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Anal. Chem. 2003, 75, 1508-1513

Analysis of Underivatized Amino Acids and Their D/L-Enantiomers by Sheathless Capillary Electrophoresis/Electrospray Ionization-Mass Spectrometry Casey L. Schultz and Mehdi Moini*

Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712

The detection, identification, and quantitation of amino acids is important in many areas of science including biological and biochemical analysis,1,2 medical diagnostics,3-5 and food analysis.3,6,7 For example, the determination of amino acids within various bodily fluids such as blood and urine is critical for routine clinical analysis. Several metabolic diseases involving amino acids, such as phenylketonurea (PKU) and tyrosinemia (elevated tyrosine), are diagnosed in the neonatal period of life through the detection of abnormal levels of amino acids in the blood or urine

of infants.5 Also, it has been found that glutamine concentration in the cerebrospinal fluid of children with meningitis decreases during the viral or bacterial infection, suggesting that this amino acid could be very useful for monitoring the growth of pathogens.3 The analysis of amino acids is also very important in the food industry where amino acids are measured to correlate flavor trends, monitor fermentation, and assess the quality of the final product.6 Another aspect of amino acid analysis that has been particularly challenging in the past is the separation and detection of amino acid enantiomers.8,9 Interest in the role of D-amino acids in mammalian systems has surged in the past decade due to findings that suggest neuronal and neuroendocrine roles of some D-amino acids.10-12 The analysis of amino acid enantiomers is also important for food quality analysis where the enantiomeric ratio of amino acids can be used as a reliable parameter to assess food quality.7 An increase in the ratio of D-amino acids to L-amino acids within foods may be indicative of extensive processing, contamination, adulteration, or aging.7 The enantiomeric ratio of amino acids within a biological sample can also be used in biological dating applications.13 Gas chromatography,14 liquid chromatography,15 GC/MS,16 and capillary electrophoresis3 have all been used for the detection, identification, and quantitation of amino acids. However, for efficient separation and sensitive detection using these methods, amino acids generally require derivatization prior to their analysis. This is a labor-intensive and time-consuming process. Recently, HPLC/MS17 and sheath-flow capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS)6,18 techniques have been introduced for the analysis of underivatized amino acids. However, because of the high liquid flow rate (>5 µL/min)

* Corresponding author: E-mail: [email protected]. Phone: (512) 4717344. Fax: (512) 471-1420. (1) Teerlink, T. J. Chromatogr., B 1994, 659, 185-207. (2) Schegg, K. M.; Denslow, N. D.; Anderson, T. T.; Bao, Y.; Cohen, S. A.; Mahrenholz, A. M.; Mann, K. In Techniques in Protein Chemistry VIII; Marshak, D. R., Ed.; Academic Press: San Diego, CA, 1997; pp 207-216. (3) Prata, C.; Bonnafous, P.; Fraysse, N.; Treilhou, M.; Poinsot, V.; Couderc, F. Electrophoresis 2001, 22, 4129-4138. (4) Vecchione, G.; Margaglione, G.; Grandone, E.; Colaizzo, D.; Cappucci, G.; Fermo, I.; D’Angelo, A.; Di Minno, G. Electrophoresis 1999, 20, 569-574. (5) Nyhan, W. L. Abnormalities in Amino Acid Metabolism in Clinical Medicine; Appleton Century Crofts: East Norwalk, CT, 1984. (6) Soga, T.; Heiger, D. N. Anal. Chem. 2000, 72, 1236-1241. (7) Marchelli, R.; Dossena, A.; Palla, G. Trends Food Sci. Technol. 1996, 7, 113-119.

(8) Verleysen, K.; Sandra, P. Electrophoresis 1998, 19, 2798-2833. (9) Kuhn, R.; Stoecklin, F.; Erni, F. Chromatographia 1992, 33, 32-36. (10) D’Aniello, A.; Lee, J. M.; Petrucelli, L.; Maddalena Di Fiore, M. Neurosci. Lett. 1998, 250, 131-134. (11) Schell, M. J.; Cooper, O. B.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2013-2018. (12) Schell, M. J.; Brady, R. O., Jr.; Molliver, M. E.; Snyder, S. H. J. Neurosci. 1997, 17, 1604-1615. (13) Lubec, G.; Lubec, B. Amino Acids 1993, 4, 1-3. (14) Amino Acid Analysis by Gas Chromatography; Zumwalt, R. W., Kuo, K. C., Gehrke, C. W., Eds.; CRC Press: Boca Raton, FL, 1987; Vols. I-III. (15) Deyl, Z.; Hyanek. J.; Horikova, M. J. Chromatogr. 1986, 379, 177-250. (16) Duncan, M. W.; Poljak, A. Anal. Chem. 1998, 70, 890-896. (17) Kwon, J.; Moini, M. J. Am. Soc. Mass Spectrom. 2001, 12, 117-122. (18) He, T.; Quinn, D.; Fu, E.; Wang, Y. K. J. Chromatogr., B 1999, 727, 43-52.

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/Lenantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-µm-i.d., 150-µmo.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.

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10.1021/ac0263925 CCC: $25.00

© 2003 American Chemical Society Published on Web 02/19/2003

associated with these techniques, their main disadvantage is a low absolute sensitivity. Several capillary electrophoresis techniques have been introduced for the on-line separation of amino acid enantiomers.8,9,19 Nonmass spectrometric techniques, however, exhibit low selectivity and are generally limited to aromatic amino acids (Tyr, Trp, Phe) if one wants to avoid time-consuming derivatization procedures. Because of their low flow rates (nL/min), sheathless CE/ESIMS techniques are highly sensitive and have proved effective in the analysis of complex biological mixtures that are available in low quantities.20 In this study, the split-flow technique for interfacing CE to MS has been applied to the analysis of underivatized amino acids. In addition, the utility of CE/ESI-MS in the analysis of real-world samples has been demonstrated by the identification of two metabolic disorders (PKU and tyrosinemia) through blood analysis. Moreover, on-line CE/ESI-MS separation of a multitude of underivatized amino acid enantiomers has been demonstrated. EXPERIMENTAL SECTION Methods and Instrumentation. The split flow method21 was used for interfacing CE (MDQ, Beckman-Coulter; Fullerton, CA) to MS (LCQ, Finnigan; San Jose, CA). For the analysis of the L-amino acid standard and the separation of amino acid enantiomers, an underivatized 130-cm-long, 20-µm-i.d., 150-µm-o.d. fusedsilica capillary was used. For the blood analysis, an underivatized 115-cm-long, 20-µm-i.d., 150-µm-o.d. fused-silica capillary was used. The outlet tips of all capillaries used were sharpened through etching with 49% hydrofluoric acid while wearing gloves and working under a fume hood. The MS was scanned in the m/z range of 74.5-250 under positive ionization mode. The CE was operated in forward polarity mode (30 kV was applied to the CE inlet electrode), and 1.1-1.5 kV was applied to the CE outlet/ ESI electrode.20 To speed up the analysis time during the separation of the 21-amino acid standard and the blood analysis experiments, and to maintain stable electrospray during the enantiomer separation, 5, 10, and 5 psi of pressure was applied, respectively, to the CE inlet.22 Reagents and Chemicals. All chemicals, other than HPLCgrade water and 49% HF (Fischer Scientific; Pittsburgh, PA), were purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification. For the analysis of the L-amino acid standards and the blood samples, a 1 M formic acid solution was used as the CE background electrolyte (BGE). For the separation of amino acid enantiomers, a 30 mM (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid (18-C-6-TCA) solution was used as the BGE/ chiral selector reagent with no formic acid added. Two standard amino acid solutions were prepared. The first one contained all 20 standard protein amino acids and was made by spiking a commercially available 17-amino acid standard solution with the remaining 3 standard amino acids (Asn, Gln, Trp). The final concentration of each amino acid in the solution was 1.00 mM. The second amino acid standard contained 21 amino acids and was made by combining an aliquot of the 20-amino acid standard with an equal volume aliquot of a 1.00 mM solution of L(19) Verleysen, K.; Bosch, T. Van den; Sandra, P. Electrophoresis 1999, 20, 26502655. (20) Moini, M. Anal. Bioanal. Chem. 2002, 373, 466-480. (21) Moini, M. Anal. Chem. 2001, 73, 3497-3501. (22) Cao, P.; Moini, M. Electrophoresis 1998, 19, 2200-2206.

Figure 1. CE/ESI-MS base peak electropherogram of the separation of the 20 standard protein amino acids using the 20-µm-i.d., 130cm-long CE capillary. No pressure was applied at the CE inlet during this separation. Forward polarity with 30-kV separation voltage was used. Approximately 400 fmol of each amino acid was injected.

canavanine, giving all 21 amino acids a final concentration of 0.50 mM. The D/L-amino acid enantiomer standard solution used in the enantiomer separation contained 11 D/L-amino acid pairs at a concentration of 0.25 mM. The blood samples were prepared by soaking a 3/16-in.-diameter dried blood spot on filter paper in 100 µL of HPLC grade water for 10 min. A 20 µL aliquot of this solution was then removed and was diluted 10 times with a solution of acetonitrile/water/formic acid (49.9/49.9/0.2; v/v). Amino acid recovery from dried blood spots has been extensively studied by government and commercial laboratories23 and was, therefore, not investigated in this study. However, since the relative intensities of the same amino acids were compared in the blood of healthy versus diseased infants, the issue of recovery has a minimal effect on the comparison. RESULTS AND DISCUSSION Analysis of Underivatized Amino Acids. Figure 1 shows the CE/ESI-MS electropherogram of the separation of the 20 standard protein amino acids using the underivatized 130-cm-long CE capillary in conjunction with 1 M formic acid as the BGE. For this experiment, ∼0.4 nL of the 20-amino acid standard solution (∼400 fmol of each amino acid) was injected. As shown in Figure 1, all amino acids were separated and detected. The use of a nonderivatized column is certainly a big advantage since capillary derivatization techniques are usually long, labor-intensive, expensive, and short-lived. In addition, the low molecular weight of formic acid ensures minimal background chemical noise and, therefore, lower detection limits. Table 1 summarizes the extracted ion detection limits and linearity correlations of the 20 standard protein amino acids except for cysteine (Cys), which was observed as its oxidized form (Cys(23) Adam, B. W.; Alexander, J. R.; Smith, S. J.; Chace, D. H.; Loeber, J. G.; Elvers, L. H.; Hannon, W. H. Clin. Chem. 2000, 46, 126-128.

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Table 1. Properties and Figures of Merit of the Twenty Standard Amino Acids

amino acid nonpolar, aliphatic R groups Gly, G Ala, A Val, V Leu, L Ile, I Met, M aromatic R groups Phe, F Tyr, Y Trp, W polar, uncharged R groups Ser, S Pro, P Thr, T Cys, C Asn, N Gln, Q positively charged R groups Lys, K His, H Arg, R negatively charged R groups Asp, D Glu, E

MW

COOH

pKa values NH3+

75 89 117 131 131 149

2.34 2.34 2.32 2.36 2.36 2.28

9.60 9.69 9.62 9.60 9.68 9.21

165 181 204

1.83 2.20 2.38

9.13 9.11 9.39

105 115 119 121 132 146

2.21 1.99 2.11 1.96 2.02 2.17

9.15 10.96 9.62 10.28 8.80 9.13

146 155 174

2.18 1.82 2.17

8.95 9.17 9.04

133 147

1.88 2.19

9.60 9.67

Cys). As is shown, the detection limits are in the low-femtomole range. The detection limits reported in Table 1 were achieved under full-scan mode (m/z 74.5-250.0) and are 3-4 orders of magnitude lower than one sheath-flow CE/MS study reported.18 The linear quantitation range for all amino acids was ∼2 orders of magnitude. The high sensitivity achieved was due to the low flow rate of the narrow CE capillary used. Since ESI is a concentration sensitive ionization technique, the lower flow rates of narrow capillaries provide higher sensitivities. From the results of Figure 1, two general features of the experimental results should be discussed. One is the order of migration of the amino acids and the other is the relative intensity of each amino acid. In CE, the electrophoretic velocity (νep) of a charged species is proportional to the electric field strength (E ) V/L, where V is the separation voltage and L is the column length). The proportionality constant is called electrophoretic mobility (µep, cm2/ V‚s) and is determined by the following equation:

µep ) νep/E ) q/6πηr

(1)

where q is the analyte’s net charge, η is the viscosity of the BGE, and r is the analyte’s ionic radius. From eq 1, it is apparent that small, highly charged species generally have high electrophoretic mobilities while large, minimally charged species generally have low electrophoretic mobilities. Table 1 summarizes some of the important properties of the 20 standard protein amino acids including their molecular weights, pK values, and pI values.24 As shown in Table 1, all amino acids have a pI > 2. Therefore, with 1510

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pI

detection limit (fmol)

correlation coefficient

5.97 6.01 5.97 5.98 6.02 5.74

14.0 13.5 3.0 2.7 2.7 2.1

0.9994 0.9997 0.9996 0.9971 0.9988 0.9935

5.48 5.66 5.89

1.7 1.0 1.0

0.9979 0.9974 0.9998

5.68 6.48 5.87 5.07 5.41 5.65

6.9 3.9 5.3 7.9 3.2

0.9993 0.9998 0.9997 0.9981 0.9999

10.53 6.00 12.48

9.74 7.59 10.76

2.9 4.1 2.4

0.9993 0.9995 0.9994

3.65 4.25

2.77 3.22

5.5 3.2

0.9962 0.9981

R group

10.07

8.18

the BGE used (1 M formic acid, pH ∼1.8), all amino acids had a net positive charge and moved toward the CE outlet under forward polarity mode. Their degree of positive charge, however, depended on their pK values. As shown in Figure 1, amino acids that were highly charged (the amino acids with basic side chains) migrated first, moderately charged amino acids (aliphatic amino acids with unionizable side chains) migrated second, and both larger amino acids (aromatic amino acids, Cys-Cys) and amino acids with a very low degree of positive charge (amino acids with acidic side chains and proline) migrated last. The second feature of Figure 1 that needs to be explained is the relative intensities of the protonated amino acids. Since approximately equal molar amounts of each amino acid were injected (∼400 fmol) and analyzed under constant BGE and ESI voltage, their relative intensities reflect their ionization efficiency under ESI. Their relative intensities can be divided into three general categories: high intensity, medium intensity, and low intensity. While a detailed explanation of the relative sensitivities of the amino acids is under investigation, the general categories observed are consistent with the proposed mechanism of ESI. Under ESI, droplets with a surface excess charge are created. The ESI equilibrium partitioning model suggests that ions within a droplet partition between the surface of the droplet (where there is much excess charge) and the interior of the droplet and that the relative abundances of ions in the mass spectrum are proportional to the relative concentrations of the different charged species at the droplet surface.25,26 This partitioning is influenced (24) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry; Worth Publishers: New York, 1993. (25) Enke, C. G. Anal. Chem. 1997, 69, 4885-4893.

Figure 2. CE/ESI-MS base peak electropherogram (top panel) of the separation of the 21-amino acid mixture using the 20-µm-i.d., 130cm-long CE capillary. To shorten the analysis time, the separation voltage of 30 kV was augmented with 5 psi of inlet pressure. As is shown, L-canavanine (Can) was baseline separated from arginine (R). Middle and bottom panels show mass spectra of arginine and canavanine, respectively.

by a number of properties of the ions and the solvent including solvation energy, ion-pairing energy, charge density, and hydrophobicity.26 This is consistent with our data in which a relatively high ESI response was observed for amino acids that have highly hydrophobic areas such as aromatic rings, and a relatively low ESI response was observed for aliphatic amino acids with small side chains (or no side chain in the case of Gly). There are more than 900 amino acids, in addition to the 20 standard protein amino acids, that exist in nature (mostly in plants) and are not typically incorporated in proteins. The ingestion of some of these non-protein amino acids can result in acute and chronic poisoning or can result in the incorporation of non-protein amino acids into proteins, thereby interfering with protein structure and function. One such amino acid is L-canavanine (see inset of Figure 2 for chemical structure), whose accidental incorporation into proteins can disrupt a broad range of fundamental life processes.27 The separation and detection of these nonprotein amino acids within a mixture of standard protein amino acids is therefore an important task. To demonstrate the selectivity of CE/ESI-MS in the detection of a non-protein amino acid within a complex amino acid mixture, a 21-amino acid mixture containing the 20 standard protein amino acids plus the non-protein amino acid L-canavanine was analyzed (Figure 2). As shown, L-canavanine was completely separated from its close analogue L-arginine, even though 5 psi of pressure had been applied at the CE inlet during (26) Sjoberg, Per J. R.; Bokman, C. F.; Bylund, D.; Markides, K. E. Anal. Chem. 2001, 73, 23-28. (27) Rubenstein, E. Medicine 2000, 79, 80-89.

Figure 3. (A) Amino acid analysis of the blood afflicted with PKU and its comparison to that of a healthy individual (inset). Analysis preformed using a 20-µm-i.d., 115-cm-long CE capillary. (B) Amino acid analysis of the blood of an infant afflicted with tyrosinemia and its comparison to that of a healthy individual (inset). Analysis preformed using a 20-µm-i.d., 115-cm-long CE capillary. To shorten the analysis time, in both experiments, the separation voltage of 30 kV was augmented with 10 psi of inlet pressure.

the separation to reduce analysis time. As shown in Figure 2, the use of long and narrow capillaries provides high separation efficiency; however, a disadvantage of using long capillaries is long analysis times. Blood Analysis and Metabolic Disorder Identification. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the detection of abnormal levels of amino acids in the blood of two newborns afflicted with metabolic disorders (one with PKU and one with tyrosinemia). In this study, the relative intensities of glutamine and phenylalanine (PKU) and glutamine and tyrosine (tyrosinemia) were compared in the blood of healthy versus diseased infants. Figure 3 shows significant elevation of phenylalanine and tyrosine, respectively, in the blood of the diseased infants. In Figure 3, the migration times were normalized to the migration time of glutamine in the normal infant’s blood sample. The results demonstrate that this technique can be applied to the qualitative analysis of amino acids in realworld samples at low concentrations and low volumes. Virtually no sample preparation (other than dilution) was required for these experiments.The use of a narrow capillary in conjunction with dilute blood minimizes memory effects and prevents column overload. It should be noted that the data presented are only qualitative and simply compare the relative amounts of the amino acids present in the blood. These analyses would have to be Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

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Figure 4. Selected ion electropherogram of the separation of 11 amino acid enantiomers using a 30 mM 18-C-6-TCA solution as the BGE/chiral selector reagent and a 20-µm-i.d., 130-cm-long CE capillary. This analysis was carried out with 5 psi of inlet pressure. This pressure was necessary to maintain stable ESI due to a lowered electroosmotic flow under 30 mM 18-C-6 TCA conditions. Approximately 500 fmol of each amino acid was injected. Inset (top left) shows the chemical structure of 18-C-6-TCA.

quantitatively validated, and the analysis time would have to be significantly reduced in order for this technique to be applicable to clinical or newborn screening laboratories. On-Line Separation of Underivatized Amino Acid Enantiomers. Another important feature of amino acid analysis is the separation and detection of D- and L-enantiomers. One advantage of CE is that many D/L-amino acid enantiomers can be separated on-line by using a BGE that contains a chiral selector. It has previously been found (and was experimentally verified under ESIMS; data not shown) that a 30 mM solution of 18-C-6-TCA provides the optimum resolution for the separation of amino acid enantiomers under CE.9 The separation of D- and L-amino acid enantiomers was, therefore, studied using a 30 mM 18-C-6-TCA solution as the chiral selector/BGE. 18-C-6-TCA is a macrocyclic polyether ring system consisting of several oxygens joined by ethylene bridges. The structure of this molecule is shown in the inset of Figure 4. The polyethylene ring forms a cavity with the oxygens roughly forming a plane on the inner side of the cavity.9 The cavity formed by the ring of 18-C-6-TCA can form complexes with cations of suitable size. The ammonium cation of an amino acid can form a complex with the polyethylene ring of 18-C-6-TCA through three +NH‚‚‚O hydrogen bonds in a tripod arrangement.28-31 The NsC* bond lies perpendicular to the plane defined by the oxygens of the polyethylene 1512

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ring system (C* represents the chiral center of the amino acid). The carboxyl group substituents of 18-C-6-TCA allow for enantiorecognition. They themselves lie perpendicular to the plane defined by the polyethylene ring system and differentially impede complexation through stereospecific steric hindrance.9 Thus, Dand L-amino acid enantiomers have different affinities for complexation with 18-C-6-TCA. Figure 4 demonstrates the separation of 11 amino acid enantiomers using a 30 mM 18-C-6-TCA solution as the background electrolyte/chiral selector reagent. Approximately 2 nL of the amino acid enantiomer solution (∼500 fmol of each amino acid) was injected. As is shown (Figure 4), almost all D/Lenantiomers are baseline separated. The 30 mM solution of 18C-6-TCA was found to be compatible with ESI and suitable as the BGE without the need for additional formic acid. However, compared to the absolute intensities of the amino acids analyzed under 1 M formic acid (Figure 1), the absolute intensities of the amino acids analyzed under 30 mM 18-C-6-TCA (Figure 4) were on the average ∼4 times lower (after normalization with respect to the amount injected in each experiment). To our knowledge, this is the first demonstration of enantiomeric separation of underivatized amino acids using CE/ESI-MS. However, the online enantiomer separation of three pharmaceutical compounds with structures similar to that of the standard amino acids (all containing amine groups located near a chiral center) has been achieved using sheath-flow CE/ESI-MS in conjunction with various cyclodextrin derivatives.32 Comparison between the results obtained in these studies (Figure 4 of this article and Figures 5-7 of ref 32) demonstrates both sensitivity (after calculating the approximate amount of sample injected in each study) and resolution advantages of using 18-C-6-TCA in conjunction with sheathless CE/ESI-MS. Moreover, the technique presented here was able to separate 11 pairs of amino acid enantiomers in one run under identical BGE/chiral selector reagent conditions (30 mM 18-C-6-TCA), while the use of different chiral selector reagents (DM-β-CD and HP-β-CD) at varying concentrations (5, 15, and 20 mM) was required to efficiently separate the three pairs of enantiomers.32 To determine which enantiomer (D or L) has a higher affinity for 18-C-6-TCA, a solution containing three pairs of amino acid enantiomers with all D-enantiomers at a concentration of 1.0 mM and all L-enantiomers at a concentration of 0.5 mM was analyzed (data not shown). It was found that the peak height of the enantiomers that eluted first were approximately half that of the peaks that eluted second. This demonstrated that in each case the L-enantiomer eluted first. It was therefore concluded that, based on their differential migration time, D-enantiomers of amino acids have a stronger affinity for complexation with 18-C-6-TCA than their L- counterparts. The on-line separation and detection of nonaromatic amino acid enantiomers without the requirement of precolumn derivatization (28) Kyba, E. P.; Timko, J. M.; Kaplan, L. J.; de Jong, F.; Gokel, G. W.; Cram, D. J. J. Am. Chem. Soc. 1978, 100, 4555. (29) Sousa, L. R.; Sogah, G. D. Y.; Hoffman, D. H.; Cram, D. J. J. Am. Chem. Soc. 1978, 100, 4569. (30) Behr, J.-P.; Girodeau, J.-M.; Heyward, R. C.; Lehn, J.-M.; Sauvage, J. P. Helv. Chim. Acta 1980, 63, 2096. (31) Behr, J.-P.; Lehn, J.-M.; Vierling, P. Helv. Chim. Acta 1982, 65, 1853 (32) Lu, W.; Cole, R. B. J. Chromatogr., B 1998, 714, 69-75.

demonstrates an important advantage of CE/ESI-MS over previously reported methods of on-line amino acid enantiomer separation.9,19

sample pretreatment (dilution). By using a 30 mM solution of 18C-6-TCA, 11 amino acid enantiomers were separated and detected with ease.

CONCLUSIONS CE/ESI-MS is a powerful technique for the analysis of underivatized amino acids. By using a nonderivatized CE capillary and utilizing the split-flow technique for interfacing CE to MS, all 20 standard amino acids plus one nonstandard amino acid (L-canavanine) were separated and detected at low-femtomole levels under full-scan mode. Because a nonderivatized column is used, the technique is very easy to implement. Blood samples were analyzed for high levels of amino acids with minimal

ACKNOWLEDGMENT Dr. Donald Chace of Neo Gen Screening (Pittsburgh, PA) is gratefully acknowledged for supplying the blood samples used in this study.

Received for review December 3, 2002. Accepted January 24, 2003. AC0263925

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