Anal. Chem. 1995, 67, 1838-1844
Capillary Zone Electrophoresis with Laserhduced Fluorescence Detection for the Determination of Nanomolar Concentrations of Noradrenaline and Dopamine: Application to Brain Microdialysate Analysis F M M c Robert,**t Lionel Bert, Luc Denoroy,* and Bemard Renaudt
Laboratoire de Neuropharmawlogie, UMR 105, CNRS et Universite Claude Bemard Lyon I, Facult6 de Pharmacie, 8 Avenue Rockefeller, 69373 Lyon Cedex 08,France, and CNRS, Service Central dAnalyse, BP 22, 69390 Vemaison, France
Determination of catecholamines by capillary zone electrophoresiswith laser-inducedfluorescence detection was performed on low-concentration samples, which were derivaW with naphthalene-2,3-dicarboxaldehyde to give highlyfluorescent compounds. When the borate concentration in the derivatization medium was decreased from 130 to 13 mM, sensitivity for noradrenaline (NA) and dopamine (DA) was greatly enhanced while resolution between these two compounds decreased. A 50 mM borate concentrationin dehtization medium was chosen since it provided maximal resolution between NA and DA, together with a high separation aciency (3.1 d o n theoretical plates per meter for DA). The injection of 2.4 nL of a NA and DA solution derivatized at M produced peaks with signal-to-noiseratio of 8:1 and 3:1, respectively, corresponding to 1.8 am01 of each catecholamine. Ihe calibration curves were linear when NA and DA solutions were derivatized at concentrations ranging from to M. This method was used to determine NA in brain extracellularfluid: a peak corresponding to a basal level of 5 x M endogeneous NA was observed in microdialysates from the medial frontal cortex of the rat, and its nature was confirmed by both electrophoretic and pharmacological validations. The catecholamines noradrenaline (NA) and dopamine @A) play a major role as neurotransmitters in central and peripheral nervous systems and probably are the most studied of all neurotransmitter substances.' In vivo techniques such as brain microdialysis have been developed for monitoring extracellular neurotransmitter concentrations in anesthetized or freely moving animals. A microdialysis probe is implanted in the brain and perfused at a low flow rate with artificial cerebrospinalfluid (CSFJ. Neurotransmitters diffuse from the extracellular fluid to the artificial CSF inside the probe through the dialysis membrane and are further analyzed in collected samples. An analytical technique suitable for microdialysis has to deal with small volumes (a few microliters collected) and with very low neurotransmitter con' CNRS et Universitk Claude Bernard Lyon I. + CNRS, Service Central d'halyse. (1) Abercrombie, E. D.; Finlay, J. M. Microdialysis in the Neurosciences; Elsevier: Amsterdam, The Netherlands, 1991; pp 253-274.
1838 Analytical Chemisrty, Vol. 67, No. 1 1, June 1, 1995
centrations, usually in the nanomolar range. Furthermore, from a neurobiological point of view, it is of interest to be able to monitor over time both catecholamines and other neurotransmitters such as amino acids or neuropeptides. Capillary zone electrophoresis (CZE) offers remarkably high separation efficiencies for the analysis of complex mixtures.2 Moreover, the small inner diameter of the capillary tube is well adapted to the analysis of extremely small volumes of sample: some nanoliters of the few microliters of the sample are injected. However, given the small amount of molecules injected into the system, a high-sensitivity detector is often required for capillary electrophoresis. In this respect, CZE coupled with laser-induced fluorescence detection (LIFD) is of particular interest since very low detection limits of 10-18-10-21 mol can be r e a ~ h e d ~and -~ various molecules such as amino acids,5peptides,s proteins? and Carbohydrates'O can be detected. The combination of brain microdialysis with CZE-LIFD has been used by Hernandez and c o - ~ o r k e r s ' ~for - ~ ~the determination of extracellular levels of glutamate, an excitatory amino acid neurotransmitter. However, one disadvantage of such detection is that many molecules of biological interest (including catecholamines) do not fluoresce with sufficient quantum yields at easily evaluated wavelength^.'^ As a consequence, a derivatization reaction with a fluorescent tag (2) Landers, J. P.; Oda, R P.; Spelsberg, T. C.; Nolan, J. A; Ulfelder, K. J. BioTechniques 1993,14, 98-111. (3) Cheng, Y. F.; Dovichi, N. J. Science 1988,242, 562-564. (4) Wu, S.; Dovichi, N. J. J. Chromatogr. 1989,480, 141-155. (5) Sweedler, J. V.: Shear, J. B.; Fishman, H. A; Zare, R N.Anal. Chem. 1991, 63, 496-502. (6) Zhang, J. Z.; Chen, D. Y.; Wu, S.; Harke, H. R ; Dovichi, N. J. Clin. Chem. 1991,37, 1492-1496. (7) Ueda, T.; Mitchell, R; Kitamura, F.; Metcalf, T.; Kuwana, T.; Nakamoto, A J. Chromatogr. 1992,593, 265-274. (8) Chan, K C.; Janini, G. M.; Muschik, G. M.; Issaq, H. J. J. Lig. Chromatogr. 1993,16, 1877-1890. (9) Lee, T. T.; Yeung, E. S. J. Chromatogr. 1992,595, 319-325. (10) Liu, J.; Shirota, 0.;Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 2302-2306. (11) Hernandez, L.; Escalona, J.; Verdeguer, P.; G m a n , N. A J Liq. Chromatogr. 1993,16, 2149-2160. (12) Hernandez, L.; Tucci, S.; Guzman, N. A,: Paez, X.J. Chromatogr.,A 1993, 652,393-398. (13) Hernandez, L.; Joshi, N.; Murzi, E.; Verdeguer, P.; Mifsud, J. C.: Guzman, N. A J. Chromatogr., A 1993,652, 399-405. (14) Olefirowicz, T. M.: Ewing, A G. In Capillay Electrophoresis. Theoy and Practice; Grossman, P. D., Colburn, J. C., Eds.; Academic Press: New York, 1992; pp 45-85.
0003-2700/95/0367-1838$9.00/0 0 1995 American Chemical Society
is needed to detect the molecules of interest. The only reported labeling of catecholaminesfor CZE analysis uses naphthalene-2,3-dicarboxaldehyde (NDA) as derivatizing reagent, in association with UV detection,15 and, recently, in association with LIFD.I6 This fluorescent compound reacts with primary amines in the presence of cyanide to produce highly fluorescentcyanovlbenzoisoindole (CBD derivatives. Using NDA as derivatizing reagent, low-concentration derivatizations (in the nanomolar range) have been performed for liquid chromatography (LC) assays of catecholamine^,'^ amino acids,18and peptidesIgas well as for CZE assays of amino acids.7r20 Moreover, this fluorescent reagent has been used for the determinations of DA and amino acids in single celW and of amino acids in single neurons18.20and in microdialysis samples.l1Zz1 This paper describes the development of a technique combming CZE-LIFD with NDA derivatization in order to measure very low NA and DA concentrations. Optimization of derivatization and separation conditionsis investigated to reach high separation efficiency and sensitivity, with optimal resolution between NA and DA. Fluorescence linearity, derivatization on microvolumes, and reproducibility are tested. The combination of microdialysis and CZE-LIFD is then reported for the monitoring of nanomolar concentrations of NA in brain extracellular fluid. EXPERIMENTAL SECTION CZE-LlFDSystem. The capillary electrophoresissystem with LIFD used (IRIS 2000, Europhor Instruments SA,now Zeta Technology S.A., Toulouse, France) is directly derived from the instrument described by Hernandez and co-workers.22 In this respect, a collinear scheme was used for fluorescence detector. The excitation was performed by a Liconix helium-cadmium laser (10 mw) at a wavelength of 442 nm. The emission intensity was measured at a wavelength of 490 nm filtered by a band pass filter, and a notch filter was used to attenuate background radiations. Fluorescence was detected by a photomultiplier tube, electrical current was generated, and relative fluorescence was then expressed in microamperes. Separations were carried out with a fused-silica capillary [72 cm x 50 pm inner diameter (i.d.); effective length 52.5 cm; Polymicro Technology] and on-column LIFD was carried out through a 5 mm wide window opened by removing the polyimide cover of the capillary. Before each electrophoretic run, the capillary was sequentially flushed (4 x 2 min) with 1 M and 0.1 M sodium hydroxide, water, and finally separation buffer. Hydrodynamic injections were made by applying vacuum (300 mmHg) at the detection end of the capillary for a fixed period of time (2 s): the injection volume was calculated as being 2.4 nL according to the Hagen-Poiseulle formula.23 Unless otherwise (15) Weber, P. L.; O'Shea, T. J.; Lunte, S. M.J. Phamaceut. Biomed. Anal. 1 9 9 4 , 12, 319-324. (16) Gilman, S. D.;Ewing, A G. Anal. Chem. 1995, 67, 58-64. (17) Kawasaki, T.; Higuchi, T.; Imai, K.; Wong, 0. S. Anal. Biochem. 1989,180, 279-285. (18) Oates, M. D.; Cooper, B. R; Jorgenson, J. W. Anal. Chem. 1990,62,15731577. (19) Mifune, M.; Krehbiel, D. K.; Stobaugh, J. F.; Riley, C. M. J. Chromatogr. 1989,496, 55-70. (20) Kennedy, R. T.; Oates, M. D.; Cooper, B. R; Nickerson, B.; Jorgenson, J. W. Science 1989,246, 57-63. (21) O'Shea, T. J.; Weber, P. L.; Bammel, B. P.; Lunte, C. E.; Lunte, S. M. J. Chromatogr. 1992,608, 189-195. (22) Hernandez, L.; Escalona, J.; Joshi, N.; Guzman, N. A. J. Chromatogr. 1991, 559, 183-196.
specified,the separation buffer used was 110 mM phosphate buffer (PH 7.0). The capillary was maintained at 25.5 "C through a Peltier effect, and the running voltage was 15 kV. In experiments performed on standard solutions, a neutral marker (ethylamine) was simultaneously injected with the sample. The instrument was controlled and data were acquired (100 points/$ with an original MacIntosh software (Inforep SARL, Fontenay-leFleury,France) developed from LabVIEW 2 software (National Instruments Corp.). Reagents. NA, DA, dihydroxybenzylamine (DHBA), ethylamine, cysteic acid, mixture of amino acids, urea, normetanephh e , boric acid, sodium tetraborate, monobasic and dibasic sodium phosphate, desipramine, and idazoxan were purchased from Sigma. NDA was from Fluka and sodium cyanide from Prolabo. Water used throughout was of HPLGgrade obtained with a Mia-Q system (Miilipore). All other chemicals were of the highest purity available. Solutions. Buffers. Sodium borate buffer (500 mM, pH 8.60) was obtained by preparing separate solutions of boric acid (500 mM) and sodium tetraborate (125 mM). These solutions were then mixed to obtain a pH 8.6 buffer. Sodium borate buffer (100 mM, pH 9.00) was similarly prepared. Sodium phosphate buffers were obtained by preparing separate 110 mM solutions of monobasic and dibasic sodium phosphate and mixing them to obtain the appropriate pH (7.00,8.85). The buffer solutionswere filtered through a 0.2 pm pore size membrane filter (cellulose nitrate) and sonicated 10 min before use. NDA. A 5 mM solution was prepared in methanol on a weekly basis and stored at 4 "C. Sodium Cyanide. A 10 mM solution was prepared in water on a weekly basis and stored at 4 "C. Amines. The 1 mM stock solutions of NA, DA, DHBA, ethylamine, and amino acid mixture were prepared in 0.1 M perchloric acid and stored at -20 "C by aliquots of 1 mL. The required working solutionswere obtained by further dilution with water or artificial CSF. Standard solutions were prepared fresh for each sample to be derivatized. Cysteic Acid. A 0.1 M solution was prepared in water on a weekly basis and stored at 4 "C. Derivatization Procedures. Unless otherwise specified, the general derivatization procedure was as follows: to 750 pL of NA and DA standard solution in water (including M ethylamine as a neutral marker and M DHBA) were added 100 pL of 500 mM borate buffer solution @H 8.6), 90 pL of 10 mM sodium cyanide solution, and 10pL of 5 mM NDA solution. M e r shaking, the reaction was allowed to proceed at ambient temperature for 15 min and then quenched by the addition of 50 pL of 0.1 M cysteic acid solution. The sample was injected 2 min later. A quenching agent (high amounts of cysteic acid) was added after the derivatization was complete in order to remove the excess of NDA and cyanide, and, therefore, to minimize the formation of side product^.'^ Moreover, because of its very high negative charge, the CBI derivative of cysteic acid had a much longer migration time than catecholamines. For the microdialysis experiment, a 0.5pL volume of 500 mM borate buffer solution (PH 8.6) and 0.45 pL of 10 mM sodium cyanide were added to 3.6 pL of dialysate collected on 0.35 pL of 0.1 M perchloric acid. Next, 0.1 p L of 5 mM NDA solution was (23) Moring, S. E. In Capillary Electrophoresis. Theory and Practice; Grossman, P. D., Colbum, J. C., Eds.; Academic Press: New York, 1992; pp 87-110.
Analytical Chemistry, Vol. 67, No. 1 7 , June 1, 1995
1839
added. After shaking, the reaction was allowed to proceed at ambient temperature for 15 min and then quenched by the addition of 0.25 pL of 0.1 M cysteic acid solution. The sample was injected 2 min later. A calibration curve was simultaneously prepared by replacing dialysate sample by NA and DA standard solution in artificial CSF. Expression of Results and Data Analysis. The electrophoretic mobility (4 was determined according to the equation p = (I!&,&) ( l / t - l/toJ,24where L is the effective length of the capillary,Lt the total length, Vthe applied voltage, t the migration time, and to, the migration time of the neutral marker measured at the onset of the large peak of neutral marker. The electroosmotic flow (i.e., the apparent electrophoretic mobility of the neutral marker) was determined by the equation pOs=/&!J Vt,, bo,= 3.6 x cm2/V-sin 110 mM phosphate buffer (PH 7.0) at 25.5 O C 1 . 2 4 The separation efficiency was estimated by calculating the theoretical plate count per meter (N) using the equation N = 5.54 (1/L)(t/Wd2,where Wh is the width at half maximum of the peak. Resolution between two sample components can be defined by the equation R = 2(tz - t l ) l ( w l + U I Z ) ? ~ where tl and h are the migration time of the two components and w1 and wz their respective baseline peak widths. Linear regression analysis was performed to test the existence of statistically signscant linearity of calibration curves. Microdialysis Experiments. Preparation and Calibration of the Dialysis Probe. A concentric microdialysis probe with an active dialysis length of 3 mm was constructed in our laboratory from regenerated cellulose dialysis tubing (Spectra/Por hollow fiber; molecular weight cutoff 6000; 225 pm outer diameter: Spectrum Medical Industries) according to the model described by Abercrombie and Finlay.1 In vitro probe recovery was determined before an in vivo experiment as previously described26and was estimated to be 51.6%for NA when the probe was perfused at 0.2 pL/min. The use of such a very low flow rate allows better recoveries and avoids marked disturbance of neural ti~sue.2~ Dialysis Probe Implantation and Collection of Dialysates. An in vivo microdialysis experiment was carried out on a male SpragueDawley OFA rat (IFFA Credo, L'Arbresle, France; 300 g) initially anesthetized with 400 mg/kg intraperitoneally (ip) chloral hydrate. Anesthesia was maintained during the experiment by administering 30 mg/kg ip chloral hydrate every hour. The rat was placed in a stereotaxic frame (David Kopf Instruments). Body temperature was maintained at 37.5 f 0.5 "C with a homeothermic blanket system (Harvard Instruments). The skull was exposed and a small hole was drilled to allow the implantation of the dialysis probe in the medial frontal cortex at the following coordinates (relative to bregma): anterior 3.2 mm, lateral 1.2 mm, and ventral 7.0 mm below the bone surface.' The inlet of the dialysis probe was connected via polyethylene tubing to the perfusion pump while the outlet (fused-silicacapillary tubing 0.075 mm i.d.; dead volume 1 pL) was inserted in a 50-pL microvial containing 0.35 pL of 0.1 M perchloric acid to prevent catecholamine oxidation. The probe was continuously perfused with artificial CSF (145.0 (24) Grossman, P. D. In Capillay Electrophoresis. Theoy and Practice; Grossman, P. D., Colbum, J. C., Eds.; Academic Press: New York, 1992; pp 111-132. (25) Grossman, P. D. In Capilhwy Electrophoresis. Theory and Practice; Grossman, P. D., Colbum, J. C., Eds.; Academic Press: New York, 1992; pp 3-43. (26) Robert, F.; Lambas-Senas, L.;Ortemann, C.; Pujol, J. F.; Renaud, B. J. Neurochem. 1993,60, 721-729. (27) Gonzalez-Mora, J. L.; Guadalupe, T.; Fumero, B.; Mas, M. Monitoring Molecules in Neuroscience; University Centre for Pharmacy: Groningen, The Netherlands, 1991; pp 66-67.
1840 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
mM NaCl, 2.7 mM KC1, 1.0 mM MgC12, 1.2 mM CaC12, 0.1 mM NaHzP04,O.l mM Na2HP04, pH 6.8) at a flow rate of 0.2 pL/min while lgmin fractions (3.6 pL) were collected. Samples were derivatized as soon as collected and immediately analyzed. The three first samples collected after probe implantation were discarded. Pharmacological Treatments. The presence of NA in dialysates was authenticated by administering drugs known to increase NA extracellular concentration:28desipramine (20 mg/kg ip dissolved in artificial CSF) and idazoxan (20 mg/kg ip dissolved in artificial CSF). Administration of drugs were timed to take into account the 1-pL dead volume of the outlet of the dialysis system. RESULTS AND DISCUSSION Since fluorescent labeling of catecholamineshas been reported in LC assays with fluorescence or chemiluminescence detection using NDA as derivatizing reagent,l7sBwe selected this precolumn derivatization method with a minor modification: sodium cyanide concentrationwas increased from 0.2 to 0.9 mM, the concentration at which reaction operates at its maximal rate30 and at the optimal ratio between cyanide and NDA $e., at least a l0-fold excess of cyanide) Choice of Separation Buffer, Our initial separation conditions were those reported by Ueda and co-workers7for the CZE separation of NDA-labeled amino acids, Le., 100 mM borate buffer (PH 9.0) as running buffer. Using these conditions,we found that NA-CBI and DA-CBI did not appear on the electropherogram during the analysis time applied (1200 s): only the CBI derivative of the neutral marker (ethylamine) was eluted (data not shown). Then, we replaced the borate buffer with a phosphate buffer in which NDA-labeled catecholamines have been shown to exhibit higher fluorescence inten~ities.'~Separation performed in such a phosphate buffer (110 mM, pH 8.85) showed that these compounds were eluted during the analysis time and exhibited a high fluorescence but were not resolved (Figure lA). When the pH of the phosphate separation buffer was decreased from 8.85 to 7.00, the resolution between NA-CBI and DA-CBI was dramatically improved (R = 1.8, Figure 1B). When compared to phosphate buffer (PH 8.85), no change in peak heights was observed at pH 7.00. Moreover, optimal conditionswere obtained when the phosphate concentration ranged between 90 and 120 mM (data not shown). Finally, we confirmed that the separation buffer must not contain borate ions since the addition of borate buffer (2.5-35 mM) in 110 mM phosphate buffer (PH 7.00) induced a pronounced decrease of NA-CBI and DA-CBI peaks heights whereas the total fluorescence emission of these derivatives (estimated by peaks areas) was constant: baseline peak widths were thus increased (data not shown). This shows that borate in the separation buffer has a great influence on peak shape and suggests that, when a highly concentrated borate separation buffer was used (see above), peaks could disappear. Influence of Borate Concentration in Derivatization Buffer. Since borate in the separation bufferhas been shown to decrease the peak height of NDA-labeled NA and DA, and since sample (28) Dennis, T.; L'Heureux, R.; Carter, C.; Scatton, B. J. Phamzacol. Erp. They. 1987,241, 642-649. (29) Kawasaki, T.; Imai, K; Higuchi, T.; Wong, 0. S. Biomed. Chromatogr. 1990, 4, 113-118. (30) De Montigny, P.; Stobaugh, J. F.; Givens, R S.; Carlson, R G.; Srinivasachar, K; Sternson, L. A; Higuchi, T. Anal. Chem. 1987,59,1096-1101. (31) Lunte, S. M.; Wong, 0. S. LC-GC 1989,7,908-916.
A
DA, NA
9 9j
4
87
4
v v
I
~
? ]
h
-1
1000
500 migration time (s)
NA
DA
10 20 30 40 50 60 70 80 90 100 110 120 130
B
M I
0
I
500 migration time (s)
Borate concentration (mM) ~-
I
1000
Figure 1. Electropherograms of M NA-CBI and DA-CBI (including M ethylamine as a neutral marker) obtained with two different separation buffer solutions: (A) 110 mM phosphate buffer (pH 8.85); (B) 110 mM phosphate buffer (pH 7.00). Conditions: see Experimental Section; current, 66 and 55 pA in (A) and (B), M NA respectively. Derivatization procedure: to 600 pL of a and DA standard solution (including M ethylamine as a neutral marker), were added 250 pL of a 500 mM borate buffer solution (pH 8.6) and 90 pL of 10 mM sodium cyanide solution. NDA and cysteic acid solutions were then added as described in the Experimental Section. Note that the changes in pH of separation buffer from (A) to ( 6 )allow a complete resolution between NA-CBI and DA-CBI.
contains borate used for derivatization,the effect of a decrease in borate concentration of the derivatization medium was investigated. The results of this experiment are presented in Figure 2 and clearly indicate that DHBA-CBI and NA-CBI peak heights (and to a smaller extent, DA-CBD were strongly increased when the borate concentration was decreased in the derivatization medium (Figure 2A). Separation efficiency (as estimated by the theoretical plate count per meter) varied in the same way (Figure 2B). Thus, at 30 mM, the NA-CBI peak height was &fold higher than at 130 mM and the theoretical plate count reached 23 million/m (corresponding to a baseline peak width of 1 s for a migration time of 800 s). In contrast, resolutions between NACBI and DA-CBI, and between NA-CBI and DHBA-CBI, decreased when the borate concentration was reduced, and the three compounds were no longer resolved for borate concentrations lower than 40 mM (Figure 2C). This fall in resolution was associated with an increase in mobility, as assessed by the lower migration time observed (data not shown). Addition of the appropriate amounts of salts (i.e., by dissolving standards in artificial CSF rather than in water), or the addition of phosphate buffer or carbonate buffer to the derivatizationmedium containing 25 mM borate, (in order to obtain samples with ionic strength equivalent to that of a sample containing 100 mM borate), did not have any Muence on the theoretical plate count of peaks (data not shown). These results strongly suggest an influence of the concentration in borate and not of the final ionic strength of the derivatized sample on catecholamine peak shapes. For the above reasons, a concentration of 50 mM borate buffer was chosen since (i) resolution was optimal, (ii) peak heights were 4and 1.7-foldhigher for DA-CBI and NA-CBI, respectively, than
10 20 30 40 50 60 70 80 90 100 110 120 130
Borate concentration (mM)
C
0.50
0.00
1 !.
I
.
1
.
..
1
.
1
.
I
.
I
.
I
.
I
.
I
.
I
.
I
I
10 20 30 40 50 60 70 80 90 100 110 120 130
Borate concentration (mM) Figure 2. Effect of variations in the concentration of borate buffer in the derivatization medium on the following: (A) NA-CBI, DACBI, and DHBA-CBI peak heights; (B) theoretical plate count per meter for NA-CBI, DA-CBI, and DHBA-CBI; (C) resolution between NA-CBI and DA-CBI, and between NA-CBI and DHBA-CBI. Theoretical plate count and resolution were calculated as explained in the Experimental Section. Conditions: see Experimental Section. Derivatization procedure: to 600 pL of a 1O-' M NA, DA, and DHBA standard solution was added a volume of 500 mM borate buffer solution (pH 8.6) varying between 25 and 250 pL (to give final concentrations of 13-130 mM); water was added to bring the final volume to 250 pL. Sodium cyanide, NDA, and cysteic acid solutions were then added as described in the Experimental Section. The pH of the derivatization medium was constant (8.6) whatever the borate concentration. Note that borate concentration in derivatization medium has a great influence on peak heights, separation efficiencies, and resolution for NA-CBI and DA-CBI.
those observed using initial conditions (130 mM borate),17and (iii) separation efficiency was still excellent (3.1 million theoretical Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
1841
Table 1. Linear Relationships between the Concentration of NA and DA and the Peak Areas for Solutions Derivatized at Concentrations of 10-6-10-9 M
derivatization A" y intercept
slope
corr ceff no. of points
min concn derivatized (M) min amt injected (amol) max concn derivatized (M) max amt injected (amol)
derivatization Ba
NA
DA
NA
DA
-0.517 7.773 107 0.999 7 10-9 1.8 10-6 1800
-0.167 4.518 x lo7 1.000 7 10-9 1.8 10-6 1800
-1.382 1.080 x lo8 0.998 5 10-9 1.8 10-6 1800
-0.654 4.988 x lo7 0.998 5 10-9 1.8 10-6 1800
a (A) Derivatizationperformed on a total volume of 1mL; (B) derivatization performed on a total volume of 10 pL. Conditions: see Experimental Section, except for (B), where all volumes for the derivatization procedure were lO@foldlower.
A -
DA NA
1
I
750
760
770
780
790
800
DA
migration time (s)
I
1
820
840
I 8M)
Ill\
I
1
I
1
880
900
920
940
migration time (5)
800
810
820 830 840 migration time (s)
850
I
Figure 3. Electropherograms of lo-' M NA-CBI, DA-CBI, and
DHBA-CBI obtained with two different borate concentrations in the derivatization medium: (A) 50 mM borate; (B) 30 mM borate. Conditions: see legend of Figure 2. Note that (i) the 50 mM concentration of borate in derivatization medium allows optimal resolution with good separation efficiencies and peak heights and that (ii) despite very high separation efficiencies (23 million theoretical plates per meter for DA-CBI), the 30 mM borate concentration gives a lower resolution. plates/m for DA-CBI). Figure 3A shows the electropherogram obtained with a borate concentration of 50 mM and illustrates the advantage of using this concentration instead of 30 mM (Figure 3B). In these conditions of derivatization and separation, NA and DA were separated not only from each other but also from amino acids or primary amines potentially present in microdialysis samples. Indeed, the injection of a standard solution containing NA and DA at low concentration M) together with loe6M normetanephrine (the only enzymatic metabolite of NA having a primary amine group) and a mixture of urea and 32 physiological amino acids, each at a concentration of 2.5 x M, showed that none of these compounds comigrated with the analytes of interest (data not shown; the amino acids tested were as follows: alanine, p-alanine, a-aminoadipic acid, a-aminobutyric acid, y-aminobutyric acid, /3-aminoisobutyric acid, arginine, asparagine, aspartic acid, citrulline, cystathionine, cystine, DOPA, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, 0-phosphoethanolamine, O-phosphoserine, proline, sarcosine, serine, taurine, threonine, tryptophane, tyrosine, and valine). 1842 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
I 820
I 840
I
I
1
I
t
860
880
900
920
940
migration time (s)
Figure 4. Electropherograms of (A) M NA-CBI and DA-CBI corresponding to 1.8 amol detected for each catecholamine and (B)
blank. Conditions: see Experimental Section. Note that the signalto-noise ratios were 8:land 3.1 for NA-CBI and DA-CBI, respectively. Reproducibility. Intra- or interassay variability may originate in the derivatization, injection, migration, or detection step. A lack of reproducibility in the migration could be due to excessive variability in the electroosmotic flow. Nevertheless, the electroosmotic flow was found to be nearly constant all along the experiments since its interassay relative standard deviation was 3.1% (n = 10). Intraassay reproducibility of NDA-labeled catecholamine determination (i.e., injection, migration and detection steps) was assessed by performing repeated injections (n = 7) of a DHBA solution derivatized at 2 x M concentration: the relative standard deviation was 7.1% for the electrophoretic mobility and 7.6% for the peak area. Interassay reproducibility was tested on M NA solutions: multiple derivatizations (n = 8) of this catecholamine followed by electrophoretic separation gave a relative standard deviation of 6.1%for the electrophoretic mobility and 8.3% for the peak area. The comparison between intra- and interassay reproductibility indicates that the variability originating in the derivatization step is weak. In summary, reproducibility of derivatization, injection, migration, and detection steps can be considered as satisfactory for biological studies.
B
A I200 nA
C
A
I200 nA
1200 nP
I
I
C
I200 nA
I200 np
I
-1i.3
,
-
-
7
-9:7
n
-7.9 -9.7 -11.3
-11.3 850 900 960 820 870 920 860 915 970 p (cm?V.s)(xld) p (cm2/V.s)(x105) p (cm2/V.s)(xd) migration time (s) migration time (s) migration time (s)
3.9
-7.9 -9.7 -11.3 -7.9
iI
-9.7
760
805
850
p ~Cm2/V.s)(xlos) migration time (s)
Figure 5. Electropherograms of (A) lo-' M NA-CBI and DA-CBI, (B) basal dialysate sample, and (C) basal dialysate sample after addition of exogeneous NA-CBI. Conditions: see Experimental Section. The NA peak is indicated by an arrow and the DA peak by an asterisk. Note the presence, in basal conditions (B), of a peak with an electrophoretic mobility 0.)corresponding to that of standard NA (A). The height of this peak was increased after addition of exogeneous NA-CBI (C).
Iinearity of Fluorescence. Limits of Detection. To assess linearity of fluorescence, 750 pL of NA and DA standard solutions in water were derivatized in concentrations varying from to M. After derivatization, the final Concentration, in a total to 7.5 x 10-lo M. Linear volume of 1mL, ranged from 7.5 x relationships between the NA-CBI and DA-CBI concentrations and the peak areas between and M were obtained with respective regression ccefficients of 0.999 and 1.000 [Table 1A: F(1,5) = 11 563, p < 0.0001; F(1,5) = 18 131, p < 0.0001, respectively]. Since the present CZE-LIFD technique was developed for the analysis of brain extracellular fluid obtained by microdialysis, the derivatization procedure had to be performed on small volumes and with standards dissolved in the perfusion fluid. We found that the derivatization rate was not affected by the higher ionic strength of biological samples when compared with standards in aqueous solution because peak areas did not vary when samples were prepared either in water or in artificial CSF (data not shown), The linearity was then tested on 7.5 p L of NA and DA standard solutions in artificial CSF, which were derivatized in concentrations ranging from to M. Linear relationships between the NA-CBI and DA-CBI concentrations and the peak areas between and M were found with regression cczfficients of 0.998 [Table 1B: F(1,3) = 1477,p < 0.0001; F(1,3) = 1629,p 0,0001, respectively]. NA and DA solution derivatized at a concentration of M produced peaks with signal-to-noise ratios of 8:l and 3:1, respec tively, with no interference from the blank (Figure 4): this concentration represented the lowest initial concentration (i.e., before derivatization) producing detectable (23u) fluorescent signals. The estimated injected volume being 2.4 nL, the corresponding detected amount was 1.8 amol for both catecholamines. Extrapolations based on the fluorescent signal from the electroM level showed that the detection limit pherogram at the (3u) for NA-CBI could be 3.75 x 10-lo M, Le., 0.7 amol detected.
\
I
-7.9 790
840
885
755
I
I
-9.7
-1 1.3
800
840
p (cm2/V.s)(xld)
p (Cm2/V.s)(xlO)
migration time (s)
migration time (s)
Figure 6. Electropherograms of (A) dialysate sample after desipramine treatment, (B) dialysate sample after desipramine idazoxan treatments, and (C) dialysate sample after animal euthanasia. Conditions: see Experimental Section. The NA peak is indicated by an arrow and the DA peak by an asterisk. For a better understanding, the x-axis scale of (C) was doubled. Note that the peak corresponding to the electrophoretic mobility 0.)of endogeneous NA (see Figure 5B) was greatly increased after desipramine, idazoxan treatments, and animal death.
+
NA and DA standard solution, derivatized at a concentration M and then diluted in the derivatization medium until 10-9 M, leads to peaks with areas similar to that produced by the direct derivatization of a M NA and DA standard solution (data not shown). These data suggest that the efficiency of the derivatization reaction in the present condition is similar at very low and at high concentration of catecholamines. Detection limits quoted here were lower than those previously reported for the analysis of catecholamines using CZE with LIFD or electrochemical detection (ED). A recent report16using a CZE LIFD assay of NDA-labeled catecholamines showed a detection limit for NA of 10 amol, corresponding to a concentration of 1.6 x lo-* M according to the injection volume used in this study. Although ED offers a greater selectivity than LIFD and does not need a derivatization procedure, the best reported concentration detection limits were 1.6 x lo-* M for NA32and 8.3 x lo-* M for DA.33 On the opposite, the present CZE-LIFD method has a limit of
Analytical Chemistty, Vol. 67, No. 1 1 , June 1, 1995
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of detection slightly higher than the best value reported for the conventional analytical technique used for the analysis of catecholamines in microdialysis samples, LC-ED, i.e., (1.5-3) x M.13
In conclusion, the results obtained in the present study on linearity and detection limits show that this method could be used for the determination of very low concentrations of molecules in microvolumes of biological samples. In Vivo Microdialysis Experiments. The analysis by CZELIFD of a dialysate sample from the medial frontal cortex, a cerebral region rich in noradrenergic terminals, showed a peak with an electrophoretic mobility corresponding to that of the peak of NA-CBI in standard solution (Figure 5A,B). Moreover, the height of this peak increased when exogeneous NA-CBI was coinjected with the dialysate sample, whereas no additional peak appeared (Figure 5C). Consequently, this peak of interest is likely to correspond to the endogeneous NA In contrast, no peak corresponding to endogeneous DA was observed in the cortical dialysate. The basal concentration of NA in cortical dialysate was found to be 5 x M. When corrected for the in vitro recovery of the probe, the basal extracellular concentration of NA in medial frontal cortex was estimated to be M. This cortical extracellular concentration is similar to the values found with LC-ED by other authors who reported concentrations varying from 2 x W9to 5 x lo-* M in anesthetized or freely moving rats.1,28b35-37 Successive admimistrationsof a reuptake blocker (desipramine) and then of an a2 adrenoceptor antagonist (idazoxan) have been shown to greatly increase extracellular NA concentration in the medial frontal cortex.28 In the present study, we report that desipramine injection (20 mg/kg ip) induces an increase in NA ~
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(32) Wallingford, R. A.; Ewing, A G. Anal. Chem. 1988, 60, 1972-1975. (33) Wallingford, R. A.; Ewing, A G. Anal. Chem. 1989, 61, 98-100. (34) Ruban, V. F. J. Chromafogr. 1993, 619,111-115. (35) Vanveldhuizen, M. J. A.; Feenstra, M. G. P.; Boer, G. J.; Westerink, B. H. C. Neurosci. Left. 1990, lZ9,233-236. (36) Gustafson, I.; Westerberg, E. J.; Wieloch, T. Ezp. Brain Res. 1 9 9 1 , 8 6 , 5 5 5 561. (37) Lavicky, J.; D u m , A J. J. Neurochem. 1993, 60, 602-612. (38) Rossetti, Z. L.; Pani, L.; Gessa, G. L. Psychopharmacology 1989, 98, 562563.
1844 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
concentration in the dialysate (+64%) 20 min after administration (Figure 6A). Idazoxan, injected (20 mg/kg ip) 70 min after desipramine administration, produced a 6.3-fold increase in NA concentration as compared with basal concentration (Figure 6B). These effects are of the same magnitude as those previously reported by Dennis and co-workers,28using similar microdialysis conditions. Samples collected just after animal euthanasia showed very high NA concentrations (1C-fold higher than basal concentrations), whereas a large peak likely to correspond to DA appeared on the electropherogram (Figure 6C). These results are in agreement with previous microdialysis studies on NA and DA release in rat cortex upon death.35,38 In conclusion, the present CZE-LIFD method appears to be quantitative since the peak area was reproducible and a plot of the peak areas of synthetic catecholamines versus sample concentration gave excellent linearity, even at the lower concentrations. Moreover, the results demonstrate the possibility of monitoring NA in microdialysis brain samples by CZE-LIFD at the nanomolar range. Further developments will be made to adapt this technique to the analysis of other neurotransmitters, such as amino acids and neuropeptides, and to enhance the temporal resolution of microdialysis. ACKNOWLEDGMENT The IRIS ZOO0 system was purchased by grants from La Region
Rhdne-Alpes, the Centre National de la Recherche Scientitique (CNRS) , and the Universite Claude Bernard Lyon I (UCBL). This research was supported by CNRS and UCBL. F.R. held a fellowship (Bourse de Docteur-Ingenieur) from CNRS. The authors gratefully acknowledge Michel Nertz from Europhor Instruments S.A. (now Zeta Technology SA, Toulouse, France) for his constant technical help, which made this work possible. They also thank Philippe Verdeguer, Franqois Couderc, and Bruno DeVandiere from Europhor Instruments S.A. for their support. Received for review June 28, 1994. Accepted March 16, 1995.a AC940648S @Abstractpublished in Advance ACS Abstracts, April 15, 1995.
