol was used as a model compound, and dog plasma was employed as the blologlcal matrix for these studles.
INTRODUCTION The rapid and accurate measurement of ultratrace levels of drugs and their metabolites in biological fluids plays a major role in the pharmaceutical development process. The measurement of plasma levels of drugs and metabolites provides information for mechanism of action and pharmacology and toxicology studies, as well as for clinical development efforts of drug substances.' Many ultratrace analysis techniques are based on chromatographic methods and usually require some form of sample preparation prior to analysis. The sample preparation step may be required to remove potential endogenous interferences and proteins. In addition, a sample concentration step is often required to raise the signal-to-noise level for the analyte to a value sufficient for accurate quantification. Liquid-liquid extraction (LLE) and more recently solidphase extraction (SPE) have been used extensively as sample preparation methods.* Although widely employed, LLE techniques have a number of potential disadvantages including the formation of emulsions, lack of totally automated instrumentation, and disposal issues around organic solvents. SPE has seen increasing use as a sample preparation techn i q ~ e . ~SPE B offers a number of attractive features including the availability of automated instrumentation and a high degree of selectivity in analyte isolation. However, for ultratrace analysis the collected SPE effluent must be removed prior to analysis. This solvent removal is a time-consuming step, often requiring hours or overnight conditions to reach dryness. Additionally, SPE still involves the use of large quantities of organic solvents, which results in significant purchase and disposal costs. Recent studies have shown that the use of supercritical fluids as an extraction medium provides a powerful alternative
to traditional extraction method^.^^^ Supercritical fluids can provide increased extraction efficiencies and give a t least 1 order-of-magnitude increase in the rate of extraction. The solvent strength of the supercritical fluid can be easily controlled by adjusting extraction pressure or temperature. Also, the supercritical fluid can be easily removed following extraction. The most commonly used supercritical fluid, COz, is cost effective from both an acquisition and disposal point of view. To date, most published supercritical fluid extraction (SFE) work has been done on solid or semisolid sample mat rice^.^ A small number of reports have dealt with the direct SFE of liquid matrices. Taylor and Hedric6v7extracted triprolidine, phenol, and phosphonate from aqueous solutions by directly passing a supercritical fluid through the matrix. Ong and co-worked extracted high levels of cholesterol from a plasma matrix directly with a supercritical fluid. An alternative approach for liquid aqueous matrices would be to remove the water before sample extraction with the supercritical fluid. Several approaches have been taken to remove water from sample matrices including freeze-drying the sample9 and using an adsorbent like anhydrous sodium sulfate.'O Additionally, Niessen et al. have used an on-line cartridge precolumn packed with XAD-2 resin to isolate an analyte from plasma prior to analysis by supercritical fluid chromatography.ll We report a new approach for the extraction of drugs at ultratrace levels from plasma which combines SFE with SPE using octadecylsilane (ODS) cartridges. The SFE/SPE procedure is similar to SPE except that a supercritical fluid is used to elute the analyte from the cartridges. The use of a supercritical fluid eliminates the time-consuming task of solvent removal incurred when conventional SPE is used for sample preparation in ultratrace analysis. Mebeverine alcohol (MEBOH; see Figure l),a metabolite of the antispasmodic drug mebeverine, was used as a model compound for these studies. The effect of extraction temperature and pressure on the recovery of MEBOH during SFE/SPE was examined to determine optimal extraction conditions. The selectivity, accuracy, and precision of SFE/SPE relative to conventional SPE were examined over a MEBOH concentration range from 10 to 250 ng/mL using a stable-isotope-based gas chromatography/mass spectrometry (GC/MS) method with selected-ion monitoring (SIM) for MEBOH analysis. EXPERIMENTAL SECTION Chemicals. Distilled/deionized water was from a Barnstead
Nanopure I1 system (Dubuque, IA). Methanol (MEOH; HPLC Grade), 2-propanol (HPLC Grade), and formic acid (Reagent Grade) were from J. T. Baker, Phillipsburg,NJ. Octane (99%+) and triethylamine (TEA: 99%) were from Aldrich Chemical, Milwaukee, Wl. Dimethylhexylamine (DMHA; Purum Grade) was from Fluka Chemical Corp., Ronkonkoma, NY. The derivatizing reagent, N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA) was from Pierce, Rockford, IL. Mebeverine alcohol and deuterium-labeled mebeverine alcohol (MEBOH-d4;see Figure 1)were synthesized at Procter & Gamble, Miami Valley Labo-
0003-2700/92/0364-0802$03.00/0Q 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992 HSCO ~ , - C H - K ( C H J , C H , - O H
I I
A
CH,CH~
6
C
D
Flgure 1. Structure of (A) mebeverine alcohol (MEBOH), (B) trimethylsllyl (TMS) derivative of MEBOH, (C) internal standard, m a beverlned, alcohol (MEBOW,), and (D) ThlS derivative of MEBOW,. The position of the "C label is shown by the asterisk on the MEBOH struchrrs. The ions used for selectedion monitoringduring the GWMS analysis are indicated for the TMS derlvatlves.
ratories. [14C]Mebeverinealcohol ([14C]MEBOH)was obtained from [14C]mebeverine (109 pCi/mg) by alkaline hydrolysis, as described previously.12 Dog plasma was obtained from Pel-Freez, Rogers, AR. SFC-grade C02and SFC-grade COzwith 5% MEOH modifier were purchased from Scott Specialty Gases, Plumsteadville, PA. SFE/SPE Recovery Studies. Dog plasma (1.0 mL), containing 0.1 mL of TEA or DMHA, was spiked with ['%]MEBOH (10,50, or 250 ng/mL). The plasma was applied to ODS cartridges (J.T. Baker, 3 mL, 500 mg), previously conditioned with 5 mL of MEOH followed by 5 mL of water/MEOH (95/5, v/v), at a flow rate of approximately 2 mL/min. The ODS cartridges were then washed with 5 mL of water/MEOH (95/5, v/v). In some cases, the cartridges were given an additional wash with 5 mL of water/MEOH (60/40, v/v). The cartridges were then blown dry with N2 The ODS packing was then removed from the cartridges and carefully transferred into 1-mL extraction vessels (Suprex, Pittsburgh, PA). Each vessel was placed in a Suprex SFE/50 extraction system with a fused-silica capillary restrictor (50 cm X 25 pm i.d.). The end of the restrictor was placed into a test tube (16 mm X 100 mm) containing 4 mL of 2-propanol at room temperature to trap the eluted [14C]MEBOH. Both C02 and c o z / 5 % MEOH were used as supercritical fluids. Extraction temperature, pressure, and time were varied to determine their effect on the recovery of ["CIMEBOH. The extent of the extraction was determined by counting the 2propanol solutions on a Packard Model 2000CA liquid scintillation analyzer (Packard Tri-Carb, Downers Grove, IL). SFE/SPE recovery was directly compared to an equivalent standard solution. Additionally, SFE/SPE recovery of ['%]MEBOH was examined without the use of amine modiiers in the plasma. Recovery was also examined when TEA was added directly to the ODS material prior to SFE/SPE, rather than added to the plasma prior to SFE/SPE. The use of a 100-mg polymeric-based octadecyl packing as the solid-phase material (Polysorb MP-1 Columns, Interaction Chemicals, Mountain View, CA) was also examined. SFE/SPE Selectivity, Accuracy, and Precision Studies. Blank plasma, blank plasma spiked with internal standard (MEBOH-d,, 50 ng/mL), and blank plasma spiked with intemal standard (50 ng/mL) and MEBOH (10,50, or 250 ng/mL) were prepared. TEA (0.1 mL) was added to 1.0-mL aliquota of each plasma sample, and the samples (1.1mL) were applied to ODS cartridges. The cartridges were washed and dried, and the ODS packing was transferred to the SFE extraction cells as described above. The SFE/SPE extraction was performed using coz/5%
803
MEOH at 350 atm and 40 "C for 10 min with a liquid flow rate of 0.3 mL/min. The effluent from the restrictor was passed through 4 mL of 2-propanol at room temperature to trap the extracted MEBOH and MEBOH-d4. After completion of the extraction, the 2-propanol was blown to dryness under N P The residue was derivatized with 0.2 mL of BSTFA at 60 "C for 15 min. The BSTFA was removed under Nz, and the resulting residue was reconstituted in 0.1 mL of octane. The reconstituted sample was analyzed by GC/MS analysis as described below. Solid-Phase Extraction Conditions. Solid-phase extraction was done using 3-mL syringe-barrel cartridges containing 500 mg of ODS (J. T. Baker). The cartridges were conditioned with 5 mL of MEOH followed by 5 mL of water/MEOH (95/5, v/v). Blank plasma, blank plasma spiked with MEBOH-d4(50ng/mL), and blank plasma spiked with MEBOH-d4 (50 ng/mL) and MEBOH (10,50,and 250 ng/mL) were applied to ODS cartridges. The total sample volume was 1.0 mL in each of these cases. The cartridges were then washed sequentially with 5 mL of water/ MEOH (95/5, v/v) and water/MEOH (60/40, v/v). In some instances, the second wash (water/MEOH, 60/40) was omitted. The cartridges were then eluted using 5 mL of water/MEOH/ TEA/formic acid (10/90/0.5/0.32, v/v/v/v). The purpose of TEA was to block active silanol sites, and that of formic acid was to reduce the ionization of acidic silanol groups on the ODs surface.'2 The effluent was collected and taken to dryness on a Speed Vac concentrator (Savant, Farmingdale, NY). The residue was then derivatized with 0.2 mL of BSTFA as described above and reconstituted with 0.1 mL of octane. The reconstituted sample was then analyzed by GC/MS analysis as described below. GC/MS Analysis. Briefly, the GC conditions involved a 0.5-pL splitless injection (1.0 min) onto a Hewlett-Packard (HP; Avondale, PA) Ultra-2 column (30 m X 0.20 mm i.d., 0.11-pm fi). A thermal program involving an initial oven temperature of 75 "C for 1min followed by a thermal ramp to 300 OC at 25 "C/min was used to elute MEBOH and MEBOH-d4. The injection port and GC/MS transfer line were held at 225 and 290 "C, respectively. MEBOH and MEBOH-d, were detected using SIM at m/z 216.0 and 220.0, respectively (see Figure 1). The GC/MS system was composed of a HP Model 58906 GC system equipped with a HP Model 7673A autosampler and a HP Model 5971A massselective detector. MEBOH standard solutions covering a concentration range from 2 to 500 ng/mL were prepared in MEOH. The standards (1.0 mL) were pipetted into test tubes containing 50 ng of MEBOH-d4,and the MEOH was removed under NP The standards were derivatized with BSTFA and reconstituted as described above. Standard curves were obtained by plotting the peak area ratio (peakarea MEBOH/peak area MEBOH-dJ versus concentration of MEBOH. The concentration of MEBOH in plasma samples was determined from the peak area ratio obtained from the sample by extrapolation from the linear regression curve.
RESULTS AND DISCUSSION Optimization of MEBOH Recovery. [WIMEBOH was used to examine the absolute recovery of the SFE/SPE and conventional SPE approaches. A high recovery of the analyte from the sample preparation step is desirable, although the use of a stable-isotope intemal standard (MEBOH-dd should correct for any laas of MEBOH. For both SFE/SPE and SPE, recovery of [14C]MEBOH was poor without the addition of an amine modifier due to silanol interactions between MEBOH and the silica. The recovery of [14C]MEBOH (50 ng/ mL) by SFE/SPE (350atm and 40 or 80 "C for 20 min) was less than 1 % without the addition of an amine modifier (TEA or DMHA) using either C02 or coz/5%MEOH as the supercritical fluid. Subsequent extraction of the ODS packing material demonstrated that the unextracted ['QCIMEBOH was still on the packing. It is common practice in the reversed-phase HPLC of basic compounds to mask the active silanol groups by adding a tertiary amine to the mobile phase. Two tertiary amines, TEA and DMHA, were investigated to determine their effect on SFE/SPE recovery of [W]MEBOH. The SFE/SPE recovery of [WIMEBOH from the ODS packing using plasma samples containing TEA or DMHA with c o 2 / 5 % MEOH as the su-
804
ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992
Table I. Absolute Recovery of ["CC]MEBOH"**
[14C]MEBOH concn, ng/mL 10.0 50.0 250
SFE/SPE 70recovery TEA DMHA 70.2 f 5.6 87.9 f 3.7 85.7 f 1.0
80.3 f 2.2 95.2 f 3.0 95.5 f 5.0
I""
i
I
SPE % recovery
101.3 f 1.1 100.2 f 3.0 100.6 f 1.5
Errors are standard deviations based on triplicate measurements. SFE/SPE conditions: supercritical fluid = CO,/MEOH (95/5), pressure = 350 atm, temperature = 40 "C, liquid flow rate = 0.3 mL/min, and extraction time = 10 min.
/ '"/
(I
percritical fluid is shown in Table I. The addition of the amine modifiers to the plasma resulted in good recovery of [ 14C]MEBOH (70-95%) for spiked plasma concentrations from 10 to 250 ng/mL. However, when pure COz was used as the supercritical fluid, recoveries of [14C]MEBOHwere less than 3%. Therefore, only c o z / 5 % MEOH was used in subsequent studies. The addition of DMHA gave a better recovery of [14C]MEBOH than TEA (Table I). The greater recovery obtained with DMHA may be due to less loss of DMHA during the Nz drying step that precedes the SFE/SPE extraction. It was also found that similar recoveries were obtained if TEA was added to the packing immediately before SFE extraction rather than into the plasma. However, all the packing may not be exposed to the modifier during the extraction without an initial static extraction step. The possible loss of extracted [ 14C]MEBOHdue to poor trapping or aerosol formation was studied by total counting of the radioactivity. Experimental resuits showed that the sum of the radioactivity in the trapping solvent and that left on the ODS packing virtually accounted for 100% of the radioactivity added to the sample. Therefore, the loss due to poor trapping is minimal. The recovery of [14C]MEBOHat the 10 ng/mL level using either TEA or DMHA was lower than obtained at higher spiked concentrations (Table I). The lower recovery at the 10 ng/mL level may be due to the inability of the supercritical fluid to overcome strong silanol interactions. An alternative approach for the plasma analysis would be to use a nonsilica-based support in the extraction cartridge to avoid the strong silanol interactions. A preliminary study with a polymeric-based support containing octadecyl groups as the solid-phase material gave quantitative recovery (100%) by SFE/SPE without the use of an amine modifier. Further investigations of this material are in progress. The recovery of [14C]MEBOH from the SPE extraction using an eluent containing 0.5% TEA is shown in Table I. Quantitative recovery of [14C]MEBOHwas obtained for all plasma concentration levels (10-250 ng/mL). The greater recovery of [14C]MEBOH by SPE relative to SFE/SPE is probably due to the water/methanol eluent being a stronger solvent than the supercritical fluid for desorbing the adsorbed MEBOH from the ODS surface. The standard deviations for triplicate extractions by SPE and SFE/SPE procedures are comparable. The time profile for the SFE/SPE of [14C]MEBOHin TEA spiked plasma from the ODS packing was studied by collecting the supercritical effluent from the cartridge in a series of Bmin intervals. The SFE/SPE recovery of [14C]MEBOH versus time, for extraction at 350 atm and 40 "C with a liquid flow rate of 0.3 mL/min, is shown in Figure 2. The extraction reached a plateau in less than 10 min under these conditions. To ensure complete extraction, however, a 10-min extraction time was used for all subsequent studies. The effect of pressure and temperature on the cumulative recovery of ['*C]MEBOH by SFE/SPE was also examined by collecting the effluent from a single extraction cell for 10
ci
0 0
5
10
20
15
25
Time (min.)
Flgure 2. Cumulative SFEISPE cumulative recovery of [ 14C]MEBOH from plasma samples applied to ODS cartrldges and extracted with C0,/5% MEOH in the presence of an amlne modifier (TEA) versus time.
'
O
so
-
i
60-
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8 K
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0
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 7, APRIL 1, 1992
805
Table 11. Analysis of MEBOH in Plasma Samples',*
found [MEBOH], ng/mL
[MEBOH],
ng/mL
1
2
10.0 50.0 250.0
10.2 47.7 226.2
10.1 47.8 226.9
10.0 50.0 250.0
9.75 47.6 238.9
9.99 49.4 234.2
% recovery mean (% RSD)
mean + SD
3
SFE/SPE 10.0
10.1 f 0.1 47.8 f 0.1 227.7 f 2.0
47.9 230.0
101.0(1.0) 95.6 (0.2) 91.1 (0.9)
SPE 9.87 51.3 236.2
9.87 f 0.12 49.4 4~ 1.8 236.5 f 2.3
98.7 (1.2) 98.8 (3.6) 94.6 (1.0)
'Errors are standard deviations based on triplicate sample preparations and analysis. *SFE/SPE conditions: supercritical fluid = CO,/MEOH (95/5),pressure = 350 atm, temperature = 40 OC,liquid flow rate = 0.3 mL/min, and extraction time = 10 min. mh: 216.0 30000 20000
10000
1
30000 20000
,,,I,
m/z: 220.0
mlz: 216.0
A
1
A A
i -
:::_E 0
mlz: 220.0
A
,,,,,,,,,
k E B Z
,,,,,,,,/
10000
::::j:i.;
,,,,,,,,,,,/
0
,,,,,,,,,,-
,
;
300001
20000
k;Eo:,,,_
d-MEBOH
l o o o o ~ A , , , ~
10000
,,,,,
0
0 8.6
0.8
9.0
9.2
-A C
=
9.4 9.6
8.6 8.8
9.0
9.2
9.4 9.6
Time (min.) Flgvr 4. selected-kn chromatograms (mlz 216.0 and 220.0) for the SFE/SPE of MBOH from (A) blank dog plasma, (B) blank dog plasma spiked wlth lntemel standard (MEBOKd,, 50 ng/mL), and (C) blank dog plasma splked wlth MEBOKd4 (50 ng/mL) and MEBOH (10 ng/mL). cation of the plasma sample. In the other case, the ODS cartridge was given an additional wash with water/MEOH (60/40, v/v). Typical SIM chromatograms obtained for samples prepared by SFE/SPE and SPE using the water/methanol(95/5) wash before elution are shown in Figures 4 and 5, respectively. Both approaches allowed interference-free detection of MEBOH and MEBOH-db However, the SIM chromatogram for the SPE approach contained several closely eluting peaks which were absent from the SFE/SPE approach. If the additional water/MEOH (60/40) wash was used, the selectivity of the SPE approach improved and wae equivalent to that obtained by SFE/SPE. Apparently, the intermediate wash with water/MEOH (60/40) removes some materials that are extractable with the SPE elution solvent but not extractable with the supercritical fluid. These results demonstrate the potential of SFE/SPE to provide different, and perhaps complementary,selectivities to those obtained with conventional SPE. DMHA was found to contain impurities which produced interferences in the GC/MS analysis by SPE and SFE/SPE. SFE/SPE Accuracy and Precision. The accuracy and precision of the SFE/SPE and conventional SPE approach were compared for the analysis of dog plasma (1.0 mL) spiked with MEBOH over a concentration range from 10 to 250 ng/mL. The results of the GC/MS analysis for triplicate SFE/SPE sample preparations of the spiked dog plasma are shown Table 11. The recovery of MEBOH relative to MEBOH-d4 from plasma by SFE/SPE ranged from 91.1 to 101.0% with RSD values of 1%or less for triplicate sample preparations. The recovery of MEBOH relative to MEBOH-d, from the plasma by the SPE method ranged from
8.6 8.8
9.0 9.2
9.4
9.6
8.6
8.8
9.0 9.2
9.4 9.6
Time (min.) Flgur 5. Selectedkn chromatograms (mlz 216.0 and 220.0) for the solid-phase extraction of MEBOH from dog plasma: (A) blank plasma, (B) blank plasma spiked with lntemal standard ( M E W 4 , 50 ng/mL), and (C) blank plasma spiked with MEBOH-d4 (50 ng/mL) and MEBOH (10 ng/mL).
94.6 to 98.8% with RSD values of 3.6% or leas (Table II). The SFE/SPE and SPE sample preparation approaches demonstrated comparable accuracy and precision for the analysis of MEBOH.
CONCLUSIONS Although the SFE/SPE approach described in this work is clearly in a development stage, there are a number of potentially attractive aspecta to this mode of sample preparation for the analysis of drugs in biological fluids. The removal of the supercritical fluid is very facile whereas the removal of the organic/aqueous eluent from SPE is often a very timeintensive step. The selectivity of the SFE extraction can be varied easily by changing the pressure and/or temperature conditions of the extraction and thereby changing the solvating power of the supercritical fluid. The selective partitioning of the anal* with the ODS packing coupled with the ability to vary the solvating power of the supercritical fluid by controlling pressure results in a powerful and selective new approach to sample preparation. Additionally, the SFE/SPE approach minimizes the cost associated with the use and disposal of organic solvents. Future areas of investigation will involve further examination of polymeric octadecyl materials, the direct use of the SPE cartridges without the removal of the packing material, and investigations into the effect of drying time and the effect of the composition and volume of the trapping solvent.
ACKNOWLEDGMENT The technical assistance of T. E. Delaney is gratefully appreciated.
006
Anal. Chem. 1992, 6 4 , 806-810
REFERENCES ( 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.
(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.
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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
