Simultaneous Analysis of Amino Acids and Carboxylic Acids by

Anal. Chem. 2010, 82, 9967–9976

Simultaneous Analysis of Amino Acids and Carboxylic Acids by Capillary ElectrophoresisMass Spectrometry Using an Acidic Electrolyte and Uncoated Fused-Silica Capillary Masataka Wakayama, Naohiro Aoki, Haruto Sasaki, and Ryu Ohsugi* Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z ) [M + 18]+ or [M + 23]+, after protonated molecule detection (m/z ) [M + 1]+) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z ) [M - 1]-), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism. Capillary electrophoresis-mass spectrometry (CE-MS) has been used for analyzing complex mixtures because CE has high separation performance,1,2 and MS has a high selectivity, based on the molecular mass to charge ratio (m/z).3 For metabolomics analyses, CE-MS systems have been used to quantify small polar metabolites in Bacillus,4 rice leaf,5 human cells,6 and so on.1,3 In these papers, amino acids and carboxylic acids were analyzed by a different analytical mode. Amino acids were separated by CE, using an uncoated fused silica capillary with an acidic buffer such * Corresponding author: (fax) +81-3-5841-8048; (e-mail) aohsugi@ mail.ecc.u-tokyo.ac.jp. (1) Monton, M. R.; Soga, T. J. Chromatogr., A 2007, 1168, 237–246. (2) Song, E. J.; Babar, S. M.; Oh, E.; Hasan, M. N.; Hong, H. M.; Yoo, Y. S. Electrophoresis 2008, 29, 129–142. (3) Ramautar, R.; Somsen, G. W.; de Jong, G. J. Electrophoresis 2009, 30, 276– 291. (4) Soga, T.; Ohashi, Y.; Ueno, Y.; Naraoka, H.; Tomita, M.; Nishioka, T. J. Proteome Res. 2003, 2, 488–494. (5) Sato, S.; Soga, T.; Nishioka, T.; Tomita, M. Plant J. 2004, 40, 151–163. 10.1021/ac1019039  2010 American Chemical Society Published on Web 11/16/2010

as formic acid.7-9 Electrophoresis was performed from the anode inlet to the cathode (normal directional electrophoresis), and electrospray ionization mass spectrometry (ESI-MS) detection was in the positive ion mode. In contrast, the separation for anionic carboxylic acids was performed with the opposite polarity, from the cathode inlet to the anode, an alkali ammonium acetate buffer was used as the electrolyte, and MS was performed in the negative ion mode.4 In this mode, when an uncoated fused silica capillary is used, negatively charged silanol groups of the capillary surface and the electrolyte buffer cause electroosmotic flow (EOF) movement toward the cathode (CE inlet side) and cause electrolyte depletion at the MS side of the capillary.10 To prevent this phenomenon, positively charged capillary, such as a Polybrene-, polydimethylsiloxane-coated, or other coated capillary is needed, instead of an uncoated fused silica capillary.10-16 CE-MS analysis of anionic carboxylic acids using an uncoated capillary has also been reported using strong EOF mobilities in normal directional electrophoresis with weak electrophoresis mobilities of the anionic compounds. In these systems, an alkali ammonium formate buffer or trimethyl amine/acetate buffer, which has strong EOF mobility, is used as the electrolyte.17,18 To reduce the migration time, pressure assistance from the CE inlet side was also applied.18 For biological metabolic analysis, a comprehensive analysis of both amino acids and carboxylic acids is valuable. For analyzing (6) Hirayama, A.; Kami, K.; Sugimoto, M.; Sugawara, M.; Toki, N.; Onozuka, H.; Kinoshita, T.; Saito, N.; Ochiai, A.; Tomita, M.; Esumi, H.; Soga, T. Cancer Res. 2009, 69, 4918–4925. (7) Lu, W.; Yang, G.; Cole, R. B. Electrophoresis 1995, 16, 487–492. (8) Soga, T.; Heiger, D. Anal. Chem. 2000, 72, 1236–1241. (9) Levandi, T.; Leon, C.; Kaljurand, M.; Garcia-Can ˜as, V.; Cifuentes, A. Anal. Chem. 2008, 80, 6329–6335. (10) Soga, T.; Ueno, Y.; Naraoka, H.; Ohashi, Y.; Tomita, M.; Nishioka, T. Anal. Chem. 2002, 74, 2233–2239. (11) Johnson, S.; Houk, L.; Johnson, D.; Houk, R. Anal. Chim. Acta 1999, 389, 1–8. (12) Soga, T.; Ueno, Y.; Naraoka, H.; Matsuda, K.; Tomita, M.; Nishioka, T. Anal. Chem. 2002, 74, 6224–6229. (13) Hagberg, J. J. Chromatogr., A 2003, 988, 127–133. (14) Mokaddem, M.; Varenne, A.; Belgaied, J.-E.; Factor, C.; Gareil, P. Electrophoresis 2007, 28, 3070–3077. (15) Soga, T.; Igarashi, K.; Ito, C.; Mizobuchi, K.; Zimmermann, H.-P.; Tomita, M. Anal. Chem. 2009, 81, 6165–6174. (16) Timischl, B.; Dettmer, K.; Kaspar, H.; Thieme, M.; Oefner, P. J. Electrophoresis 2008, 29, 2203–2214. (17) Sawada, H.; Nogami, C. Anal. Chim. Acta 2004, 507, 195–202. (18) Harada, K.; Fukusaki, E.; Kobayashi, A. J. Biosci. Bioeng. 2006, 101, 403– 409.

Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

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these metabolites simultaneously with a single CE instrument, the replacement of the electrolyte17,18 or the derivatization of the samples19 is required. In some cases, a commercial coated capillary or a capillary-coating treatment10-15 was needed with correct storage conditions for subsequent analysis to be successful. In normal directional electrophoresis using an acidic electrolyte, good separation of amino acids has been demonstrated.8 However, the possibility of analyzing anionic carboxylic acids in an acidic electrolyte had not been previously investigated. Analyte migration rates in CE-MS are governed not only by effective electrophoretic mobility and EOF, as in typical CE separations, but also by the additive effects of the pressure exerted by sheath liquid and gas flow.20 These two additional mobility flows are mixed at the outlet side of CE and the tip of the ESI-MS. Thus, there is a high possibility of analyzing carboxylic acids in an acidic electrolyte using these CE-MS-specific mobility factors instead of using pressure assistance or pump-like mobility by high EOF electrolyte. In this study, we established a separation method for CE-MS analysis for both amino acids and carboxylic acids using a single uncoated fused silica capillary with a single acidic electrolyte. By changing the polarity of ESI-MS at the time lag of the detection time between cationic and anionic compounds, high-resolution analysis of both amino acids and carboxylic acids was achieved with only a single CE-MS run. EXPERIMENTAL SECTION Chemicals and Reagents. Methionine sulfone (MetS), which was purchased from Sigma-Aldrich (St. Louis, MO), and piperazine-1, 4-bis-(2-ethanesulfonic acid), sesquisodium salt (PIPES), which was purchased from Dojindo (Kumamoto, Japan), were used as the internal standards for quantitation and as the relative migration time standard. All other reagents (Tables 1 and 2) were obtained from Kanto Chemical (Tokyo, Japan) and Sigma-Aldrich. All analytical standard sample solutions (10 mM solution, Tables 1 and 2) were prepared with LC grade water (Kanto Chemical), and stored in a refrigerator. All these analytical standards have a charge of one. Thus, each m/z of compound is directly correlated with the molecular weight of each compound. Dimethyl sulfoxide (DMSO) and glucose were used for the EOF marker in positive ion mode and in negative ion mode, respectively. Instrumentations. CE-MS experiments were performed using a Beckman P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA) with a Finnigan TSQ Quantum Discovery Max quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA). To connect the CE to the MS, a P/ACE MDQ external detector adaptor (Beckman Coulter) and a CEspecific ESI nozzle (Thermo Fisher Scientific Japan, Yokohama, Japan) were used. The ESI nozzle was mounted on an Ion-Max type (Thermo Fisher Scientific) ionization source. The CE was controlled by 32 Karat software (Beckman Coulter). For MS analysis, N2 gas was supplied from an N2 gas generator M12ES (System Instruments, Tokyo, Japan), and the sheath liquid flow was supplied by a Shiseido Nanospace SI-2 pump (Shiseido, Tokyo, Japan). The control and quantitation of MS were (19) Yang, W.-C.; Regnier, F. E.; Adamec, J. Electrophoresis 2008, 29, 4549– 4560. (20) Huikko, K.; Kotiaho, T.; Kostiainen, R. Rapid Commun. Mass Spectrom. 2002, 16, 1562–1568.

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performed using TSQ Quantum 1.2 and Xcalibur 1.4 (Thermo Fisher Scientific), respectively. CE-MS Conditions. CE separation was performed on a bare fused silica capillary (GL Sciences, Tokyo, Japan) measuring 50 µm i.d. × 100 cm total length. The polyimide coating of the MS side capillary tip (3 mm) was removed. To make the flow of the electrophoresis stable, the capillary tip of both the CE inlet side and the MS side were set to the same height. Prior to the initial separation, the capillary was disconnected from the MS, and flushed with, in order, methanol, 1 M HCl, 0.1 M NaOH, and the electrolyte for 12 min each at 30 psi (2000 hPa). Prior to each injection, the capillary was flushed with the electrolyte for 5 min at 30 psi. For positive mode detection of anionic carboxylic acids, capillary flushing using 5 mM ammonium formate for 3 min at 30 psi was performed prior to flushing with the electrolyte. Quantitative CE analyses were performed with normal directional electrophoresis at +30 kV (anode at the CE inlet and cathode at the MS side) using 1 M formic acid electrolyte. To evaluate the performance of separation for the standard solution mixture, ESI-MS detection was performed in both positive and negative ion modes. In positive ion mode, the sample mixture was injected at 0.7 psi (50 hPa) × 18 s (∼ 15 nL). In negative ion mode, the mixture was injected at 0.7 psi × 30 s (∼ 25 nL). During analysis, the capillary temperature was maintained at 25 °C. During the injection of the sample and the stabilization of the electrophoretic voltage, the electrospray voltage was turned off to prevent electrokinetic introduction of the analyte. For ESI-MS detection, the sheath liquids were finally optimized to 10 µL min-1 flow in 50% (v/v) methanol-water. The electrospray needle was set to +3.5 kV in positive mode and to -3 kV in negative mode. The sheath gas flow (N2) was finally set to 25 psi (103 kPa). The heated ion-transfer capillary was maintained at 240 °C. Ion sweep gas, auxiliary gas, source collision-induced dissociation and collision energy were not applied. The spectrometer was scanned from m/z 50 to 370 in a 0.5 s/scan monitoring mode. In our instruments, because the electrode is shared between the CE outlet electrode and ESI-MS probe, the total voltage of electrophoresis (V ) VCE - VMS) was calculated by the differences of CE voltage (VCE) and ESI-MS voltage (VMS). The total charge of electrophoresis (E ) V/L) was calculated by dividing the total voltage (V) by the capillary length (L). The apparent mobility (µapp,i ) L2/tiV) of each amino acid and carboxylic acid was determined by migration time (ti), applied total voltage (V), and capillary length (L).21 The effective electrophoretic mobility (µeff ) µeof - µapp,i) was calculated by the difference of the apparent mobility of EOF marker (µeof ) µapp,eof) and those of each compound (µapp,i). These mobilities of each compound peak were analyzed by electrolyte concentration, sheath gas pressure, and sheath liquid flow. For simultaneous analysis in both positive and negative modes ESI-MS, the sample was injected at 0.7 psi (50 hPa) × 30 s (∼ 25 nL), and the ESI polarity was changed from positive to negative 30 min after the injection. During the change in ESI polarity, the ESI voltage was stopped for 60 s, and the electrophoretic voltage was also stopped for 30 s after stopping the ESI (21) Landers, J. Introduction to Capillary Electrophoresis; in Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, 3rd ed; Landers, J., Ed.; CRC Press: Boca Raton, FL, 2008; pp 3-74.

Table 1. Performance of Positive/Negative Mode CE-MS for Amino Acids and Carboxylic Acidsa

compounds

adduct ion

N, N-diaminobutane Ethanolamine Carnosine Ornithine Lysine Arginine Histidine β-alanine γ- aminobutyrate Glycine Alanine Sarcosine R-aminobutyrate Cystathionine Serine Valine Homoserine Isoleucine Leucine Asparagine Threonine Methionine Glutamine Proline Glutamate Tryptophan Phenylalanine Tyrosine Aspartate Methionine sulfone Hydroxyproline Phenylglycine Phosphoethanolamine Urea Taurine DMSO (EOF marker) Glucose (EOF marker) Shikimate +NH4 +Na Succinate +NH4 +Na +K Lactate Glycerate Malate +NH4 +Na +K Isocitrate +NH4 +Na +K Pyroglutamate Citrate +NH4 +Na +K Fumarate Tartrate +NH4 +Na Cis-Aconitate +NH4 +Na PIPES

m/z 89/ 62/ 227/ 133/ 147/ 175/ 156/ 90/ 104/ 76/ 90/ 90/ 104/ 223/ 106/ 118/ 120/ 132/ 132/ 133/ 120/ 150/ 147/ 116/ 148/ 205/ 166/ 182/ 134/ 182/ 132/ 152/ 142/ 61/ 126/ 79/ 181/ 175/ 192/ 197/ 119/ 136/ 141/ 156/ 91/ 107/ 135/ 152/ 157/ 172/ 193/ 210/ 215/ 230/ 130/ 193/ 210/ 215/ 230/ 116/ 151/ 168/ 173/ 175/ 192/ 197/ 303/

relative migration time

87 0.417/ N.D. 60 0.557/ N.D. 225 0.590/ 0.582 131 0.594/ 0.586 145 0.603/ 0.591 173 0.620/ 0.610 154 0.636/ 0.622 88 0.637/ N.D. 102 0.665/ N.D. 74 0.718/ N.D. 88 0.778/ N.D. 88 0.812/ N.D. 102 0.822/ N.D. 221 0.847/ 0.837 104 0.850/ 0.844 116 0.851/ 0.845 118 0.856/ 0.852 130 0.866/ 0.865 130 0.876/ 0.865 131 0.887/ 0.879 118 0.890/ 0.884 148 0.907/ 0.901 145 0.911/ 0.901 114 0.912/ 0.902 146 0.925/ 0.911 203 0.927/ 0.923 164 0.936/ 0.924 180 0.955/ 0.944 132 0.969/ 0.960 180 1.000/ 1.000 130 1.004/ 1.009 150 1.032/ 1.026 140 1.575/ 1.625 59 1.641/ N.D. 124 1.695/ 1.771 77 1.710/ N.D. 179 N.D./ 1.780 173 N.D./ 1.782 1.710/ 1.710/ 117 N.D./ 1.802 1.724/ 1.724/ 1.724/ 89 N.D./ 1.827 105 N.D./ 1.860 133 N.D./ 1.878 1.787/ 1.78/ 1.787/ 191 1.796/ 1.889 1.796/ 1.796/ 1.796/ 128 1.804/ 1.897 191 1.849/ 1.947 1.849/ 1.849/ 1.849/ 114 N.D./ 2.001 149 N.D./ 2.010 1.897/ 1.897/ 173 N.D./ 2.022 1.902/ 1.902/ 301 1.952/ 2.084

% RSD of migration % RSD of area time (n ) 6) ratio (n ) 6) 0.31/ 0.31/ 0.35/ 0.31/ 0.29/ 0.32/ 0.36/ 0.32/ 0.41/ 0.42/ 0.38/ 0.40/ 0.39/ 0.44/ 0.41/ 0.41/ 0.42/ 0.44/ 0.44/ 0.44/ 0.45/ 0.46/ 0.46/ 0.43/ 0.44/ 0.46/ 0.42/ 0.47/ 0.50/ 0.35/ 0.54/ 0.53/ 0.81/ 0.82/ 0.82/ 0.83/ / / 0.83/ 0.83/ / 0.82/ 0.82/ 0.82/ / / / 0.84/ 0.84/ 0.84/ 0.86/ 0.86/ 0.86/ 0.86/ 0.85/ 0.90/ 0.90/ 0.90/ 0.90/ / / 0.91/ 0.91/ / 0.92/ 0.92/ 0.93/

0.44 0.27 0.62 0.53 0.83

0.73 0.64 0.59 0.49 0.73 0.80 0.68 0.69 0.60 0.55 0.68 0.52 0.56 0.61 0.54 0.47 0.61 0.55 0.44 0.74 0.88 0.74 0.51

0.53

0.47 0.51 0.57

0.40

0.43 0.67

0.70 0.58

0.71

0.76

4.7/ 2.0/ 5.2/ 4.0/ 4.3/ 4.2/ 4.7/ 4.8/ 4.3/ 2.5/ 2.7/ 4.4/ 3.5/ 3.5/ 2.7/ 3.4/ 4.2/ 5.0/ 1.9/ 1.8/ 2.9/ 3.0/ 2.3/ 3.8/ 3.5/ 3.9/ 2.9/ 4.1/ 4.7/ 4.3/ 1.7/ 2.7/ 5.4/ 9.5/ 7.9/ / / / 8.9/ 3.8/ / 6.1/ 5.4/ 5.6/ / / / 5.1/ 7.6/ 7.8/ 9.9/ 6.0/ 9.0/ 8.7/ 3.6/ 3.8/ 7.7/ 4.1/ 4.6/ / / 13.7/ 12.3/ / 7.2/ 6.0/ 9.7/

7.1 3.1 1.6 6.6 9.9

7.1 2.4 7.4 6.4 8.9 9.6 7.6 9.1 8.0 8.8 9.2 9.2 8.7 9.8 6.0 4.1 4.0 6.3 10.0 6.0 5.8

9.6

7.4

9.1 7.5 6.3

8.4

6.8 8.6

9.6 9.2

7.7

8.3

linearity LOD (µM) 2.5/ 1.0/ 0.5/ 0.5/ 1.0/ 2.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 1.0/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 0.5/ 1.0/ 1.0/ 0.5/ 0.5/ 0.5/ 1.0/ 0.5/ 1.0/ 5.0/ 1.0/ / / / 50.0/ 200.0/ / 100.0/ 5.0/ 200.0/ / / / 5.0/ 200.0/ 200.0/ 200.0/ 5.0/ 200.0/ 200.0/ 25.0/ 200.0/ 5.0/ 200.0/ 200.0/ / / 200.0/ 100.0/ / 2.5/ 200.0/ 200.0/

correlation (R2, n ) 5)

0.988/ 0.994/ 2.5 0.997/ 5.0 0.993/ 2.5 0.991/ 0.5 0.995/ 0.5 0.995/ 0.996/ 0.995/ 0.995/ 0.994/ 0.995/ 0.997/ 0.5 0.996/ 0.5 0.995/ 2.5 0.997/ 0.5 0.997/ 0.5 0.993/ 2.5 0.997/ 2.5 0.985/ 0.5 0.993/ 1.0 0.997/ 2.5 0.996/ 2.5 0.995/ 0.5 0.994/ 0.5 0.994/ 0.5 0.996/ 0.5 0.995/ 0.5 0.994/ 0.5 / 2.5 0.995/ 5.0 0.996/ 0.5 0.995/ 0.992/ 0.5 0.959/ / / 2.5 / / / 2.5 / / 0.987/ / 2.5 / 0.5 / 2.5 / 0.942/ / / 2.5 / 0.898/ / / 2.5 / 2.5 / 0.982/ / / 10.0 / 2.5 / / / 5.0 / 0.972/ / 2.5 /

0.998 0.978 0.986 0.982 0.981

0.984 0.985 0.983 0.985 0.989 0.981 0.994 0.982 0.991 0.984 0.983 0.980 0.989 0.981 0.987 0.981 0.980 0.981 0.991 0.986

0.986

0.983

0.982 0.983 0.985

0.987

0.993 0.982

0.990 0.996

0.981

range (µM) 5-200/ 2.5-200/ 1-200/ 1-200/ 2.5-200/ 5-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 2.5-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 1-200/ 2.5-200/ 2.5-200/ 1-200/ 1-200/ / 2.5-200/ 1-200/ 2.5-200/ 10-200/ 2.5-200/ / / / / / / / 10-200/ / / / / 10-200/ / / / 10-200/ / / / / 10-200/ / / / / / / / 5-200/ / /

5-200 10-200 5-200 1-200 1-200

1-200 1-200 5-200 1-200 1-200 5-200 5-200 1-200 2.5-200 5-200 5-200 1-200 1-200 1-200 1-200 1-200 5-200 10-200 1-200 1-200

5-200

5-200

5-200 1-200 5-200

5-200

5-200 5-200

25-200 5-200

10-200

a

Each left side value indicates the value for positive mode, and each right side value indicates the value for negative mode. N.D., Not detected.


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Table 2. Performance of ESI Polarity Changing Mode CE-MS linearity compounds

m/z

% RSD of migration time (n ) 6)

N, N-diaminobutane Ethanolamine Carnosine Ornithine Lysine Arginine Histidine β-alanine γ-aminobutyrate Glycine Alanine Sarcosine R-aminobutyrate Cystathionine Serine Valine Homoserine Isoleucine Leucine Asparagine Threonine Methionine Glutamine Proline Glutamate Tryptophan Phenylalanine Tyrosine Aspartate Methionine sulfone Hydroxyproline Phenylglycine Phosphoethanolamine Urea Taurine DMSO (EOF marker) Glucose (EOF marker) Shikimate Succinate Lactate Glycerate Malate Isocitrate Pyroglutamate Citrate Fumarate Tartrate Cis-Aconitate PIPES

89 62 227 133 147 175 156 90 104 76 90 90 104 223 106 118 120 132 132 133 120 150 147 116 148 205 166 182 134 182 132 152 140 59 124 77 179 173 117 89 105 133 191 128 191 114 149 173 301

0.43 0.56 0.57 0.59 0.68 0.74 0.66 0.68 0.63 0.62 0.72 0.75 0.74 0.69 0.65 0.61 0.57 0.73 0.71 0.69 0.66 0.71 0.66 0.83 0.81 0.81 0.76 0.75 0.74 0.71 0.81 0.71 0.53 N.D. 0.69 N.D. 0.66 0.67 0.62 0.51 0.70 0.57 0.51 0.50 0.50 0.57 0.55 0.56 0.55

voltage. As the electrophoretic voltage also affects the ESI voltage and polarity, the stopping of electrophoresis is required in our instruments. After stabilization of the electrophoretic voltage, a negatively charged ESI voltage was applied. To achieve stable CE separation, the total voltage of electrophoresis was maintained at the same value during both positive and negative ion modes of ESI-MS detection. Extraction from Pineapple Leaf Samples. Pineapples (Ananas comosus) were grown in pots with a light-dark cycle of 14 h (06:00-20:00) at 25 °C and 10 h (20:00-06:00) at 20 °C. Mature leaves were collected and stored in liquid nitrogen at 06:00, 11: 00, 16:00, and 22:30, respectively. In each time points, six individual samples were used for analysis. The samples were extracted using the method described by Sato et al.5 Leaves (50 mg fresh weight) were ground using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) at 1800 rpm for 20 s with cooling, and were extracted using 9970


% RSD of area ratio (n ) 6)

LOD (µM)

correlation (R2, n ) 5)

Range (µM)

ESI polarity

9.8 6.9 9.8 8.4 7.9 4.4 5.5 4.7 9.0 5.6 5.8 5.7 3.7 7.3 7.8 5.7 4.9 10.2 9.3 2.9 5.9 4.4 8.5 6.5 4.8 2.9 4.4 5.9 4.2 3.5 4.9 3.2 5.8

1.0 1.0 0.5 1.0 1.0 1.0 0.5 1.0 1.0 1.0 0.5 0.5 0.5 0.5 1.0 0.5 0.5 0.5 0.5 1.0 0.5 0.5 1.0 1.0 0.5 0.5 0.5 1.0 1.0 0.5 1.0 0.5 0.5

0.992 0.985 0.994 0.993 0.999 0.997 0.999 0.982 0.998 0.994 0.995 0.986 0.999 0.990 0.989 0.993 0.989 0.995 0.983 0.996 0.984 0.982 0.997 0.998 0.991 0.996 0.993 0.998 0.998

2.5-200 2.5-200 1-200 2.5-200 2.5-200 2.5-200 1-200 2.5-200 2.5-200 2.5-200 1-200 1-200 1-200 1-200 2.5-200 1-200 1-200 1-200 1-200 2.5-200 1-200 1-200 2.5-200 2.5-200 1-200 1-200 1-200 2.5-200 2.5-200

0.999 0.982 0.987

2.5-200 1-200 1-200

8.5

0.5

0.993

1-200

6.3 4.2 9.4 7.4 8.3 5.7 9.8 8.7 2.9 3.5 10.6 4.3

2.5 0.5 2.5 2.5 2.5 2.5 0.5 2.5 10.0 2.5 1.0 2.5

0.988 0.991 0.981 0.992 0.988 0.996 0.988 0.993 0.989 0.999 0.985

5-200 1-200 5-200 5-200 5-200 5-200 1-200 5-200 25-200 5-200 2.5-200

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

250 µL of methanol and 250 µL of water (including internal standard) with vortex mixing. The supernatant was transferred to a 5 kDa ultrafilter (Millipore, Bedford, MA), and then centrifuged at 12 000g for 40 min and the filtrate was analyzed by CE-MS.

RESULTS AND DISCUSSION CE-MS Separation in Different Acidic Electrolytes. In CE, the choice of electrolyte strongly affects the separation pattern. Thus, we first investigated the possibility of the separation of both amino acids and carboxylic acids using various concentrations of formic acids and acetic acids as the electrolyte. As the concentrations of more than 1 M cause the CE current to exceed 40 µA at 26.5 kV m-1 CE run, we analyzed the electrolyte at concentrations below 1 M.

Figure 1. Apparent mobilities (A) and effective mobilities (B) of amino acids and carboxylic acids at various concentrations of formic acid electrolyte. Conditions: electrolyte, various concentrations of formic acid; sheath liquid, 50% methanol at 10 µL min-1; sheath gas pressure, 20 psi; applied voltage, 26.5 kV m-1.

With formic acid electrolytes, rough separations of both amino acids and carboxylic acids were achieved at concentrations above 10 mM. From 10 to 250 mM of formic acid, the apparent mobilities of most analytes were decreased (Figure 1A), while the separation of neighboring peaks of neutral and acidic amino acids became possible. Between 100 and 250 mM of formic acid, current drop during separation frequently occurred. The lowest apparent mobility was observed around 250 mM of formic acid. Above this concentration, the apparent mobility was increased. Good separation of neighboring peaks, such as isocitrate and citrate (Supporting Information Figures S1, S2), and isoleucine and leucine was observed in higher concentrations of electrolytes, especially in 1 M formic acid. With acetic acid electrolytes, rough separations were achieved above 50 mM. By increasing the concentration to 1 M, apparent mobilities were decreased in all the analytes as the apparent mobilities changed patterns in 10 to 250 mM of formic acid. As good separation of neighboring peaks was not observed below 1 M of acetic acid, we chose 1 M formic acid as the best electrolyte for the simultaneous analysis of both amino acids and carboxylic acids. Increasing the concentration of the electrolyte increases the ionic strength of electrolyte, and in the case of acidic electrolyte, the pH becomes lower. In most studies on ionic strength effects on separations, pH > 2 has been used for analysis.22,23 Above pH

) 2, the silanol of the capillary surface is negatively charged.24 In general, when the ionic strength of the separation buffer increases, the thickness of the ionic double layer also increases, causing the decrease of the EOF and, hence, the increase of the analysis time.21 In our analysis, decreases in apparent mobility were observed in 10 to 250 mM of formic acid (Figure 1A) and in 50 mM to 1 M of acetic acid. These concentrations of electrolytes were pH > 2; thus, this phenomenon was a general characteristic of ionic strength. The effective electrophoretic mobilities were changed in these conditions (Figure 1B). This indicates the existence of some dissociated compounds. Hence, in 100 and 250 mM of formic acid, current drop was often observed. Moreover, in 250 mM to 1 M of formic acid, the apparent mobility was increased by the order of formic acid concentration increase (Figure 1A). The effective electrophoretic mobilities were stable in 250 mM to 1 M of formic acid (Figure 1B). Under these conditions, the pH was nearly 2.5 or less. Thus, the silanol of the capillary surface was not, or was rarely, charged, and the dissociated molecules, including formic acid, were restricted in the order of acidic electrolyte concentration increase. The ionic strength effect under a low pH state was not fully studied, but these alterations cause the apparent mobility increase in 250 mM to 1 M of formic acid. CE-MS Separation under Various Sheath Gas Pressures. For a 1 M formic acid electrolyte with a 50% methanol sheath liquid at 10 µL min-1 flow, the sheath gas pressure affects the

(22) Koval, D.; Kasicka, V.; Zuskova´, I. Electrophoresis 2005, 26, 3221–3231. (23) Allison, S. A.; Pei, H.; Baek, S.; Brown, J.; Lee, M. Y.; Nguyen, V.; Twahir, U. T.; Wu, H. Electrophoresis 2010, 31, 920–932.

(24) Li, S. Capillary Electrophoresis: Principles, Practice, And Applications; Journal of Chromatography Library; Elsevier Science Ltd: Amsterdam, Netherlands, 1992; pp 6-9.


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Figure 2. Apparent mobilities (A) and effective mobilities (B) of amino acids and carboxylic acids under various sheath gas pressures. Conditions: electrolyte, 1 M formic acid; sheath liquid, 50% methanol at 10 µL min-1 flow; applied voltage, 26.5 kV m-1.

Figure 3. Apparent mobilities (A) and effective mobilities (B) of amino acids and carboxylic acids in various sheath liquid flows. Conditions: electrolyte, 1 M formic acid; sheath liquid, 50% methanol at various flows; sheath gas pressure, 20 psi; applied voltage, 26.5 kV m-1.

apparent mobility of all analytes (Figure 2A). For both the amino acids and carboxylic acids, the apparent mobility was accelerated by an increase in sheath gas pressure, especially above 10 psi. 9972


This indicates that the sheath gas is a strong acceleration factor in the simultaneous analysis of amino acids and carboxylic acids in an acidic electrolyte, and can reduce the migration times of

Figure 4. Positive mode CE-MS electropherogram (m/z ) 50-370 and total ion current (TIC)) for standard mixture of amino acids and carboxylic acids. Each standard contained 200 µmol L-1. Conditions: electrolyte, 1 M formic acid; sheath liquid, 50% methanol at 10 µL min-1; sheath gas pressure, 25 psi; applied voltage, 26.5 kV m-1.

anionic carboxylic acids species. However, a higher sheath gas pressure (above 30 psi) also caused a decline in the signal/noise (S/N) ratio and bad separation of neighboring peaks. Thus, we chose 20-25 psi of sheath gas pressure as the best choice in our analysis. Despite the apparent mobility being affected by sheath gas (Figure 2A), the effective electrophoretic mobility was not affected by sheath gas (Figure 2B). EOF and effective electrophoretic mobility depend upon the separation voltage. Sheath gas pressure is not voltage dependent and has a similar mobility of pressure assistance. Below a sheath gas condition of 15 psi, we confirmed that the assistance of 0.5 psi pressure was equivalent to a sheath gas changing of 15 psi in positive ion mode detection of normal directional electrophoresis. However, in negative ion mode, sheath gas was required for stable electrophoresis and ionization. Especially, with a low sheath gas pressure (below 5 psi), electrophoretic voltage could not be applied. Pressure assistance consumes the lifetime of the CE pump. Therefore, sheath gas control is advantageous in term of pressure assistance control from the

CE inlet side because sheath gas is essential for ionization, and enhances apparent mobilities. CE-MS Separation in Various Sheath Liquids. The sheath liquid composition influences ionization efficiency in CE-MS. In particular, an ammonium-containing sheath liquid made ammonium adducts at high intensities in positive ion mode. The volatile alcohols with low concentrations of acids, bases, or salts have been often used in previous studies.10-18 However, in our analysis, 50% methanol showed better signal intensity than 5 mM formic acid containing sheath liquid. Because the sheath liquid met 1 M formic acid electrolyte at the endmost of the capillary, 50% methanol was enough for ionizations. Although we tried to apply 50% 2-propanol sheath liquid, the high background signal was observed in positive mode. The effect of sheath liquid composition on migration time has been reported limitedly.25 We analyzed the effects of the sheath liquid flow rate on mobility by using 50% methanol sheath liquid. (25) Lin, X.; Gerardi, A. R.; Breitbach, Z. S.; Armstrong, D. W.; Colyer, C. L. Electrophoresis 2009, 30, 3918–3925.


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Figure 5. Negative mode CE-MS electropherogram (m/z ) 50-370 and total ion current (TIC)) for standard mixture of amino acids and carboxylic acids. Each standard contained 200 µmol L-1. Conditions: electrolyte, 1 M formic acid; sheath liquid, 50% methanol at 10 µL min-1; sheath gas pressure, 25 psi; applied voltage, 33.0 kV m-1.

Even at different flow rates, the changes in sheath liquid showed no obvious effect on apparent mobilities (Figure 3A), and also no effect on effective electrophoretic mobilities (Figure 3B). We optimized the sheath liquid flow rate at 10 µL min-1, as demonstrated in the previous study,8 because lower flow rates (

Simultaneous Analysis of Amino Acids and Carboxylic Acids by

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