Anal. Chem. 1987, 59, 1872-1874
1872
CORRESPONDENCE Measurement of Electroactive Antibiotic Drugs in the Bloodstream of Rats with a Catheter Electrode Sir: In vivo monitoring of catecholamines, serotonine, and metabolites in rat brain tissue has been feasible with voltammetry for a few years (I, 2). But indwelling electrodes for continuous monitoring of therapeutic drugs in vivo have not been described (3). Voltammetry in blood using electrochemical transducers has enabled the blood oxygen tension to be determined. Several transducers have been described for oxygen. They are generally platinum electrodes based on the original Clark type electrode (4-6). In addition ascorbic acid, cystein, and glutathion gave some reproducible oxidation waves in blood serum and were tested in vitro (7,8). In this paper, we report the performances of an indwelling catheter that was introduced in the common carotid artery of living rats near the aortic arch. This electrode was a small carbon rod (0.5 mm diameter) in a polyethylene tube. This new transducer enabled the drug concentrations to be monitored by differential pulse voltammetry (DPV) after intravenous injection of antibiotics over a few hours in an useful therapeutical range (5 X to M depending of antibiotics)
EXPERIMENTAL SECTION
Voltammetry, Chemicals, and Electrolytes. Differential pulse voltammetry was performed with a conventional apparatus (PRG-5. Tacussel) using the following fixed parameters: scan rate, 40 m V d ; pulse amplitude, 100 mV; one pulse each 0.1 s. The calibration was based upon the peak height after subtraction of the residual current. An external calibration method was employed with standard solutions of drugs (5, 10,50, 100, 250, 500, and 1000 Wg-rnL-') in phosphate-buffered saline at 37 OC. Metronidazole, cefsulcdin, nimoraz.de, and chloramphenicol succinate were purchased from pharmaceutical laboratories. All miscellaneous compounds were analytical grade pure (Merck). High-Performance Liquid Chromatography. HPLC was used to assay drugs in samples of plasma in a series of rats used for comparison of both methods. For metronidazole, nimorazole, and chloramphenicol, 0.1 mL of rat plasma was extracted with 0.3 mL of ethyl acetate, then 0.2 mL of the supernatant was evaporated and 0.15 mL of the mobile phase added to the residue, and 0.05 mL was injected into the chromatograph. For cefsulcdin, 0.1 mL of rat plasma was extracted by 0.08 mL of methanol on a rotor-type agitator (5 min) and then centrifuged (10 min, lOOOg, 4 "C); 0.04 mL of the extract was injected into the chromatograph. The column was a pBondapak-C18 (30 cm to 10 pm) and the absorbance detector (M 640, Waters) was set at 280 nm for chloramphenicol, 313 nm for metronidazole and nimorazole, and 254 nm for cefsulodin. For chloramphenicol the mobile phase (MeOH/H,O/acetic acid 37/63/1) was pumped through at 1.3 mlemin-'. For cefsulodin the mobile phase (acetonitrile/ammonium acetate buffer 0.02 M, 3.5/96.5, pH 7.4) was pumped through at 3 rnl-min-'. For metronidazole and nimorazole the mobile phase (MeOH/phosphate buffered solution 5 X M, 20/80, pH 7.4) was pumped through at 2.5 mL-min-'. Standard solutions of drugs were prepared in pooled rat plasma without drugs.
Preparation of the Catheter Electrode. A carbon rod (external diameter 0.5 mm; Reynolds; length 2-3 mm) was introduced in a polyethylene tube (internal diameter 0.58 mm; outer diameter 0.96 mm; length 15-20 cm) with epoxy cement in its inner wall. A copper wire (200 W r n diameter) with a drop of graphite powder in polyester resin at one end was sealed to the carbon rod. The other end of the copper wire was sealed to the polyethylene tube with araldite. The electroactive part of the catheter was beveled with a cutter to facilitate introduction into the common carotid artery of the rat (Figure 1). A conventional Ag/AgCl reference electrode (a bare AgCl coated Ag wire) and an auxiliary platinum electrode were miniaturized (0.5 mm diameter, 1 mm length) and placed together in a Teflon tube. Animal Preparation. The experiments were conducted on Sprague-Dawley adult rats (mean weight 280 g). The animals were anesthetized with urethane (1.25 gkg-') given intraperitoneally as a 25% solution. Under these conditions the animals were anesthetised during all the measurements. One experiment was performed on an awake animal which was under light ether anesthesia during implantation of the catheter electrode. We used a variation of the chronic cannula implantation technique (10) to insert the catheter electrode into the right common artery. The right jugular vein was cannuled to inject drugs. In one experiment a cannula was also inserted in the left common carotid to obtain arterial blood near the catheter electrode for HPLC determinations. The cannulae were tunneled subcutaneouslv and brought out at the back of the neck. The reference and the auxiliary electrodes were implanted subcutaneo~~sly at the back of the neck.
RESULTS A N D DISCUSSION High-Performance Liquid Chromatography. Under the described conditions cefsulodin was eluted a t 6 min. The limit of sensitivity of the assay was 0.1 pgmL-' for plasma. Intraand interassay variation was below 10%. For metronidazole and nimorazole the retention times were 6 and 15.6 min, respectively. The limit of sensitivity was 0.5 pgmL-' for both of these drugs and intra- and interassay variations were below 6%. Chloramphenicol succinate and chloramphenicol eluted at 7.3 and 6.3 min, respectively. The limit of sensitivity was 0.5 pg.mL-' for both components. The intra- and interassay variations were below 5%. No metabolites of chloramphenicol, nimorazole, and metronidazole were found in rat plasma ( I 1-1 3 ) . Voltammetry. The diffusion coefficient for oxygen in phosphated buffered saline was the same as in blood a t 37 "C ( 1 4 ) . Thus we considered that the diffusion coefficient of drugs was the same in blood as in the phosphate-buffered saline. The potential range in reduction was as wide in the buffer as in the blood (-1.5 V vs. Ag/AgCl on 12.5 A full scale). In drug-free blood or buffer the oxygen peak was a t a variable potential between -0.5 and -0.7 V vs. Ag/AgCl. This peak was wide (0.4-0.5 V width) and its height depended on the oxygen concentration. At a fixed negative potential on the voltammetric wave the oxygen tension of blood or buffer could he evaluated as precisely with oxygen transducers. Peaks of
(9).
A classical method (high-performance liquid chromatography) and voltammetry were compared for two drugs (chloramphenicol and cefsulodin). A double catheterization of the carotids enabled the concentrations of metronidazole obtained with HPLC and voltammetry to be compared in the same rat. An awake animal was studied after intramuscular injection of nimorazole.
0003-2700/87/0359-1872$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
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Figure 1. (A) Scheme of the catheter electrode. (B) Implantation of the catheter electrode via the common carotii artery to the aortic arch
of rats.
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Figure 3. (A) Pharmacokinetics of cefsulodin after an iv injection in rats (125 mg-kg-'), HPLC determinations in plasma with standard deviations (n = 5) (0).Voqammetric determinations In the bloodstream of one rat (*). (B) Pharmacokinetics of chloramphenicol after an iv
injection (170 mg-kg-') of chloramphenicol succinate in rats. HPLC concentrations of chloramphenicol and chloramphenicol succinate in plasma were added (0).Voltammetric determinations in blood stream of one rat (M).
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2006
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B
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Figure 2. In vivo voltammograms in the bloodstream of rats: (A) lower
trace (1) obtained before injection and upper trace (2) after injection of metronidazole (-1.1 V, 95 pg-ml-l); (B) peak due to cefsulodin (-1.2 V, 83 pg-mL-I); (C) peak due to chloramphenicol (-1.0 V 9 0 pgmL-'); (D) peak due to nimorazole (-0.9 V, 88 pg-mL-'). The first peak (-0.4 to -0.7 V) was due tq arterial oxygen. drugs were well separated from the oxygen peak (-1.1 V vs. Ag/AgCl for metronidazole,-1.2 V vs. Ag/AgCl for cefsulodin, -1.0 V vs. Ag/AgCl for chloramphenicol and chloramphenicol succinate, -0.9 V vs. Ag/AgCl for nimorazole) (see Figure 2). In vitro and ih vivo voltammograms were exactly identical. The width of peaks varied from 0.45 to 0.6 V. The limit of sensitivity in vitro was 5-10 pg-mL-' depending on the catheter electrodes. The reproducibility of measurements was between 7 and 11 % in vitro. These catheter electrodes had a life span of several months in vitro, but in vivo the life span varied from 30 min to 3 or 4 h. In fact in vivo the life span depends on the method of implantation during anesthesia and on the blood clotting around the tip during the experiment. Two observations indicated the "death" of the catheter electrode in vivo: Firstly, the width, height, and potential of the oxygen peak changed; secondly, electrical artifacts and increasing residual currents appeared. These facts indicated clotting on the electrode surface. Rats could be injected with heparin or other anticoagulants to prolong the "life" of the electrodes. Pharmacokinetics. In Figure 3A the on-line pharmacokinetics of cefsulodin after an intravenous injection (125 mg/kg) was compared to HPLC determinations of the drug. Cefsulodin is a third generation cephalosporin which is not metabolizable and is excreted only in the urine (15). Binding to plasma rat proteins measured by equilibrium dialysis is low (33%). The correlation of both techniques was excellent (R = 0.998, n = 6). The similar results obtained by HPLC and voltammetry are explained by the fact that the drug t o serum-albumin complex in plasma is rapidly and highly reversible (17). In Figure 3B the concentrations of chloramphenicol and chloramphenicol succinate obtained by HPLC were added; in fact the prodrug and the drug are equally potent and presented the same NOz group for reduction. Thirty minutes after injection of the drug (170 mgkg-') all chloramphenicol succinate was hydrolyzed to chloramphenicol. The correlation of both methods was good ( R = 0.996, n = 5). Binding of chloramphenicol to rat proteins measured by
Figure 4. (A) Pharmacokinetics of metronidazole (140 mg-kg-') (") in one rat with double catheterization after an intravenous injection. The
second catheter in the other caroti was used to 6 ?w plasma. The last six measurements were correlated with HPLC cuterminations. (B) An awake rat was injected intramuscularly with nimorazole (140 mg-kg-I) and measurements with the catheter electrode were done during diurnal activity of the rat without perturbations. equilibrium dialysis was low (25%) and did not interfere in the voltammetric assay. In Figure 4A after injection of metronidazole (140 mgkg-') plasma samples were drawn every 15 min by the other carotid; HPLC and voltammetric measurements were well correlated on the same animal (R = 0.998, n = 6). Metronidazole is an unbound drug and no metabolite was observed in plasma (16). The measurements were carried out after injection of nimorazole (140 mgkg-' im) in an awake rat. No perturbations of the signal and no physiological reactivity of rats were observed. The blood was collected at the end of the experiment. HPLC and voltammetric determination a t this time gave similar values according to the standard deviations of both methods. In conclusion, the carbon-rod catheter electrode described here can be used to assay electroactive drugs in the bloodstream. Measurements were on-line and did not perturb animals. Antibiotics seemed to be a good choice for the application of this catheter electrode: the therapeutic range was usually higher than the limit of sensitivity of the electrode; furthermore new antibiotics are generally not metabolizable drugs. In this preliminary work an interesting fact is that I have demonstrated in vivo that, as suggested by others (In, the concentration of the total drug and the diffusible drug was the same for all studied antibiotics in the bloodstream. ACKNOWLEDGMENT We thank J. Alix for translation and A. Sitbon for typing the manuscript. Registry No. Metronidazole, 443-48-1;cefsulodin, 62587-73-9; chloramphenicol, 56-75-7; nimorazole, 6506-37-2.
Anal. Chem. 1987, 5 9 , 1874-1879
1874
LITERATURE C I T E D (1) Adarns, R. N. Anal. Chem. 1978, 4 8 , 1126A-1137A. (2) Stamford, J. A. Brain Res. Rev. 1985, 10, 119-135. (3) Pinkerton, T. C.; Lawson, 8. L. Clin. Chem. (Winston-Salem, N . C . ) 1982, 28, 1946-1955. (4) Meldrum, S. J.; Watson, B. W.; Becker, G. A. Biomed. Eng. 1973, 471 -479. (5) Nilsson. E.; Edwall, G.; Larsson, R.; Olsson. P. Scand. J. Clin. Lab. Invest. 1981, 4 1 , 557-563. ( 6 ) Wise, R . R.; Naylor, A. W. Anal. Biochem. 1985, 146, 260-264. (7) Pradac, J.; Prodacova, J.; Koryta, J.; Ossendorfova, N. Experentia , Suppl. 1971, 78, 367-373. (8) Koryta, J. Medlcal and Biological Applications of Nectro-chemical Devices; Wlley: New York, 1980; Chapter 7. (9) Goodman Gllrnan, A.; Goodman, L. S.; Gilman, A. The Pharmacological Basis of Therapeutics; Macmillan: London, 1980; Section X I I . (10) Popovic, V.; Popovic, P. J. Appl. Physiol. 1980, 15, 727-728. (11) Nilsson-Ehle, I.; Ursing, 6.; Nilsson-Ehle, P. Antimicrob, Agents Chemother. 1981, 19, 754-760. (12) Wheeler, I. A.; De Meo, M.; Halula, M.; George, L.:HeseRine, P. Antimicrob. Agents Chemother. 1978. 13, 205-209.
(13) Oseekey. K. 8.;Rowse. K. L.; Kostenbauder, H. K. J. Chrom., Biomed. Appl. 1980, 182, 459-464. (14) Baumgartl, H.; Lubbers, D. W. Polarographic Oxygen Sensors; Springer-Verlag: Berlin, 1983; Chapter 1.4. (15) Tanayama. S.; Yoshida, K.; Kanai, Y. Antimicrob. Agents Chemother. 1978, 14, 137-143. (16) Meulemans, A.; Vicart, P.; Mohler, J.; Henzel, D.; Vulpillat, M. Chemotherapy (Basel) 1988, 32, 486-493. (17) Ogren, S.;Cars, 0. Scand. J . Infect. Dis., Suppi. 1985, 44, 34-40.
Alain Meulemans Laboratoire de Biophysique Facult6 de MBdecine Xavier Bichat 16 rue H. Huchard Paris 75018, France
RECEIVED for review December 16, 1986. Accepted April 3, 1987.
Electrothermal Vaporization and Laser- Induced Fluorescence for Screening of Polyaromatic Hydrocarbons Sir: Recently, several groups have reported on the use of electrothermal atomizers for the study of the ultraviolet absorption of molecular vapors (1-6). Thompson and Wagstaff used ultraviolet spectrometry to monitor organic pollutants in water (I). They found that by monitoring the absorption a t one or more wavelengths as a small amount of sample was vaporized from 35 to 900 "C, they could rapidly characterize many types of substances based on peak shapes, intensities, and appearance times. The higher temperatures obtained with the graphite furnace allowed the rapid characterization of samples often too difficult to analyze by gas chromatographic methods, such as heavy oils, tar residues, and resinous materials. This technique has also been used by Tittarelli et al. for the identification of crude oils spilled in seawater (2),for the identification of organic and inorganic pigments ( 3 ) ,and for recording the vapor spectra of several aromatic hydrocarbons ( 4 ) . The latter work involved using the graphite furnace to produce a steady vapor supply while scanning over the absorption spectrum. Several sample aliquots were generally necessary to acquire the entire spectrum. The use of photodiode arrays for fast spectral acquisition was suggested for the programmed heating studies of molecular compounds. Subsequently, the graphite furnace was combined with a diode array detector for the ultraviolet investigation of crude oils, pigments, and polymers (6). Thermal cycles from 150 to as high as 2750 O C were run while monitoring the ultraviolet spectra. Both atomic and molecular emission and absorption occurred at temperatures exceeding loo0 O C and emission was evidenced in the occurrence of negative absorbance peaks. The collected spectra were plotted as a three-dimensional representation of the vaporization cycles to produce spectral "fingerprints" characteristic of the vaporized oils or pigments. The simultaneous measurement of more than one parameter in this way provided enhanced selectivity over conventional spectroscopic techniques, especially where spectra were too broad banded to be useful for analytical measurement of similar compounds. While the graphite furnace has been used as a vaporization source for laser-excited atomic fluorescence, it has not been widely applied to the laser-induced fluorescence of molecular vapors. In this work, laser-induced fluorescence of molecular vapors produced by electrothermal vaporization was investigated. The capabilities and behavior of the laser-induced fluorescence of polyaromatic hydrocarbons (PAHs) produced 0003-2700/87/0359-1874$0 1.50/0
in a graphite furnace were evaluated in terms of sensitivity and potential use as a fingerprinting technique for particulate matter and other environmental samples. A new sampling technique was developed which allowed the direct collection of samples within the graphite rods of the furnace system. The advantages and limitations of this technique as a routine screening technique will be discussed. EXPERIMENTAL SECTION Apparatus. A block diagram of the various detection systems used in this work is shown in Figure 1. Fluorescence could be excited from opposite directions by either a lumonics TE 861s excimer laser or a Molectron UV 24 N2laser passing directly above the graphite furnace. Either laser was brought t o a focus a few millimeters beyond the vapor produced by the furnace. Lenses on either side of the furnace were used to form a 1:l image of the fluorescence onto the entrance slit of a 0.35-m Heath monochromator (EU-700) and a 0.2-m ISA flat field spectrograph (UFS-200). Apertures were used t o reduce stray light and background emission from the furnace. Limits of detection for several PAHs were obtained by using an R1414 photomultiplier tube (Hamamatsu Corp.) with the Nzlaser as the excitation source. A photodiode was used to trigger the boxcar. Fluorescence was detected at the wavelength of maximum emission for the particular PAH. The ISA flat field spectrograph had a dispersion of 24 nm/mm and a spectral range of 200-800 nm. The spectrograph was mounted to a Princeton Applied Research OMA detector (either a 1205D SIT or an OMA I11 1421 linear photodiode array): the grating could be accessed and rotated back and forth to shift the spectral region of interest onto the detector. Graphite Furnace System. The graphite furnace is illustrated in Figure 2. Two water-cooled brass blocks were mounted on opposite sides of a round phenolic block and were connected beneath the phenolic block to an SCR 20-250 power supply (ElectronicMeasurements). A groove in the center of these brass electrodes supported the graphite rod which contained the sample. Electrodes to fit both 1/4 in. diameter and 3/s in. diameter graphite rods were constructed. The in. rods were solid throughout, except that a small 2-mm circular depression approximately in. deep was drilled out to hold the sample. For this type rod, argon carrier gas was connected underneath the phenolic block and fed through a burner head to flow upward past the graphite rod. Room air currents were found to grossly affect the reproducibility of signal measurements; a glass cover with quartz windows was used to minimize this problem. The 3/8 in. diameter rods were hollow, with a small hole drilled through the middle of the top for sample introduction. The carrier gas was made to 0 1987 American Chemical Society
