Anal. Chem. 1989, 61 432-435
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Determination of Naphthalene-2,3-dicarboxaldehyde-Labeled Amino Acids by Open Tubular Liquid Chromatography with Electrochemical Detection Mary D. Oates and James W. Jorgenson* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Naphthalene-2,3-dlcarboxaldehyde (NDA) has been Investlgated as a new derlvatlzlng reagent for the electrochemical detectlon of tagged amino acids. Gradlent elutbn ahwed for the separatlon of 18 NDA-derlvatlzed amino ackls on an open tubular liquid chromatography column In less than 50 mln. Gradient elutbn and electrochemloal detection were found to be compatible. A detection lknlt of 36 am01 was obtained for the asparaglne-NDA derlvatlve. The usefulness of this technique for quantltatlon was demonstrated by the analysis of the NDA-tagged hydrolysis products from bovlne chymotrypslnogen.
INTRODUCTION Amino acid analysis by liquid chromatography has been an important technique for many years. The development of derivatizing reagents that tag amino acids was a critical step in the evolution of this method. Tagging, which can be performed either pre- or postcolumn, is necessary so that all the amino acids can be sensitively detected with a single detection method. The vast majority of derivatizing reagents that have been developed have employed spectroscopic modes of detection. The classical method of amino acid analysis, f i s t proposed by Spackman, Stein, and Moore ( I ) , involves separation by ion-exchange chromatography followed by postcolumn derivatization with ninhydrin. The lack of sensitivity inherent in this technique, which relies upon visible absorption for detection, leads to the development of other, more sensitive derivatizing reagents. These, which include dansyl chloride (2),fluorescamine (3),and phenyl isothiocyanate (41, were also developed with spectroscopic detection in mind. Recently, o-phthalaldehyde (OPA), which reacts with primary amines in the presence of a thiol to form intensely fluorescent products, has come into wide use as a tagging reagent (5,6). The OPA derivatives have been shown to be electrochemically active as well as fluorescent (7,8).Despite certain advantages of electrochemical detection over various optical methods, including simplicity of design, low cost, and lower detection limits than available with UV absorbance, OPA is the only derivatizing reagent that has found even minimal use for the electrochemical detection of tagged amino acids. Although OPA has proven to be quite useful for the analysis of primary amines @-lo), it has an important drawback in that the isoindole derivatives formed are highly unstable ( 9 , 1 1 , 1 2 ) , creating difficulties when precolumn derivatization is attempted. In an effort to overcome this problem, a group of researchers at the University of Kansas developed naphthalene-2,3-dicarboxaldehyde(NDA) as a new fluorogenic reagent for the determination of primary amines (23-15). They have reported good results for the separation of 18 NDA-tagged amino acids in 60 min. They have also mentioned that NDA-labeled amino acids are not only fluorescent but also electroactive (16). However, to our knowledge there are no published reports that explore this aspect of the reagent. The goal of this work, therefore, was to examine the feasability of open tubular liquid chromatography with electrochemical 0003-2700/89/0361-0432$01.50/0
detection for the determination of NDA-tagged amino acids. Due to the wide range of polarities of the derivatized amino acids, it was necessary to make use of gradient elution. Although many have reported that gradient elution with electrochemical detection is problematic at best (17) and perhaps impossible (18,19),in this work no difficulty was encountered when concentrations typically found in routine analyses were used.
EXPERIMENTAL SECTION Apparatus. The chromatographic system used in this work is shown in Figure 1. It is similar to what has been previously described (20),except that modifications were necessary in order for gradient elution to be used. The helium gas pressure that was formerly used for solvent delivery has been replaced with a Waters 600E multisolvent delivery system (Milford, MA). The flow rate of the open tubular column is typically 60 nL/min, while the lowest flow rate available from the high-performance liquid chromatography (HPLC) pump is 10000 nL/min. A splitting system was therefore developed to divert the majority of the mobile phase from the column. Split-flow gradient elution systems have been successfully applied elsewhere (21,22). In this case, a splitting tee was placed after the capillary column. A section of fused silica tubing was inserted into the tee and drew off most of the flow from the column. By proper choice of the length and inner diameter of the fused silica, the desired splitting ratio can be obtained. The tubing used in this work had an inner diameter of 100 pm and was 41 cm long. The pump was used at a flow rate of 1.0 mL/min, generating a pressure at the head of the column of approximately 400 psi. The analytical column had a 15-pm i.d., was 119 cm long, and had octadecylsilanebound to ita porous glass walls (23). A second splitting capillary was added to the injection valve. When an injection is made, valve 2 is opened and valve 1is positioned so that sample can be injected from a syringe into the reservoir tee. Turning valve 1 in this way cuts off the flow of mobile phase from the pump to the chromatographic system. This flow is entirely diverted into the second splitting capillary. With the dimensions of the two splitting capillaries identical to each other, the pressure on the system remains constant at all times. This allows for reproducible injections. After the tee is filled with sample, which requires approximately 0.5 mL, valve 2 is closed and the pump is reconnected to the system by changing the position of valve 1. Mobile phase then flows into the tee and forces a few nmliters of the sample onto the column. After 5 s, valve 1 is repositioned, valve 2 is opened, and the remaining sample is flushed to waste by manually rinsing the injection loop with a syringe. Finally, valve 2 is closed, valve 1 is changed so that mobile phase again flows onto the system, and the sample begins to move down the column. The electrochemical detector used here has been described previously (20,24).The working electrode, a carbon fiber 9 pm in diameter and 0.8 mm long, was inserted into the outlet end of the column. All chromatograms were obtained in the amperometric mode with an electrode potential of 0.9 V versus a Ag/AgCl reference electrode. Data were acquired at one point per second through the use of a microcomputer. A Model 427 current amplifier (KeithleyInstruments, Inc., Cleveland, OH) with a 300-ms rise time and a Model 3341 low pass filter (Krohn-Hite Corp., Avon, MA) set at 10 Hz were also used. Voltammetric waves for all the amino acids were determined by scanning the electrode from 0 to 1 V at a rate of 1 V/s (24). The data acquisition rate in the voltammetricmode was 100 points per second, 0 1989 American Chemical Society
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1 - Phe
I
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TEE (splitting) VALVE 2
LA% lw7-T 9 D k
SPLITTING CAP #2
sample syringe
waste
T
2 nA
waste
VALVE 1
1
SPLITTING CAP #1
TEE
(reservoir)
waste
Figure 1. Schematic diagram of the chromatographic system. the rise time was 30 ms, and the Krohn-Hite filter was not used. Reagents. HPLC grade methanol and acetonitrile (Fisher Scientific Co., Fair Lawn, NJ) were used as received. All water used was purified by a Milli-Q water purification system (Millipore, Bedford, MA). Amino acids and bovine chymotrypsinogen were obtained from Sigma (St. Louis, MO). NDA was purchased from Molecular Probes, Inc. (Eugene, OR), while reagent grade sodium cyanide was obtained from Aldrich (Milwaukee, WI). Solutions. Buffers. Borate buffer (pH 9.5, 0.01 M) was prepared by dissolving boric acid in water and adding sodium hydroxide until the desired pH was reached. Phosphate buffer (pH 7.0,0.05 M) was made by diluting phosphoric acid in water and adjusting the pH with sodium hydroxide. Mobile Phases. Mobile phases were prepared by adding 1mM ethylenediaminetetraacetic acid (EDTA) to the pH 7.0 phosphate buffer, filtering through a 0.22-pm nylon filter (Fisher), and then mixing with filtered acetonitrile. Stock Solutions. Amino acid stock solutions M) were dissolved in water and stored in the refrigerator. They were prepared fresh weekly. Methanolic solutions of NDA (0.1 M) were prepared daily, while sodium cyanide (0.1 M) was dissolved in water and used for up to a month. Procedures. Deriuatization of Amino Acid Standards. This procedure follows that published by de Montigny et al. (14). To mixed solutions of amino acid standards, 182 pL of 0.1 M cyanide and enough borate bufferlmethanol (80120) to make a final volume of 1mL were added, followed by 182 pL of 0.1 M NDA. In every derivatization only the concentrations of the amino acids varied. The concentrations of cyanide and NDA remained constant and allowed for at least a 10-fold excess of the derivatizing reagents over the total amino acid concentration. Derivatization was allowed to proceed for 30 min at room temperature. Immediately prior to injection the tagged mixture was diluted 1:l with the initial mobile phase. Protein Hydrolysis. Two milligrams of bovine chymotrypsinogen were accurately weighed and then dissolved in 1 mL of constant boiling 6 N HCl (Pierce, Rockford, IL). The hydrolysis proceeded for 24 h at 110 O C in the HC1. Prior to hydrolysis, liquified phenol (88% (v/v)), which improves the yield of tyrosine (W), was added. The HCl was evaporated after hydrolysis by use of heat (80-90 "C) and a stream of nitrogen. The residue was dissolved in 4 mL of water and derivatized exactly as described for the amino acid standards.
RESULTS AND DISCUSSION Electrochemistry. The majority of the 18 NDA-tagged amino acids investigated demonstrated voltammetric waves such as that shown for phenylalanine in Figure 2. The peak potential for these derivatives was in the vicinity of 0.8 V. A voltage of 0.9 V, on the plateau, was thus chosen as the working potential for amperometric detection. The two amino acids that display native electrochemical activity, tyrosine and tryptophan, have slightly different waves. These are also shown in Figure 2. Amino Acid Analysis. A gradient separation was developed for mixtures of 18 NDA-labeled amino acids. The initial mobile phase consisted of 98% 0.05 M phosphate buffer (pH 7.0) with 1 mM EDTA present (solvent A) and 2% acetonitrile (solvent B). The composition was changed linearly
0.0
0.5
1.0
E(V) vs. Ag/AgCI
Flgure 2. Voitammograms of 0.1 mM phenylalanine, tyrosine, and tryptophan. Scan rate was 1 V s-' and the electrode length was 0.8 mm. 6
T 0.5nA
19
1
0
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24 32 TIME Cminutes)
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Flgure 3. Chromatogram of 18 NDA-tagged amino acid standards at 5X M each. Norleucine is present at 1 X M. Peak X is likely due to ammonia present in the water used. The peak numbers correspond to those listed in Table I. See the text for chromatographic conditions. over the first 10 min of the separation to 80% A and 20% B. During the next 20 min a linear gradient to 70130 A/B was carried out. Finally, the composition was changed linearly to 50150 A/B over the next 20 min. This gradient provided sufficient resolution while reducing the time necessary for separation of the 18 amino acids to under 50 min. A gradient separation of a mixture of tagged standards is shown in Figure 3. Although many have reported difficulties when using gradient elution with electrochemical detection (17-19), no such problems were encountered here when dealing with concentrations that are typical for routine analyses. Variation in the retention times of the amino acid derivatives was examined by determining the relative standard deviation as a percentage of the average of the retention times (RSD%) on 4 days within a 1-week period. The average RSD% was 2.36%, ranging from 1.20% for the serine derivative to 4.69% for the glutamic acid derivative. No difficulties were encountered in identifying any of the derivatives due to these small variations in retention times. One problem that was encountered involved the peak that elutes immediately after norleucine. It is large and a t times interfered with the quantitation of isoleucine, phenylalanine,
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Table I. Amino Acid Composition of Bovine Chymotrypsinogen
8
peak
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?, 9
1
12
1
III
0
2
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Flgure 4. Chromatogram of the NDA-tagged hydrolysis products of bovine chymotrypsinogen. The peak numbers cwespond to the amino acids listed in Table I. Derivatired ammonia likely results in peak X.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
amino acid
concn, M calcd found
Asp 4.02 X 2.85 X Glu 2.62 X 1.67 X Asn (included in Asp) Ser 4.89 x 10” 2.90 x Gln (included in Glu) His 3.50 X 10“ 2.35 X 10“ Thr 4.02 X 2.80 X Gly 4.02 X 3.10 X Ala 3.84 X lo-& 2.91 X Tyr 7.00 X 10” 5.85 X 10” Arg 7.00 X 10” 4.76 X 10” Val 4.02 X 10” 3.10 X 10” Met 3.50 X 10” 2.99 X 10” Trp (not determined) Ile 1.75 X 1.32 X Phe 1.05 X 7.66 X 10” Leu 3.32 X 2.57 X norleucine (internal std) Lys 2.45 X 1.80 X
no. of residues % diff actual found” (residues) 23 15
21.5 12.6
-6.5 -16
28
21.9
-22
2 23 23 22 4 4 23 2
1.78 21.2 23.4 22.0 4.42 3.60 23.4 2.26
-11 -7.8 f1.7
10 6 19
9.98 5.79 19.4
-0.20 -3.5 +2.1
14
13.6
-2.9
+10 -10 +1.7 f13
Based on the assumption of 22 alanine residues.
For further details consult the text. leucine, and norleucine. By derivatization of an ammonium chloride solution with NDA, it was determined that this peak is likely due to ammonia dissolved in the water used. The gradient was adjusted to minimize the overlap of the ammonia peak with the amino acid peaks. Protein Hydrolysis. The usefulness of open tubular liquid chromatography/electrochemical detection (OTLC/EC) for the quantitation of NDA-tagged amino acids was evaluated by use of bovine chymotrypsinogen. This protein consists of 245 residues and was hydrolyzed and derivatized as described earlier. Prior to hydrolysate analysis, calibration curves were constructed by derivatizing and analyzing four standard mixtures of amino acids, with concentrations of 1 x lo4, 5 x lo“, 1 X and 5 x M of each amino acid. To facilitate quantitation, an internal standard, norleucine, was included in each amino acid mixture. Norleucine was chosen because it is available in high purity and is adequately resolved from the other peaks. The concentration of the internal M in all runs. The standard was held constant a t 1 x ratio of the peak area for each amino acid to the area of the norleucine peak in that run was found and these were plotted versus the amino acid concentration values to obtain the calibration curve for each amino acid. The correlation coefficients for these calibration curves ranged from 0.9957 to 1.000 for all the amino acids. I t was necessary to electrochemically clean the electrode between each chromatographic run by using a method developed in this laboratory (26). A triangular potential wave from 0 to 1.8 V a t 1 V/s is applied to the electrode for 30 s. Cleaning the electrode in this manner was needed because the NDA derivatives appear to foul the electrode. After two chromatographic runs containing all the amino acid derivatives examined in this paper at a concenM, the area of the peaks decreased by an tration of 1 x average of approximately 15%. A third run yielded a similar result. Once the calibration curves had been determined, the protein hydrolysate was dissolved in water, norleucine was added, and derivatization was performed as described earlier. A chromatogram obtained from the NDA-tagged hydrolysis products of bovine chymotrypsinogen is shown in Figure 4. Tryptophan is almost totally destroyed during acid hydrolysis, and therefore could not be determined. Serine is partially destroyed (and so not included in the absolute average deviation determined for the protein analysis), while asparagine and glutamine are converted to aspartic and glutamic acids,
respectively. The ratio of the peak area of each amino acid to the area of the norleucine peak was calculated and this value was used to determine the concentration of the amino acids present in the protein hydrolysate. The results obtained from one analysis of chymotrypsinogen are presented in Table I. All the concentrations determined are low compared to their predicted values, as might be expected. To determine the number of residues of each amino acid present, the experimentally determined concentration of alanine was divided by the actual number of alanine residues present. The other concentrations were divided by this number t o yield the number of residues of each amino acid found. The absolute average deviation from the known amino acid composition for this particular hydrolysate was 6.6%. Two analyses of other chymotrypsinogen hydrolysates had absolute average deviations of 6.1% and 9.6%. An advantage of using electrochemical detection rather than fluorescence for NDA-tagged amino acids can be seen in Table I. It is possible to quantitate lysine electrochemically, while the lysine derivative exhibits a low quantum efficiency and thus low response in fluorescence. This is due to intramolecular quenching in the doubly labeled derivative and seriously restricts the fluorescence detection limit for lysine relative to the other amino acid derivatives (14). No solution to this problem with fluorescence detection has been published. Detection Limit. The detection limit was determined for the asparagine derivative of NDA. With a signal to root mean square noise ratio of 3, the detection limit was determined to be 5 X lo4 M, or 36 amol injected onto the column. This is approximately an order of magnitude lower than the minimum detectable quantity (430 m o l ) that was obtained with laser-induced fluorescence for the same NDA derivative by other researchers (13). The improvement in the mass detection limit seen in this work can be attributed to the use of a capillary column rather than a conventional LC column. The smaller sample volumes that can be used with capillary columns as well as the decreased sample dilution that occurs contribute to the improved detection limits seen in these columns with small (15 pm) inner diameters. Figure 5 shows a chromatogram of the asparagine-NDA derivative at 5 X 10” M. The gradient used is the same as that described earlier. Base-line drift is evident a t this sensitivity and undoubtedly adversely affects the detection limit. However, at concentrations more commonly used, such as lo-’ M and above, the drift becomes negligible.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
ACKNOWLEDGMENT The authors wish to thank Waters Associates (Milford, MA) for the loan of the 600E multisolvent delivery system. Registry No. Asp, 56-84-8; Glu, 56-86-0; Am, 70-47-3; Ser, 56-45-1;Gln, 56-85-9;His, 71-00-1;Thr, 72-19-5;Gly, 56-40-6;Ala, 56-41-7;Tyr, 60-184; Arg, 74-79-3;Val, 72-184; Met, 63-683; Trp, 73-22-3;ne, 73-32-5; Phe, 63-91-2;Leu, 61-90-5;Lys, 56-87-1;NDA, 7149-49-7.
0.03 nA
1
i
ASN
LJ
I
0
8
435
LITERATURE CITED
,
1
,
16
24
32
I
40
48
TIMECminutes)
Figure 5. Chromatogram of 5 X 10" M NDA-asparagine derivative.
Other Considerations. We have found two basic difficulties with amino acid analysis by NDA. One was mentioned earlier and involves the large peak due to ammonia, which can coelute with other peaks. By adjustment of the gradient used, the ammonia peak can be avoided to some extent, but it is always present and a potential interferent. The second problem occurs when the final reagent, NDA, is added to the derivatizing mixture. In all cases for standard mixtures of amino acids and protein hydrolysates, a yellow precipitate formed within several minutes of adding the NDA. This precipitate had to be removed before the mixture could be injected. The cause and identity of the solid are unknown. Despite these problems with the tagging reagent, open tubular LC with electrochemical detection has proven to be a viable technique for the analysis of amino acids. Eighteen amino acids can be detected and quantified with detection limits in the attomole range. In addition, electrochemical detection and gradient elution have been shown to be highly compatible. An important trend in protein analysis has been the ability to determine the amino acid composition of ever smaller quantities of protein. Present technology permits these analyses at the low picomole level (27,28),but a need to work with still smaller quantities of proteins is evident. The keys to this endeavor will likely lie in the development of smaller scale systems for sample handling and analysis. Recently, a microinjector capable of injecting less than 1nL onto an open tubular column was developed in this laboratory (29,30).A future goal of the work with NDA derivatives of amino acids described here is to combine it with the use of such a microinjector to permit amino acid analysis of proteins at the low femtomole level.
Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 30, 1190-1206. Dejong, C.; Hughes, G. J.; van Wieringen, E.; Wilson, K. J. J. Chromet w r . 1982. 24 1 . 345-359. Ribenstein; M.; Chen-Kiang, S.; Stein, S.; Undenfriend, S. Anal. Blochem. 1979, 95. 117-121. Koop, P. R.; Morgan, E. T.; Tarr, G. E.; Coon, M. J. J . Biol. Chem. 1982, 257, 8472-8480. Roth, M. Anal. Chem. 1971, 43, 880-882. Simons, S. S., Jr.; Johnson, D. F. J. Am. Chem. SOC. 1976, 9 8 , 7098-7099. Joseph, M. H.; Davies, P. J. J. Chromatogr. 1983. 277, 125-136. Allison, L. A.; Mayer, G. S.; Shoup, R. E. Anal. Chem. 1984, 56, 1089-1096. Lindroth, P.; Mopper, K. Anal. Chem. 1979, 5 1 , 1667-1674. Davis, T. P.; Johnson, H. D.; Williams, C. H. J. Chroamtogr. 1979, 162, 293-310. Hogan, D. L.; Kraemer, K. L.; Isenberg, J. 1. Anal. Biochem. 1982, 127, 17-24. Turneii. D. C.; Cooper, J. D. H. Clin. Chem. 1982, 2 8 , 527-531. Roach, M. C.; Harmony, M. D. Anal. Chem. 1987, 5 9 , 411-415. de Montigny, F.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. 0.; Srlnivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 5 9 , 1096-1101. Matuszewski, B. K.; Givens, R. S.; Srinlvasachar, K.; Carlson, R. G.; Higuchi, T. Anal. Chem. 1987, 5 9 , 1102-1105. Higuchi, T.; de Montigny, P.; Repta, A. J.; Stobaugh, J. F.; Sternson, L. A.; Eur. Pat. App. EP 199432 A2, 1986. Stulik, K.; Pacakova, V. CRC Crit. Rev. Anal. Chem. 1984. 14, 297-35 1. Rucki, R. J. Talanta 1980, 27. 147-156. Lores, E. M.; Bristol, D. W.; Moseman, R. F. J. Chromatogr. Sci. 1978, 16. 358-362. Knecht, L. A.; Guthrie, E. J.; Jorgenson. J. W. Anal. Chem. 1984, 5 6 , 479-482. van der Wal, Si.; Yang, F. J. HRC CC, J. High Resoiut. Chromatogr. Chromatogr. Common. 1983, 6 . 216-217. Yang, F. J. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. l98SS9 , 84-88. St. Claire, R. L., 111 Ph.D. Thesis, University of North Carolina, Chapel Hill, North Carolina, 1986. White, J. G.; St. Claire. R. L., 111; Jorgenson, J. W. Anal. Chem. 1986, 5 8 , 293-298. Darbre, A. Practical Protein Chemistry; Wiiev: New York, 1986: Chapter 8. St. Claire, R. L., 111; Jorgenson, J. W. J. Chromatogr. Sci. 1985, 2 3 , 186-19 . .. . .1. .. Bdlingmeyer, B. A.; Cohen, S. A.; Tarvin, T. L. J. Chromatogr. 1984, 336.93-104. Cohen, S. A.; Bidlingmeyer, B. A.; Tarvin, T. L. Nature 1988, 320, 769-770. Kennedy, R. T.; St. Claire, R. L., 111; White, J. G.; Jorgenson, J. W. Mikrochim. Acta 1988, 1987(11),37-45. Kennedy, R. T.; Jwgenson, J. W. Anal. Chem. 1988, 6 0 , 1521-1524.
RECEIVED for review July 8, 1988. Accepted November 23, 1988. Support for this work was provided by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and by a grant from Glaxo, Inc.
