Detection of Peptides by Precolumn Derivatization with Biuret Reagent

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capi...
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Anal. Chem. 1999, 71, 987-994

Detection of Peptides by Precolumn Derivatization with Biuret Reagent and Preconcentration on Capillary Liquid Chromatography Columns with Electrochemical Detection Hong Shen, Steven R. Witowski, Brendan W. Boyd, and Robert T. Kennedy*

Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25µm inner diameter packed with base-resistant, polymerbased reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-µL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus. Neuropeptides are a group of intercellular signaling molecules present in the central nervous system that function as neurotransmitters, neuromodulators, and neurohormones. Over 80 peptide neurotransmitters have been identified, and they have been implicated in a wide variety of physiological processes including memory and learning, pain transmission, and appetite control. In addition, defects in the regulation of peptides are associated with several diseases including Alzheimer’s and Parkinson’s. An important approach to studying the regulation and role of peptides involves their measurement in samples collected by methods such as microdialysis.1-12 Microdialysis permits samples to be taken from living animals with temporal resolution, allowing the cor* Corresponding author: (phone) 352-392-9839; (fax) 352-392-4582; (e-mail) [email protected]. (1) Boarder, M. R.; Weber, E.; Evans, C. J.; Erdelyi, E.; Barchas, J. J. Neurochem. 1983, 40, 1517. (2) Kendrick, K. M. J. Neurosci. Methods 1990, 34, 35. (3) Consolo, S.; Baldi, G.; Russi, G.; Civenni, G.; Bartfai, T.; Vezzani, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8047-8051. (4) Maidment, N. T.; Siddall, B. J.; Rudolph, V. R.; Erdelyi, E.; Evans, C. J. Neuroscience 1991, 45, 81-93. 10.1021/ac9809837 CCC: $18.00 Published on Web 01/22/1999

© 1999 American Chemical Society

relation between extracellular peptide level and behavior, stimuli, or pharmacological manipulations. Direct measurement of neuropeptides is difficult because they tend to be present at trace levels in microdialysates and other biological samples. Peptide concentrations in brain extracellular fluid are usually less than 1 nM, and since the microdialysis probe only recovers a fraction of the extracellular concentration, peptides must be detected at even lower concentrations. As a result of these low levels, most peptide measurements in microdialysates have been made by radioimmunoassay (RIA), which has picomolar or better concentration detection limits. Although RIA allows detection at the recovered concentration, RIA mass detection limits usually prevent good temporal resolution for monitoring peptide concentration changes. The low temporal resolution arises since absolute recovery, defined as mass removed per unit time by the sampling probe, is typically 15-50 amol/min for peptides whereas the mass detection limit for RIAs is 100-500 amol.4,5 In addition, RIAs are usually limited to measurement of one analyte at a time unless they are coupled to HPLC with fraction collection, which is a laborious, time-consuming procedure. Studies of the role of peptide neurotransmitters would be facilitated by methods that allow detection of multiple peptides with better mass sensitivity, and hence better temporal resolution, than RIA. We have recently described a method for high-sensitivity detection of electroactive peptides in microdialysates based on capillary LC with electrochemical detection (EC) at C-fiber microelectrodes.13 In this method, samples as large as 1 µL are injected onto packed capillary columns (inner diameter (i.d.) 25 µm) under conditions that allow preconcentration of the peptide. (5) Maidment, N. T.; Evans, C. J. In Microdialysis in the Neurosciences; Robinson, T. E., Justice, J. B., Eds.; Elsevier: New York, 1991; pp 275-304. (6) Maidment, N. T.; Brumbaugh, D. R.; Rudolph, V. D.; Erdelyi, E.; Evans, C. J. Neuroscience 1989, 33, 549-557. (7) Mesquita, S. D.; Beinfeld, M. C.; Crawley, J. N. Prog. Neuro-Psychopharmacol. Biol. Psychiat. 1990, 14, S5-S15. (8) Levine, J. E.; Meredith, J. M.; Vogelsong, K. M.; Legan, S. J. In Microdialysis in the Neurosciences; Robinson, T. E., Justice, J. B., Eds.; Elsevier: New York, 1991; pp 305-326. (9) Kendrick, K. M. In Microdialysis in the Neurosciences; Robinson, T. E., Justice, J. B., Eds.; Elsevier: New York, 1991; pp 327-348. (10) Kendrick, K. M. J. Neurosci. Methods 1990, 34, 35-46. (11) Guzman, N. A.; Hernandez, L. Adv. Curr. Res. Protein Chem. 3rd 1989, 203-216. (12) Advis, J. P.; Guzman, N. A. J. Liq. Chromatogr. 1993, 16, 2129-2148. (13) Shen, H.; Lada, M. W.; Kennedy, R. T. J. Chromatogr., B 1997, 704, 4352.

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The large preconcentration effect improves the concentration detection limit ∼100-fold. With mass detection limits of 10-40 amol for small peptides, the method allows for more frequent sampling and hence better temporal resolution than what is possible by RIA. Extension of this method to nonelectroactive peptides requires a method of derivatizing peptides at trace concentrations. One possible route to peptide derivatization for electrochemical detection is to use the biuret reagent as described in a series of articles by Weber’s group.14-22 In this elegant method, peptides react with Cu2+ under basic conditions to form a complex that can be detected electrochemically on the basis of oxidation of Cu2+-peptide to Cu3+-peptide. This derivatization method is unique in that the reaction does not require primary amines but rather involves the peptide backbone. As a result, the method is highly selective for peptides and allows detection of peptides, such as those with posttranslational modifications to the N-terminus, which are undetectable by reagents that act on amines. Weber’s group has carefully explored the chemistry of this method and found that a wide range of bioactive peptides with 3-18 amino acids can be detected with detection limits in the 6-100-fmol range corresponding to 60 pM to 1 nM for 100-µL injections on a microbore HPLC column.19,20 Given the apparent success of the biuret reagent for HPLCEC, microbore LC-EC, and capillary electrophoresis with electrochemical detection,23 we have begun to explore the combination of this method with the preconcentration/capillary LC method we have used with electroactive peptides. Through this work we have tested the limits of biuret derivatization and discovered that it is well-suited for analysis at low-picomolar concentrations because stable products can be rapidly formed at low concentrations. Equally important, the biuret reagent does not produce any background peaks of consequence, which greatly facilitates lowconcentration detection. As a demonstration of the method, we measure peptides in dialysates from the rat supraoptic nucleus (SON) of the hypothalamus. The high mass sensitivity of the method allows 5-min sampling times. EXPERIMENTAL SECTION Chemicals and Reagents. Unless specified otherwise, all chemicals were purchased from Sigma (St. Louis, MO). All buffers were made fresh and filtered with 0.22-µm-pore size hydrophobic Teflon filter membrane (MSI, Westboro, MA) before use. The Teflon-based filter enabled the filtration of basic solutions and removed some hydrophobic impurities. (14) Tsai, H.; Weber, S. G. Anal. Chem. 1992, 64, 2897-2903. (15) Warner, A. M., Weber, S. G. Anal. Chem. 1989, 61, 2664-2668. (16) Tsai, H.; Weber, S. G. J. Chromatogr. 1991, 542, 345-350. (17) Tsai, H.; Weber, S. G. J. Chromatogr. 1990, 515, 451-457. (18) Tsai, H.; Chen, J. G.; Woltman, S. J.; Weber, S. G. Anal. Chem. 1995, 67, 541-551. (19) Chen, J. G.; Weber, S. G. Anal. Chem. 1995, 67, 3596-3604. (20) Chen, J. G.; Woltman, S. J.; Weber, S. G. J. Chromatogr., A 1995, 691, 301-315. (21) Woltman, S. J.; Alward, M. R.; Weber, S. G. Anal. Chem. 1995, 67, 541551. (22) Woltman, S. J.; Chen, J. G.; Weber, S. G.; Tolley, J. O. J. Pharm. Biomed. Anal. 1995, 14, 155-164. (23) Deacon, M.; O’Shea, T. J.; Lunte, S. M.; Smyth, M. R. J. Chromatogr., A 1993, 652, 377-383.

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Preparation of Capillary Reversed-Phase Columns. Capillary columns were prepared by previously described techniques24 using 15-20-cm lengths of 25-µm-i.d., 360-µm-outer diameter (o.d.) fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) as the column blanks. Columns were slurry-packed with 5-µm Astec C-18 (Alltech, Deerfield, IL) reversed-phase particles (slurries consisted of 10 mg of packing material in 3 mL of acetonitrile) at 2000-3000 psi using a pneumatic amplifier pump (Catalog No. 1666, Alltech). Capillary LC System Operation. The capillary LC system has been described in detail elsewhere.13 The system allows injection volumes from 5 nL to 1 mL. To load 1 mL onto the column requires 3 mL of sample to quantitatively fill the loop and for complete rinsing. Loading large-volume samples can be slow; therefore, during loading the pressure was 4000 psi to ensure fast sample loading while the separation was carried out at pressure generally below 1000 psi to achieve better resolution and detection. These pressures correspond to flow rates of 2.4 nL/s for sample loading and 0.6 nL/s during separation. Gradient Separation. The aqueous portion of the mobile phase (solvent A) was 40 mM sodium carbonate buffer in 0.75 mM sodium potassium tartrate and 0.25 mM cupric sulfate adjusted to pH 10.5 with sodium hydroxide. The organic phase (solvent B) was prepared by mixing 40% (v/v) aqueous buffer with 60% acetonitrile. The mobile phase was degassed by helium sparging before and during use. To maintain the best detection limits and minimal drift from large-volume injections, the aqueous buffer was prepared fresh and filtered every day. In addition, after loading, the column was rinsed with 5% solvent B-95% solvent A for 2 min before gradient elution started. Gradient elution started at 5% solvent B, linearly changed to 35% solvent B in 5 min, kept at this composition for 2 min, and then stepped back to 100% solvent A. The Cu2+ concentration used in the mobile phase was important for achieving high sensitivity. At higher concentrations, an increase in background noise was observed with no increase in signal. At lower concentrations, the signal was degraded possibly because of loss of complex during separation. Biuret Complex Formation. In initial characterization studies, biuret complexes were formed by dissolving peptides in the aqueous mobile phase (1-2 mL final volume) and then heating at 60 °C for 20 min in a glass vial. For dialysate analysis and microvolume testing, samples were held in a polypropylene microvial. The vial was formed from a 20-µL polypropylene pipet tip by sealing the end with a flame. For in vivo experiments, 2.5 µL of dialysate was collected in a microvial and 0.50 µL of biuret reagent was added. The biuret reagent consisted of 3.75 mM sodium potassium tartrate and 1.25 mM cupric sulfate in 40 mM borate at pH 10.5. The solution was then incubated in a water bath at 60 °C for 8 min immediately before the sample loading. (Samples were allowed to cool to ambient temperature before injection.) The amount of time allowed for incubation was necessary to achieve the maximum signal for bradykinin, the slowest reacting peptide tested. Longer times led to a slow decrease in signal for all peptides, possibly (24) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128-1135.

due to adsorptive or oxidative losses of the complex.30 Electrochemical Detection. A 9-µm-diameter, 1.2-mm-long carbon fiber electrode was used as the detector electrode25,26 and was inserted into the outlet of the column using a micropositioner. The outlet of the column was mounted in a cell containing 100 mM NaCl as electrolyte and fitted with a Ag/AgCl reference electrode. Unless stated otherwise, the detector electrode potential was set at +0.60 V versus this reference using a battery and voltage divider. The detector current was amplified by a SR570 current preamplifier (Stanford Research Systems, Sunnyvale, CA). Data were collected using a 386 personal computer (Gateway, Sioux City, SD) and data acquisition board (AT-MIO-16F-5, National Instruments, Austin, TX). Software was developed inhouse using LabWindows (National Instruments). Sweeping the electrode potential between -0.40 and 1.6 V for four cycles at 1 V/s between chromatographic runs enhanced reproducibility. Hydrodynamic voltammograms (HDVs) were collected using the LC-EC system. In experiments, a dual detector was used to compare the detection of biuret complex at glassy carbon and carbon fiber electrodes. In these experiments, a 4.6-mm-bore C-18 reversedphase column was used and the eluent was split with a tee to both a glassy carbon thin-layer cell and capillary tubing with a carbon fiber inserted as described above. Both electrodes were held at +0.80 V versus an Ag/AgCl reference electrode and current was collected using a EI-400 bipotentiostat (Ensman Instrumentation, Bloomington, IN). Capillary Electrophoresis. A capillary electrophoresis (CE) instrument with both UV and EC detectors was assembled for experiments to characterize the complexes. The CE column was 30 cm long with 50-µm-i.d. and 360-µm-o.d. fused-silica tubing. A window was prepared by burning off a 1-cm section of polyimide ∼8 cm from the outlet end. This capillary was mounted in a Spectra 100 UV-visible detector (Spectra-Physics, San Jose, CA) set at 210 nm to allow simultaneous detection of both native and Cu complexes of peptides. The outlet was connected to a 2-cm length of the same type of fused-silica tubing by a Nafion joint, which was grounded, as described elsewhere.27,28 The C-fiber detector electrode was inserted into the outlet of the connector tubing and operated as for the LC-EC experiments. For electrophoresis, the capillary was rinsed first with 1.0 M HCl, then with 1.0 M NaOH, and finally with electrophoresis buffer for 10 min each prior to a series of experiments. The column was rinsed with buffer between separations. Electrophoresis buffer consisted of 10 mM sodium borate with 3 mM potassium sodium tartrate with pH adjusted to 11.0 with NaOH. All peptide samples were prepared in electrophoresis buffer with various concentrations of cupric sulfate added. Peptides were injected electrokinetically by 5000 V for 2 s. For the separation, +15 kV (40 µA) was applied at the inlet. (25) Kawagoe, K. T.; Zimmerman, J. B.; Wightman, R. M. J. Neurosci. Methods 1993, 48, 225. (26) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479482. (27) O’Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E. J. Chromatogr. 1992, 593, 305-312. (28) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989, 61, 98-100. (29) Ludwig, M.; Landgraf, R. Brain Res. 1992, 576, 231-234. (30) Margerum, D. W. Pure Appl. Chem. 1983, 55, 23-34.

Surgical Preparation and Procedures. Male SpragueDawley rats weighing 300-450 g were anesthetized with subcutaneous injections of 100 mg/mL chloral hydrate at a dose of 4.0 mL/kg. Booster injections of 2.0 mL/kg were given every 30 min until the animal no longer exhibited limb reflex. After surgery, the rat was kept unconscious with subcutaneous administration of 1.0 mL/kg chloral hydrate as needed. Once the rat was secured in the stereotaxic apparatus, the microdialysis probe was placed in the SON at the coordinates 1.1-mm posterior to bregma, 1.7mm lateral to midline, and 9.1 mm below the surface of the skull.29 Basal level chromatograms were taken until they stabilized, which was typically 2 h after implantation of the dialysis probe into the brain. Microdialysis sampling was performed using CMA/10 probes (CMA/Microdialysis, Acton, MA) made from polycarbonate membrane with a 20-kDa cutoff. The concentric probes had a 0.5mm diameter and 4-mm tip length. Per manufacturer specifications, the probe was rinsed with ethanol and then buffer for 30 min each before use. Artificial cerebral spinal fluid (aCSF) used for microdialysis perfusion consisted of 145 mM NaCl, 2.68 mM KCl, 1.01 mM MgSO4, and 1.22 mM CaCl2. The high-K+ perfusate solutions for stimulation experiments consisted of 2.62 mM NaCl and 145 mM KCl with other salts the same as aCSF. It was found that an overnight rinse with aCSF buffer further decreased impurity peaks in chromatograms of dialysate. During experiments, the probe was perfused with aCSF using a microliter syringe pump (Harvard Apparatus 553206, South Natwick, MA) at 0.5 µL/min. At these flow rates, the relative recovery of the dialysis probe was 55-59% for the different peptides at 37 °C. For in vivo monitoring, dialysate samples were collected every 5 min and immediately stored at -4 °C. Samples were thawed immediately before derivatization and analysis. RESULTS AND DISCUSSION Detection of Derivatized Peptides at C-Fiber Electrodes. Previous work with HPLC-EC detection of biuret complexes has demonstrated that both precolumn derivatization and postcolumn derivatization are possible;15,16,19 however, most work has emphasized postcolumn derivatization primarily because of difficulties associated with performing separations at the high pH needed for complex formation and detection. The difficulty of working with postcolumn reactors in capillary separations prompted us to pursue precolumn derivatization. The advent of polymer-based HPLC supports tolerant of high-pH mobile phases made this strategy feasible. In preliminary experiments, we examined the determination of several neuropeptides by reversed-phase capillary LC-EC with C-fiber electrodes using the biuret reagent with precolumn derivatization.16 The peptides tested were (molecular mass in daltons, primary sequence given in parentheses): des-tyr-enkephalin (410 Da, GGFM), bradykinin (1060 Da, RPPGFSPFR), substance P (1347 Da, RPKPQQFFGLM-NH2), met-enkephalin (574 Da, YGGFM), vasopressin (1084 Da, C(YFQN)CPRG-NH2), oxytocin (1007 Da, C(YIQN)CPLG-NH2), and neurotensin (1673 Da, pELYENKPRRPYIL). These peptides were chosen because they are all potential transmitter peptides or metabolites. Some of the peptides are not natively electroactive while others are possibly electroactive based on the presence of tyrosine (Y) residues. As expected from these experiments, we observed that Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

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Figure 1. Hydrodynamic voltammograms for 0.5 µM bradykinin (A), 0.5 µM vasopressin (B), and 5 µM neurotensin (C). Circles are for injection of biuret complexes with Cu2+ in the mobile phase and open squares are for native peptides with no Cu2+ in the mobile phase. Injection volumes were estimated as 5 nL. Chromatographic conditions are given in the Experimental Section.

all nonelectroactive peptides tested were rendered detectable by biuret derivatization. An example is illustrated by the HDV for bradykinin shown in Figure 1A. Surprisingly, however, we observed that sensitivity of the electroactive peptides tested was not enhanced by derivatization. A typical example is shown in Figure 1B, which illustrates that HDVs for vasopressin and Cu2+vasopressin complex were virtually identical. This was the response we observed for most peptides; however, for neurotensin we observed that the use of Cu2+ actually suppressed the signal for peptide at lower potentials (see Figure 1C). In general, we would expect an increase in anodic signal for electroactive peptides upon derivatization that represents the sum of detection of the electroactive residue and Cu2+ within the complex.14,19,20 Given that previous studies using glassy-C electrodes had not observed a lack of enhancement, we investigated whether this effect could be related to the electrode material by performing experiments in which eluent from a chromatograph was split and detected by both a glassy-C electrode and a C-fiber microelectrode (see Experimental Section). For these experiments we used metenkephalin and des-tyr-met-enkephalin since this pair offered a simple peptide in which the effect of tyrosine could be observed. We found that both electrodes could detect des-tyrosine enkephalin only in the presence of Cu2+ and could detect met-enkephalin without Cu2+. In the presence of Cu2+ however, the signal for metenkephalin increased 105 ( 8% at the glassy-C electrode but was only increased 5 ( 4% at the C-fiber detector. Thus, the lack of enhancement was specific to the C-fiber electrode. Similar lack of enhancement was seen by cyclic voltammetry. These data show that the C-fiber can detect Cu2+-peptide complexes and tyrosine residues; however, having both Cu2+ and tyrosine simultaneously present in a peptide affects electrochemical sensitivity. A possible explanation for this effect is that the presence of Cu2+ modifies the electrode surface altering the electrochemical kinetics for either Cu2+ or tyrosine. Indeed, we observed that, in the presence of Cu2+, the voltammetric surface waves for C-fiber electrodes changed considerably as they were exposed to Cu2+. Further experimentation would be required to understand these effects and why they are specific to the C-fiber electrode. A second issue we explored was the anomalous behavior of the neurotensin-Cu2+ complex. Of all the Cu2+-peptide complexes tested, it was the only one that did not yield a distinct wave in the region of 0.5-0.6 V. It was also the only tyrosine-containing peptide that did not yield comparable signals for native and Cu2+ complex form. To determine whether this behavior was related to the chemistry of complex formation, we performed experiments 990 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

Figure 2. CE of vasopressin with simultaneous UV and electrochemical detection. Traces on the left are UV and those on the right are the corresponding electrochemical detection. Cu2+/peptide ratio in the sample was 0:1 (A), 0.5:1 (B), and 2:1 (C). Vasopressin concentration was 100 µM. All other conditions are given in the Experimental Section. “V” indicates free vasopressin peak and “Complex” indicates peaks believed to be the Cu2+-vasopressin complex.

in which complexes were formed and then separated by CE with dual detection (UV and electrochemical). CE provides a convenient method to separate formed complexes and then test their electroactivity individually. Figures 2 and 3 illustrate a series of electropherograms for vasopressin (Figure 2) and neurotensin (Figure 3) mixed with different ratios of Cu2+. As shown in the electropherograms for vasopressin, addition of Cu2+ results in formation of a single new peak in both the UV and EC detector traces which we take to be a Cu2+-peptide complex (Cu2+ by itself did not produce this peak and the Cu2+ did not affect electroosmotic flow). These electropherograms illustrate several important points. First, the complex formed is stable enough to survive the several minutes required to transverse the column without Cu2+ in the buffer since a sharp, well-defined band is apparent for the complex. Second, the longer migration time of the Cu2+ complexes suggest that they have a greater mobility in toward the anode which could be due to a smaller size or greater negative charge. A greater negative charge can be rationalized by considering that addition of Cu2+ displaces more positive charge, in the form of H+, than is added by Cu2+.30 Since the peak areas for the complex are similar to those obtained

Figure 3. CE of neurotensin with simultaneous UV and electrochemical detection. Traces on the left are UV and those on the right are the corresponding electrochemical detection. Cu2+/peptide ratio in the sample was 0:1 (A), 0.5:1 (B), and 2:1 (C). Neurotensin concentration was 500 µM. All other conditions are given in the Experimental Section. “N” indicates free neurotensin peak and “Complex” indicates peaks believed to be the Cu2+-neurotensin complex. The peak at 130 s in the electrochemical trace appears to be an impurity in the neurotensin that is only detected by amperometry.

for free peptide by electrochemistry, we can also conclude that the sensitivity is largely unchanged for the addition of Cu2+, in agreement with the HDV data. Thus, this experiment further confirms that complex is actually formed yet detector sensitivity is similar to the native peptide. In comparing the results for vasopressin to those for neurotensin (Figure 3), several differences are apparent. Vasopressin appears to form a quantitative complex with the Cu2+ whereas neurotensin does not. For example, in comparing the UV traces, free neurotensin is detectable even at the greatest ratio of Cu2+peptide whereas free vasopressin is absent at a 1:1 ratio of Cu2+peptide. Furthermore, the neurotensin complex peaks broaden and tail, suggestive of dissociation of the complexes during the separation. Combined, these observations indicate that neurotensin has a lower affinity for Cu2+ than does vasopressin. Weaker binding between the peptide and Cu2+ may be expected to decrease the signal due to Cu2+ oxidation since this reaction is achieved only by stabilization of the Cu3+ brought about by binding with the peptide. The weaker binding may be due to the lack of a free amine on the N-terminus of neurotensin as this has previously been shown to inhibit complete formation of complexes.19,20 A second important difference is that vasopressin clearly forms a single complex with Cu2+ but neurotensin appears to form at least two complexes (see UV traces). Formation of multiple complexes may be the result of the larger peptide being able to accommodate more than one Cu2+.19,20 The two complexes do not appear as distinct peaks in the EC trace because of the longer time to travel to the EC detector, which allows extra diffusional

band broadening as well time for the complex to dissociate. The formation of multiple complexes is not likely to be a factor in the LC-EC experiment as such a large excess of Cu2+ is present that the main product should be the maximally derivatized neurotensin. It is apparent from the EC traces, however, that a weaker signal is obtained for the Cu2+ complex than free neurotensin. Obtaining smaller signals suggests loss of the tyrosine signal in addition to lack of added signal for Cu2+. The loss of tyrosine signal for the complex may be indicative of a structure that decreases access of tyrosine to the electrode surface. Related to this conclusion, we observed that as the native peptide, neurotensin was already the least sensitively detected peptide, suggesting less access of the electrode to the two tyrosines in neurotensin. Thus, from these experiments we conclude that, at C-fiber electrodes, the biuret reagent allows undetectable peptides to be detectable similar to what has been observed with glassy-C electrodes. In contrast, the biuret reagent does not enhance the signal for tyrosine-containing peptides at C-fiber electrodes. This may be due to a variety of factors including susceptibility to alteration of the electrode surface by Cu2+. Finally, some tyrosinecontaining peptides, such as neurotensin, may display a weaker signal upon biuret derivatization due to the inability to form strong complexes and formation of complexes that inhibit tyrosine oxidation. Detection Potential for LC-EC. With the pilot experiments completed, we began to optimize separation and detection of bradykinin, vasopressin, oxytocin, and neurotensin by LC-EC. These peptides were chosen since they were all expected to be present in the SON. The first issue considered was the optimal potential for detection. The detection potential with the highest signal-to-noise ratio (S/N) was found to be +0.60 V. Although the HDVs in Figure 1 illustrate that higher signal could be obtained at higher voltages, a large increase in background and noise at around +0.70 V in the presence of Cu2+ dominated the S/N preventing use of higher potentials. Interestingly, the optimal potential used here is lower than the optimal potential used for measurements of native met-enkephalin in a more acidic mobile phase.13 Thus, although the biuret chemistry does not seem to provide an advantage in terms of sensitivity for electroactive peptides, the use of high pH allows a lower potential to be used, which should aid selectivity in detection. Other factors that were optimized for high sensitivity detection include the Cu2+ concentration in the mobile phase and the time of derivatization as outlined in the Experimental Section. LC-EC with Preconcentration. Detection of peptides by electrochemistry at levels below 1 nM requires preconcentration. Successful preconcentration requires the following: (1) samples be injected under conditions where k′ is sufficiently high that they do not migrate down the column during injection causing broad peaks, and (2) derivatization chemistry that does not produce extraneous compounds that are also preconcentrated and give rise to background peaks. Such background peaks are often the ultimate determiner of detection limits in derivatization schemes. The first requirement entails using samples dissolved in weak solvent, such as aqueous buffers when reversed-phase columns are used, and use of columns with good sample capacity. Biuret derivatization is well-suited for use with preconcentration because it does not require organic solvents, allowing the sample Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

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Figure 4. Chromatograms illustrating effect of on-column preconcentration. Peaks are for 500 amol of vasopressin (V), 500 amol of bradykinin (B), 2 fmol of oxytocin (O), and 5 fmol of neurotensin (N). Conditions for injection are 10 nL of 50 nM V, 50 nM B, 200 nM O, and 500 nM N (A), 100 nL of 5 nM V, 5 nM B, 20 nM O, and 50 nM N (B), and 1.0 µL of 500 pM V, 500 pM B, 2.0 nM O, and 5 nM N (C). All peak areas and widths for a given compound are within 7% for the different injection conditions.

to be injected in purely aqueous solvents. In addition, we found that derivatization does not produce any extra background peaks that are retained during preconcentration, resulting in surprisingly clean blank chromatograms even for large-volume injections. These points are illustrated by the data in Figure 4, which shows chromatograms of the same mass of peptide but with different concentration and injection volumes. As the data show, the peaks are not broadened as a result of the larger injection volumes and essentially the same peak areas are detected for each case, which is expected if all of the peptide is captured during preconcentration. The lack of broadening is remarkable when it is considered that the injection volume of 1 µL corresponds to ∼10 column volumes. This is indicative of the high k′ achieved during injection. The only prohibitive factor to using larger volumes is the longer time required for injection and the volume of sample available. The only negative effect of using larger injection volumes on the chromatogram is the larger baseline drift and the broad band at 500 s. The drift is apparently not due to the biuret derivatization as it is observed without the addition of Cu2+. Rather, the drift is strongly related to quality of solvents used to make the sample solvent and mobile phase. We found that the magnitude of the drift varied from day to day despite precautions of using freshly purified water and filtering. The band at 500 s appears to result from the biuret reagent. The large difference in the shape of the drift compared to the analyte peaks allows it to be removed by high-pass filtering. Figure 5 illustrates a chromatogram with the analytes at 100 pM to 1 nM concentrations treated with a median filter31 to remove the broad drift bands. The chromatograms are compared to an injection of a blank containing the same solution but no peptides. The featureless background allows confident assignment of peaks even at low concentrations. On the basis of these peaks, we calculate detection limits for vasopressin, bradykinin, oxytocin, and neurotensin of 7, 5, 20, and 59 pM, respectively, with 1-µL injection volumes. These concentration detection (31) Moore, A. W.; Jorgenson, J. W. Anal. Chem. 1993, 65, 188-196.

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Figure 5. High-sensitivity detection of biuret peptide complexes by capillary LC-EC. Chromatograms are from injection of 1 µL of 100 pM bradykinin (B), 100 pM vasopressin (V), 400 pM oxytocin (O), and 1 nM neurotensin (N). (A) Raw chromatogram. (B) Chromatogram after median filtering. (C) Blank after median filtering. Chromatographic conditions are given in the Experimental Section.

limits correspond to mass detection limits in the 5-59-amol range. The mass detection limits are typical for capillary LC-EC with columns in this size range. With 1-µL sample loading, the standard deviation of the signal of six consecutive analyses was 5-7% for all analytes. Calibration curves were linear up to at least 1 µM concentration. The low concentration detection limits illustrate that biuret derivatization is remarkable, not only for the minimal increase in background peaks but also for the ability to derivatize at low concentrations. A frequently observed problem with other derivatization reagents is lack of satisfactory reactions at trace level concentrations in addition to production of extraneous background, both of which limit derivatizations to higher concentrations. In Vivo Measurements. To illustrate the potential of this method for trace level analysis in biological samples, we examined the determination of neuropeptides in microdialysate collected in vivo. Chromatograms from dialysate samples collected at 5-min intervals (2.5-µL samples at 0.5 µL/min flow rates, 1 µL actually injected onto the column) during basal conditions and during stimulation by addition of high concentrations of K+ to the perfusion media are shown compared to standards in Figure 6. The data show several detectable peaks in the region of the peptide standards that we were using. Peaks matching the retention time of vasopressin and bradykinin were clearly observed and resolved in both basal and stimulated conditions. When sample was not treated with Cu2+, the peak identified as bradykinin was not detected whereas a peak matching native vasopressin was detected. These requirements for Cu2+ are further evidence supporting the identification of the peaks. A cluster of overlapping peaks appears in the dialysate chromatograms around the retention time of oxytocin making it impossible to determine that peptide. No attempt was made to match neurotensin because of

Figure 6. Capillary LC-EC chromatograms from dialysate compared to standards. Each chromatogram is an injection of 1 µL of (I) standards, (II) dialysate during stimulation with high K+, and (III) dialysate during basal conditions. Peaks for 1 nM vasopressin (V), 1 nM bradykinin (B), and 4 nM Oxytocin (O) are indicated in the standards. Peaks matching the migration time for V and B are shown in the dialysate. Traces on the left are the entire chromatograms while traces on the right are the sections with the majority of peaks after median filtering to remove the drift associated with the gradient and large volume injection.

the poorer sensitivity for this compound. In addition to the standards, several other peaks appeared in the chromatograms. Interestingly, some these unidentified peaks changed in level with stimulation. Given the selectivity of the detection method, it is reasonable to hypothesize that these peaks represent detection of peptide neurotransmitters which we have yet to identify. With in vivo samples we also observed a degradation of detection limit by ∼5-fold for vasopressin and bradykinin. This appeared to result from both an increase in the background drift (compare the drift of the standards in Figure 5 to that for the in vivo measurements) and some broadening of the peaks during injection. Both of these effects are related to the complexity of the sample. For example, the presence of many other retained compounds may saturate or affect the stationary phase at the head of the column and cause a perturbation in the preconcentration of the peptides. The high sensitivity of the method allowed analysis of fractions collected over just a 5-min time scale. Figure 7 illustrates monitoring of the vasopressin and bradykinin peaks with 5-min temporal resolution. The basal vasopressin level was 410 ( 120 pM, which is in good agreement with previously measured levels by RIA of 550 ( 150 pM (both concentrations corrected for relative recovery).32 Additionally, the average vasopressin increase during potassium stimulation was 2.1-fold over that of basal level, again in agreement with literature reports of a 2.4-fold increase.32 Interestingly, bradykinin concentration did not change significantly with K+ stimulation, indicating that it is not released by depolarization. Such a result calls into question the neurotransmitter role of bradykinin in this brain region. Bradykinin had not previously been monitored in the brain in vivo. CONCLUSIONS Several issues need to be addressed with future work in this area. First, the peaks in the dialysate chromatograms were only (32) Landgraf, R.; Ludwig, M. Brain Res. 1991, 558, 191-196.

Figure 7. In vivo monitoring of vasopressin (A) and bradykinin (B) in the rat SON with 5-min sampling frequency. Bar indicates stimulation by application of high-K+ buffer (corrected for dead time of system). Each point is the average from four animals with (1 standard deviation given by the error bar.

identified by migration time, detectability by EC, and dependency on Cu2+ for detection; therefore, more detailed chemical information would be required to identify the peaks with full confidence. The advent of high-sensitivity mass spectrometry methods may make this possible.33,34 Second, in terms of general applicability, we have observed a lack of signal enhancement for tyrosinecontaining peptides that was specific to the carbon fiber electrode. Perhaps development and application of a microglassy carbon electrode may alleviate this problem. Third, more effective use of sample (only 1 of 2.5 µL collected was injected) would allow improved temporal resolution since smaller fractions could be collected for the same sensitivity. Despite the room for improvement outlined above, the results have demonstrated several useful and important features of the method. The biuret reagent allows derivatization down to trace concentrations, lower than typical derivatization reagents, and is compatible with extensive preconcentration. These advantages are in addition to the previously noted advantages of not requiring amino groups and excellent selectivity for peptides.14-22 Combining this method with preconcentration on capillary LC columns allows trace concentrations to be detected even with modest sample volumes (1 µL). The high mass sensitivity of capillary LC-EC, combined low concentration detection limits possible with preconcentration, provides the opportunity to detect peptides in (33) Andren, P. E.; Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 867-869. (34) Emmett, M. R.; Andren, P. E.; Caprioli, R. M. J. Neurosci. Methods 1995, 62, 141-147.

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microdialysates with considerable improvements in temporal resolution and multianalyte capability over that achieved by RIA. Besides dialysate analysis, the high sensitivity of this method may prove useful in other applications as well. For example, analysis of small samples such as single cells often requires care to prevent dilution of the sample. Use of clean derivatization and on-column preconcentration would allow dilution of the cell to manageable volumes while still taking advantage of the high mass sensitivity afforded by capillary LC-EC.

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ACKNOWLEDGMENT This project was supported by a grant from the National Science Foundation and National Institute of Neurologic Disorders and Stroke. R.T.K. received support as a Presidential Faculty Fellow.

Received for review December 14, 1998. AC9809837

September

1,

1998.

Accepted