Anal. Chem. 1994,66, 2669-2674
Determination of Amino Acids and Peptides by Capillary Electrophoresis and Electrochemical Detection at a Copper Electrode Jlannong Ye and Richard P. Baldwin' Department of Chemistryl University of Louisville, Louisvillel Kentucky 40292
Electrooxidation of amino acids and peptides at Cu electrodes has been shown to provide an attractive method for detection and quantitation of these compounds after separation by capillary electrophoresis (CE). As has been previously seen in analogous liquid chromatography applications, the use of Cu electrodes permitted the direct detection of amino acid species in CE at constant applied potential and without derivatization. Strongly alkaline media, typically 50-100 mM NaOH, were required for the electrode process to proceed optimally; but such media were found to be suitable for useful CE separations. The electrode used was a 127-~m-diameter Cu disk operated in a wall-jet configuration. The detection limits so obtained varied from 1 to 10 fmol for most amino acids to a few hundred femtomoles for those containing hydrophobic sidechains. The electrodes could be used continuously for periods of at least 3-4 weeks with no apparent loss of response. Practical applicationsdemonstratedincluded the determinationof amino acids in human urine, of the dipeptide aspartame in diet soft drinks, and of pentapeptide products of a solid-phase synthesis procedure. Over the past two decades, one of the most active areas of electroanalytical research has been the development of amperometric detection systems for flow injection analysis and high-performance liquid chromatography. During this period, numerous electrochemical (EC) detection schemes have been developed and have been shown to offer high sensitivity and unusual selectivity for analytes that are electroactive at modest potentials. As a consequence, EC detection has become the method of choice for the determination of many easily oxidized compounds such as catechols, phenols, thiols, and aromatic amines.' As capillary electrophoresis (CE) has become established as a separation technique of comparable importance, EC approaches initially formulated for use in liquid chromatography have begun to be adapted for CE applications as welL2 The first such CE-EC system, introduced in 1988 by Wallingford and E ~ i n gutilized ,~ carbon fiber microelectrodes for the oxidation and detection of catechols. Subsequently, most CE-EC studies have maintained this focus."8 Very recently, however, attention has turned to a wider range of (1) Kissinger, P. T. In Luboratory Techniques in Electroanalytical Chemistry; Kissinger,P. T., Heineman, W. R., Eds.; Dekker: New York, 1984; pp 611635. (2) Curry, P. D., Jr.; Engstrom-Silverman, C. E.; Ewing, A. G. Electroanalysis 1991, 3, 587-596. (3) Wallingford, R. A,; Ewing, A. G. Anal. Chem. 1988.60, 258-263. (4) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989, 61, 98-100. ( 5 ) Olefirowicz, T. M.; Ewing, A. G. J . Chromarogr. 1990,499, 713-719.
0003-2700/94/0366-2669$04.50/0
0 1994 American Chemical Society
analytes and therefore to consideration of electrode materials that might have wider applicability. For example, the use of Hg electrodes has been reported for the detection of thiols by CE-EC,9 and carbohydrates have been determined in CE by pulsed amperometric detection at Au electrodes.lOJ1 This work suggests that the wide variety of electrode systems developed for use in chromatography-including novel electrode materials, chemically modified electrodes, and even dual and array electrode configurations-might be usefully extended to CE. An electrode material of immediate interest for CE-EC in copper which, in combination with strongly alkaline media, has been used in liquid chromatography for the detection of both carbohydrate compounds12-16and amino acids, peptides, and proteins. 17,18 Although these compounds are not generally electroactive at conventional carbon electrodes,19 they undergo electrocatalytic oxidation at Cu directly without derivatization. Furthermore, such detection at Cu can be carried out stably at constant potential in contrast to Pt and Au, where pulsed potential operation is required. Very recently, Zare and coworkers investigated the use of Cu electrodes in CE-EC for the determination of simple carbohydrates.20 In this application, the electrocatalytic behavior of the Cu system in CE was exactly the same as that seen previously in liquid chromatography usage. Furthermore, although the process was restricted to high-pH buffers, mono- and disaccharides were able to be separated and detected facilely at constant potential and at high sensitivity. A fairly obvious but nevertheless important extension of this work would be to examine the capabilities of CE-EC with Cu electrodes for the determination of amino acid compounds. To our knowledge, this specific approach, involving electrocatalytic oxidation at high p H , has not yet been investigated for amino acids and peptides in CE. Some years ago, (6) Huang, X.;Zare, F.N.; Sloss, S.;Ewing. A. G. Anal. Chem. 1991,63, 189-
192.
(7) OShea, T. J.; Greenhagen, R. D.; Lunte, S.M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992, 593, 305-312. (8) Sloss, S.;Ewing, A. G. Anal. Chem. 1993,65, 577-581. (9) OShea, T. J.; Lunte, S . M. AMI. Chem. 1993, 65, 247-250. (IO) O'Shu\,T. J.; Lunte, S.M.; LaCoune, W .R.Anal. Chem. 1993,65,948-951. (11) Lu, W.; Cassidy, R. M. Anal. Chem. 1993,65, 2878-2881. (12) Prabhu, S.V.;Baldwin, R. P. Anal. Chem. 1989, 61, 852-856. (13) Luo, P.; Prabhu, S. V.;Baldwin, R. P. Anal. Chem. 1990, 62, 752-755. (14) Zadeii, J. M.; Marioli, J.; Kuwana, T. Anal. Chem. 1991, 63, 649653. (15) Xie, Y.; Huber, C. 0. Anal. Chem. 1991, 63, 1714-1719. (16) Mannino, S.;Rossi, M.; Ratti, S . Electroanalysis 1991, 3, 711-714. (17) Luo, P.; Zhang, F.;Baldwin, R.P.Anal. Chem. 1991, 63, 1702-1707. (18) Luo, P.;Baldwin, R. P. Electroanalysis 1992, 4, 393-401. (19) Dou, L.; Mazzeo, J.; Krull, I. S . BioChromatography 1990, 5, 74-96. (20) Colon, L. A,; Dadoo, R.;Zare, R. N. Anal. Chem. 1993, 65,476481.
Analytical Chemistry, Voi. 66, No. 17, September 1, 1994 2008
Engstrom-Silverman and Ewingzl reported the detection of opened. In addition, the outlet end of the capillary was always maintained at ground. A 1-in.-diameter plastic vial served these compounds at Cu electrodes via their complexing,rather than electrocatalytic, properties. In their method, which was both as the cathode compartment of the CE instrument and based on one developed earlier for liquid ~ h r o m a t o g r a p h y , ~ ~ - ~ as ~ the electrochemical cell for the EC detection. Before insertion into the vial through a small slot cut into its side, the the normal oxidation of the Cu electrode itself is inhibited in phosphate and carbonate buffers by the formation of an outlet end of the capillary was made as flat as possible by use insulating Cu(I1) film; anodic currents occur for amino acids of a fiber cleaver (Newport Corp., Irvine, CA). The because of their ability to form Cu(I1) complexes and thereby electrophoresis medium was either 50 or 100 mM NaOH, enhance the anodic dissolution of the electrode. While this and sample injection was made by electromigration. It is complexation approach was shown to permit femtomole-level important to note that reproducible amino acid and peptide detection of a few amino acids and dipeptides,21 a full migration times were achieved only when care was taken to evaluation of the method's capabilities has not been reported ensure that the NaOH electrophoresis medium was freshly subsequently. In addition, the band-broadening and lowered prepared and thoroughly degassed. Apparently, the presence efficiencies observed for even dipeptides suggest that its of low concentrations of C02 markedly decreased the elecextension to more interesting polypeptide analytes may be trophoresis current in effect and correspondingly increased very limited. Finally, liquid chromatography studies comparthe observed migration times. This effect has been reported ing the Cu electrode's performance under both the complexpreviously for carbohydrates by Colon et a1.20 ation and electrocatalysis mechanisms suggest that the latter The details of the EC detection system employed have should provide the more sensitive and widely applicable been described p r e v i o u ~ l y . The ~ ~ specific working electrode detection approach.17Js Therefore, in this work, we have used here was a 127-pm-diameter Cu magnet wire (Newark investigated the analytical performance of the Cu electrode, Electronics, Chicago, IL) whose side areas were covered with operated in the electrocatalytic mode, for CE-EC of amino a nonconductive coating. The electrode was constructed by acid species. In particular, we have included practical inserting the Cu wire into a disposable polypropylene pipet tip examples of this approach for the determination of amino (No. 21-278-5 1, Fisher Scientific Co.) with 3-5" protruding acids in urine, of the artificial sweetener aspartame in diet out and then sealing it in place with epoxy glue (GC Electronics, drinks, and of the cyclic pentapeptide products of a solidRockford, IL). The pipet tip was secured onto an Oriel Corp. phase peptide synthesis procedure. (Stratford, CT) Model 14901 micropositioner. The electrode was arranged in a wall-jet configuration in which the Cu wire was inserted into the cathode compartment of the CE EXPERIMENTAL SECTION instrument and then positioned up against the capillary outlet. Reagents. Most amino acids and peptides were purchased In this configuration, the CE effluent impinged directly onto from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific the disk-shaped electrode and then flowed radially outward Co. (Pittsburgh, PA) and wereused as received without further across its surface. Also placed into this compartment were purification. Cyclic pentapeptides, prepared by standard solida Ag/AgC1(3 M NaCl) reference electrode and a platinum phase synthetic were obtained from A. F. Spatola wire counter electrode. Control of the applied potential and of the Universityof Louisville Department of Chemistry. Stock measurement of the resulting current were carried out with solutions of samples and of the NaOH separation electrolyte a Bioanalytical Systems (West Lafayette, IN) Model LC-3 were prepared with deionized water. Prior to CE analysis, amperometric detector. Upon installation of a new Cu the required sample solutions were made up fresh by serial electrode in the CE-EC system, no special pretreatment dilution in the separation electrolyte. Urine samples were procedures or other operations were performed other than obtained from healthy male volunteers and stored in a applying the desired working potential and waiting roughly refrigerator for no more than 24 h before use. These samples 10 min for the background current to stabilize. Typically, were always injected into the CE directly without any the same Cu electrode, once installed, could be used for periods pretreatment. of 3-4 weeks without removal from the CE system. Apparatus. Separations were carried out on a CE instrument built in-house with a 30-kV high-voltage power supply RESULTS AND DISCUSSION (Model 30B, Hipotronics Inc., Millerton, NY) and an 80-cm Electrochemistry. The general electrochemical behavior length of 25-pm-i.d., 360-pm-0.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ). In order to reduce of amino acids, peptides, and proteins at Cu electrodes in the likelihoodof operator contact with high voltages, the entire strongly basic solution has been described p r e v i ~ u s l y . ~The ~J* capillary, the electrolyte reservoirs for CE, and all electrodes results of these investigations showed that, in cyclic voltamwere enclosed in a Plexiglas box equipped with a safety switch metry, all amino acids gave an increase in anodic current wired to shut down the power supply whenever the box was between +0.4 and +0.8 V vs Ag/AgCl. The voltammograms themselves varied somewhat from case to case in terms of the (21) Engstrom-Silverman, C. E.; Ewing, A. G . J . Microcolumn Sep. 1991,3, 141amount of oxidation current seen and the exact potential at 145. which it occurred. For glycine, for example, the oxidation (22) Kok, W. Th.;Henckemp, H. R.; Bos, F.; Frei, R. W. Anal. Chim. Acta 1982, 142, 31-45. wave was well-shaped and obviously resolved from the (23) Kok, W. Th.; Brinkman, V. A. Th.; Frei, R. W. J . Chromatogr. 1983, 256, voltammetric background while, for some of the others, the 17-26. (24) Stulik, K.; Pacakova, V.; Jokuscies. G . J. Chromatogr. 1988, 436, 334-337. current appeared very close to the solvent oxidation wave. (25) Stulik, K.; Pacakova, V.; Le, K.; Hennissen, B. Talanta 1988, 35, 4 5 5 4 6 0 . (26) Darlak, K.; Romanovskis, P.; Spatola, A. F. Proceedings of 13th American Peptide Symposium; Hodges, R., Smith, J., Us.; ESCOM: Leiden, in press.
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Analytical Chemistty, Voi. 66, No. 17, September 1, 1994
(27) Ye, J.; Baldwin, R . P. Anal. Chem. 1993, 65, 3525-3527.
2
Table 1. CE-EC ot Ambo Ackk at the Cu Electrode. migration detection sensitivr peak* amino acidc time (min) limitd (fmol) (pA/p ) 1 2 3
T 1
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Figure 1. Electropherogram of amlno acids: (1) Arg (50 ptvl), (2) Trp (50 pM), (3) Lys (200 pM), (4) His (50 pM), (5) Met (200 pM), (6) Thr (50 FM), (7) Asn (50 pM), (8) Ser (50 pM), (9) Ala (100 pM), (10) Giy (100 MM), and (11) Tyr (50 MM). Electrophoresis medium, 50 mM NaOH; working electrode, 127 pm Cu at +0.60 V vs AgIAgCi; separation voltage, 20 kV; injection, eiectromigration (20 kV, 3 s).
(The cases of tyrosine, tryptophan, and cysteine are unique in that each of these amino acids may be electroactive even at conventional electrodes.) For large peptides and even proteins, the cyclic voltammetry response at the Cu electrode was qualitatively similar to that of the individual amino acids. Electrochemical behavior for Cu electrodes placed in flow injection and ion-exchange chromatography detection systems mirrored that seen in CV. For the 50 mM NaOH electrolyte used here, constant-potential amperometric detection of amino acid, peptide, and protein analytes was possible at potentials of + O S V and higher. CE-ECof Amino Acids and Peptides. Shown in Figure 1 is an electropherogram recorded at a Cu electrode for a laboratory-prepared mixture containing 11 different amino acids. The applied potential was +0.60 V vs Ag/AgCl, a value which gave the maximum signal-to-noise ratio for detection. The electrophoresis medium, 50 mM NaOH, was selected in order to optimize the resolution obtained for this particular test mixture; and its composition could be changed somewhat in order to alter the separation characteristics as long as the OH- concentration is kept high enough that the electrocatalysis proceeds at an acceptable level. When other amino acids in addition to those shown in the figure were examined, only cysteine, glutamic acid, and aspartic acid failed to give a response under these electrophoresis conditions. Because these three compounds are known to be oxidizable at the Cu electrode and have been detected with good sensitivity in analogous liquid chromatography applications,17J8 it was concluded that their migration times must have been longer than the 90-min period that the CE was allowed to continue in this experiment. Inview of theextra negativechargecarried by each of these amino acids, it was not unexpected that this should be the case as their migration rate (toward the anode)
arginine tryptophan lysine phenylalanine histidine glutamine leucine isoleucine methionine valine threonine asparagine proline serine alanine glycine cystine tyrosine
10.3 17.3 17.6 18.3 18.7 18.9 19.1 19.1 19.3 20.1 20.3 20.7 20.8 22.7 23.5 28.7 29.8 34.0
1.6 0.8 6.4 640 1.6 240 160 160 6.4 240 3.2 3.2 16 1.6 64 6.4 16 0.8
2.5 5.8 0.6 0.006 2.5 0.02 0.03
0.03 0.4 0.02 2.4 2.1 0.2 2.9 0.2 0.6 0.2 5.2
For amperometric detection at +0.60 V vs Ag/AgCl in 0.050 M NaOH; electrophoresis conditions as in Figure 1. b Peak numbers are the same as those used for peak referencing in Fi ure 1. For glutamic acid, aspartic acid, and cysteine, no response was o%served for migration times as long as 90 min. The injection volume was 1.6 nL. e Correlation coefficients were always greater than 0.97 and usually greater than 0.99.
could be close to or even surpass that of the electroosmotic flow. The analytical results for the amino acids in Figure 1 and for others not included in the test mixture are summarized in Table 1. As far as detection limits (signal/noise = 3) were concerned, a wide variation in capabilities was observed. Femtomole quantities (or micromolar concentrations) were able to be determined for most of the amino acids except for those with nonpolar hydrocarbon side chains-phenylalanine, leucine, isoleucine, valine, and alanine. In addition, glutamine also failed to give a particularly favorable response. This trend matches quite well the relative responses seen for the amino acids at the Cu electrode in liquid ~hromatography.'~ Peak heights varied linearly over a 2-3 order of magnitude range of concentrations above the compounds' detection limits. These detection limits and linear ranges are comparable to, and usually better than, those reported for Cu electrode detection by the complexation approach.21 As shown in Figure 2 (and illustrated further in applications discussed in a later section), CE-EC using the Cu electrode was also effective in the separation and detection of small peptides. The sample employed in this figure consisted of a mixture of oligoglycines containing up to six glycine units. Although experimental conditions (0.10 M NaOH, 25-kV separation voltage, and +0.65-V detection potential) were adjusted somewhat from those used earlier for single amino acids, it is interesting that the currents obtained for these peptides were approximately as large as that for glycine itself. Furthermore, there was no decrease in response at all as the size of the peptide was increased from diglycine to hexaglycine. Rather, the currents actually increased for the larger compounds-an observation which bodes well for extension of the Cu electrode approach to important oligo- and polypeptides. It is important that the peaks in both Figures 1 and 2 were always well-shaped and showed little band-broadening atAnalytical Chemistry, Voi. 66, No. 17, September 1, 1994
2671
1
Table 2. Long-Term Stabllky of Cu Ehctrode Response'
2
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TIME lminl Figure 2. Electropherogram of peptides: (1) Glyglyamide (25 pM), (2) hexaglycine (250 pM), (3) pentaglyclne (250 pM), (4) tetraglycine (250 pM), (5) trlglyclne (250 pM), and (6) diglyclne (500 pM). Electrophoresismedium, 100 mM NaOH; working electrode, 127 pm Cu at +0.65 V vs AglAgCI; separation voltage, 25 kV; Injection, eiectromlgration (10 kV, 5 s).
tributable to the Cu electrode system. With both sample systems, the migration times observed for the individual analytes were always inversely proportional to the square root of the species' mass-to-charge ratios. For the amino acids, the number of theoretical plates, calculated from the peak width at half-height, ranged from nearly 60 000 for glycine to more than 100000 for serine and asparagine. For the peptides, the calculated plate numbers were virtually the same as that seen for glycine itself. These results are noteworthy for two reasons. First, the EC system employed here was based on a wall-jet configuration,2' which allows the use of much larger electrodes than has normally been the case for CE-EC. This makes electrode construction and alignment a comparatively simple process and greatly enhances the stability and reproducibility of the detector response. Second, no degradation in efficiency was seen as the size of the peptide was increased. This is in direct contrast to Cu electrode detection performed in the complexation mode, where even dipeptide peaks exhibited significant tailing attributed to slow complexation kinetics2* Finally, a very attractive aspect of the Cu electrode/CEEC approach was its long-term ruggedness. In common practice, the same electrode surface wasused, without removal or any special treatment, for periods from up to 1 week to as long as 1 month. In general, the response obtained for a specific amino acid or peptide tended to decrease quite slowly over time. For example, currents obtained for assays involving 11 individual amino acids over a period of 1 week are summarized in Table 2. In this case, the response differences seen over this time were in the range of only 2-10%. By contrast, the short-term reproducibility seen for repeated injections at a fresh Cu electrode was also in the 5-10% range. This level of reproducibility, which was similar to that reported by Colon et al.*O for carbohydrates, appeared to be related largely to the uncertainty of the injection process. Applications. In view of the capabilities shown above for detection of amino acids and peptides by CE-EC at the Cu 2672
initialc
finald
% change
arginine tryptophan lysine histidine methionine threonine asparagine serine a1anine glycine tyrosine
1.25 (5.4) 1.39 (7.3) 1.03 (4.9) 1.38 (4.8) 0.69 (6.9) 1.12 (8.1) 1.26 (7.7) 1.38 (5.8) 0.16 (9.9) 0.42 (10.3) 0.91 (9.2)
1.19 1.28 0.98 1.32 0.63 1.09 1.14 1.24 0.15 0.41 0.93
-4.8 -7.9 -4.9 -4.3 -8.7 -6.9 -9.5
-10.1 -6.3 -2.4 +2.2
*
I l
amino acidb
AnalflicalChemistry, Vol. 66,No. 17, September 1, 1994
CE-EC conditions were the same as for Figure 1. Concentrations were as follows: tryptophan, tyrosine 250 pM; arginine, histidine, threonine, asparagine, serine, 500 pM; alanine, glycine, 1 .O mM; lysine, methionine, 2.0 mM. c Current levels indicated represent the average of at least four injections at a freshly prepared Cu electrode. Numbers in parentheses correspond to the relative standard deviation (96) for this series of measurements. Currents indicated represent the levels observed for a typical CE-EC run with the same Cu electrode after one week of continuous use.
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Figure 3. Electropherograms of (A) urine sample and (B) amino acid standards: (1) Lys, (2) His, (3) Met, (4) Thr, (5) Asn, (6) Ser, (7) Gly, and (8)Tyr. Electrophoreslsmedium, 50 mM NaOH; working electrode, 127 pm Cu at +OB0 V vs AglAgCI; separationvoltage, 20 kV; injection, electromigration (20 kV for 3 5).
electrode, it seemed desirable to complete this study by examining some applications of the approach with a few realistic samples containing these compounds. To this end, we selected three different analysis situations: the determination of individual amino acids in human urine, the quantitation of the dipeptide aspartame (aspartic acid-phenylalanine methyl ester) in diet beverages, and the analysis of the product mixture resulting from a solid-phase peptide synthesis procedure. Amino Acids in Urine. Figure 3 (curve A) illustrates a typical electropherogram obtained at a Cu electrode for a urine sample from a healthy male volunteer. Also shown for comparison (curve B) is the electropherogram of a laboratory-
Table 9. 8knuItaneow Detmlnatlon of EbM Amlno A M s In Ullne Sampkr.
amount (mg) 1 2 3 4 normal rangeb (adults) (mg/24 h)
LYS
His
43.8 (11) 64.2 (6.5) 52.6 (5.8) 155 (9.6) 12.M5.0
87.7 (4.6) 50.2 (1.0) 43.9 (1.5) 70.1 (6.3) 38.7-256.0
Met 14.9 (4.1) 12.4 (2.5) 11.9 (1.2) 17.3 (2.5) 1.5-1 1.7
Thr 15.6 (1.1) 7.3 (0.2) 7.6 (0.7) 18.5 (2.4) 2.3-36.3
Asn 16.5 (2.7) 32.1 (1.0) 37.6 (1.1) 60.1 (5.6) 34-92
Ser 27.9 (3.3) 17.8 (0.7) 13.8 (0.8) 30.3 (3.8) 2.5-54.0
GlY
TYr
28.9 (2.0) 21.2 (0.4) 22.5 (1.0) 35.5 (4.4) 54.5-152.0
3.3 (1.5) 6.3 (0.1) 8.7 (0.8) 11.7 (1.2) 3.4-40.5
Quantities shown represent the amount of each amino acid present in urine over 24-h period, assuming the daily urine volume to be lo00 mL. Shown in parentheses are the standard deviations of four injections for each sample; assume 1000 mL/24 h as urine volume. From reference 28.
prepared mixture of eight amino acids, each of whose migration times matched that of an unknown peak from the urine sample. Other than this comparison of migration times, no specific measures were taken for the sake of this study to verify unequivocally the assignment of the corresponding sample peaks to these amino acids. Therefore, it is possible that other urine constituents might have contributed at least in part to the CE-EC responses. However, if this possibility was neglected, the concentrations of these eight amino acids could be determined from calibration curves generated from standard solutions. The results of such a procedure for four different urine samples are shown in Table 3 along with an estimate of the normal range at which each of the amino acids is expected to be found. In most cases, the CE-EC method gave results that were at normal or near-normal levels. In the few instances in which this was not the case, no additional work was done to confirm whether, in fact, the sample definitely contained the amino acid at an abnormal level or whether the analysis approach used may be oversimplified. However, the potential applicability of CE-EC with Cu electrodes to the analysis of physiological samples was clearly indicated. Of particular note was the fact that no sample treatment at all had to be applied to the urine samples prior to injection into the CE instrument. Further, the same electrode surface was employed continuously for 3-4 weeks of work with the urine samples with no significant deterioration of the electrode response seen. During this period, the only adjustment made in the CE-EC system was that the electrodecapillary alignment was checked visually from time to time and was corrected manually if called for. With the exception of arginine, whose peak was not able to be easily resolved from those of other oxidizable urine constituents, the amino acids not included among the eight in the text mixture were either those not normally present in urine at high levelsZ8or not detected very sensitively at the Cu electrode (see Table 1). Numerous peaks in addition to the eight assigned above to specific amino acids were in clear evidence in the urine electropherograms, mostly at relatively short migration times. The species responsible for most of these peaks were not identified but quite possibly include peptides and/or carbohydrate compounds as both of these groups of compounds are known to undergo oxidation at the Cu electrode under the conditions in effect in this study. In addition, urine also may contain measurable quantities of conventionallyelectroactive species (e.g., ascorbic acid) which might be responsible for some of the observed peaks. (28) Handbook of Clinical Laboratory Data, 2nd 4.;Faulkner, W.R.,King, J. W.,Damn, H.C.,Us.; CRC Press, Inc.: Cleveland, OH,1968.
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Flgure 4. Electropherograms of (A) an aspartame standard (5.0 pM), (B) Diet MountainDew, (C) Diet 7Up, and (D) Diet Pepsi. Electrophoresis medium, 50 mM NaOH; working electrode, 127 pm Cu at +OB9 V vs Ag/AgCl; separation vottage, 25 kV; Injection, electromlgration (25 kV for 3 5).
Aspartame. Aspartame is the trade name commonly applied to a C-terminal modified dipeptide, aspartic acidphenylalanine methyl ester, which enjoys wide popularity as a low-calorie artificial sweetener in food products. CE-EC experiments performed on aspartame itself indicated that its response at the Cu electrode was qualitatively similar to that seen for individual amino acids. However, under ordinary analysis conditions (e.& those in effect in Figure 4), the magnitude of the response for aspartame was less than that seen for most of the amino acids. Thus, a somewhat higher detection potential (+0.69 V vs Ag/AgCl) was employed for aspartame. Even so, the detection limit was only 150 fmol injected (or 0.10 mM for the 1-2-nL injection volume Analvtical Chemistry, Vol. 66, No. 17, September 1, 1994
2673
employed). Nevertheless, as shown in Figure 4, this response was more than adequate to permit the assay of the aspartame content of diet soft drinks-Diet Pepsi, 1.24 (f0.04) mg/mL; Diet Mountain Dew, 2.71 (fO.ll) mg/mL; Diet 7-Up, 2.77 (f0.09)mg/mL. In each case, the aspartame peak was easily identified and resolved from all other sample constituents giving a response at the Cu electrode. This occurred despite the fact that the only treatment applied to the samples prior to injection was a 2-fold dilution with the 50 mM NaOH separation electrolyte in order to bring the aspartame levels down to a more workable range. Although the substituents giving rise to the additional peaks in the electropherograms in Figure 4 have not been identified, it is possible that some are due to the presence of low levels of carbohydrates, which are also readily detected at the Cu electr~de.~OJ~ Peptide Analysis. In recent years, a research area that has experienced intense interest and activity has been the synthesis of peptides and peptide analogs including complex mixtures known as peptide libraries.z9 Cyclic peptide libraries, in particular, represent a promising approach for drug discoveriesbut can present special analytical challengesz6To facilitate such studies, it is important to have sensitive and selective assay methods available to determine the composition and purity of the resulting product mixtures. Figure 5 shows the CE-EC at a Cu electrode for the product solution following
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TIME ( m i n ]
7 Flgwr 5. Electropherogram of cyclic pentapeptkles: (1) Leu-Tip I Asp-FreVal(O.5 mg/mL) and (2) Leu-Tyr-Asp-Pro-Val (0.5 mg/mL). Electrophoresismedlum, 100 mM NaOH working electrode, 127 pm Cu at +0.60 V vs Ag/AgCi; (separation voitage, 25 kV, Injection, electromigratlon(10 kV for 5 s).
peptides. In view of the presence of Trp or Tyr in both of the peptides synthesized, it is likely that CE detection approaches involving UV-visible absorption or fluorescence might also be applicable to this specific set of peptides. However, EC I a solid-phase synthesis of two cyclic pentapeptides @-Leudetection based on the use of the Cu electrode should be more I I L-TrpD/L-Asp-D-Pro-L-Val and D-Leu-L-Tyr-D/L-AspD-Pro- universal in its operation in that it does not require the presence of specific UV-absorbing amino acids in the synthesized 1 L-Val). The two principal peaks seen by CE were able to be sample. assigned as indicated to these intended products by comparison to CEs obtained for purified standards of these peptides ACKNOWLEDGMENT obtained after wet chemical and HPLC workup of the sample The authors thank A. F. Spatola for supplying the cyclic mixture. Although two optical isomers (from D,L-As~)of pentapeptide samples and for related discussions. This work each peptide were present in the synthesis, no attempt was was supported by the National Science Foundation through made here to achieve any degree of enantiomeric separation, Grant EHR-9108764 of the Kentucky Advanced EPSCoR and presumably, both of the principal CE peaks seen with the Program. Cu electrode correspond to a mixture, as yet not quantitatively defined, containing both the D - A s and ~ L - A s isomers ~ of the Received for review December 1, 1993. Accepted May 18, 1994.@ (29) June, G.; Btck-Sickinger. A. G. Angew. Chem., In?. Ed. Engl. 1992,31,367383.
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AMWaIChemisW, Vol. 66, No. 17, September 1, 1994
Abstract published in Advance ACS Abstracrs, July 15, 1994.
