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A novel two-dimensional separation method, which hy- phenated chromatography and electrophoresis, was de- veloped for analysis of Bacillus subtilis ...
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Two-Dimensional Separation Method for Analysis of Bacillus subtilis Metabolites via Hyphenation of Micro-Liquid Chromatography and Capillary Electrophoresis Li Jia,*,† Bi-Feng Liu,† Shigeru Terabe,† and Takaaki Nishioka‡

Graduate School of Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan, and Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

A novel two-dimensional separation method, which hyphenated chromatography and electrophoresis, was developed for analysis of Bacillus subtilis metabolites. Micro-liquid chromatography (LC) with a monolithic silica-ODS column was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Concentration strategies, namely, dynamic pH junction and sweeping, were selectively employed to interface the two dimensions, which proved to be beneficial for the detection of metabolites. For system evaluation, an artificial sample containing 54 standard metabolites was separated according to their hydrophobicity by micro-LC with gradient mode. The early-eluting fractions were separated by capillary zone electrophoresis in combination with dynamic pH junction, while the late-eluting fractions were separated by sweeping micellar electrokinetic chromatography. The middle fractions were analyzed by both modes of CE. Under the optimum conditions, all the components in the artificial sample could be well resolved. The method was applied to profile B. subtilis metabolites. Some crucial metabolites were identified. This method provided great potential for resolving complex biological samples containing compounds having different characteristics. The metabolome refers to the entire complement of metabolites that are related to the genome. Metabolomics can be defined as a comprehensive analysis of metabolites present in a living cell.1 Since the metabolome can reveal the connection of biochemical networks and provide a system-level understanding of the cell, it has attracted increasing attention in the biological field. Compared to the genome, the metabolome is dynamic and highly variable with cell types, genes, environment, and history. In addition, the fact that more than 1000 metabolites exist in the cell complicates * To whom correspondence should be addressed. Tel: +81-791-58-0173. Fax: +81-791-58-0493. E-mail: [email protected]. † Himeji Institute of Technology. ‡ Kyoto University. (1) Raamsdonk, L. M.; Teusink, B.; Broadhurst, D.; Zhang, N.; Hayes, A.; Walsh, M. C.; Berden, J. A.; Brindle, K. M.; Kell, D. B.; Rowland, J. J.; Westerhoff, H. V.; Van Dam, K.; Oliver, S. G. Nat. Biotechnol. 2001, 19, 45-50. 10.1021/ac035039b CCC: $27.50 Published on Web 01/20/2004

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the analysis.2,3 Hence, metabolome analysis has become a new challenge for separation scientists with the advent of the postgenome era. Because of the large number and low concentration of many intracellular metabolites and the changes in their concentrations with environment and cell history, it is impossible to analyze the intracellular metabolite profile in one run using a single chromatographic or electrophoretic technique. A major point is that a single-dimensional separation method has limited peak capacity. According to the mathematical model introduced by Giddings,4 the peak capacity of a multidimensional separation system is the product of the peak capacity of its components. Therefore, a multidimensional separation system is a more practical choice in order to separate as many metabolites as possible. Felinger discussed the problems of peak resolution and overlap by means of statistical methods and by using Fourier analysis of the complex chromatogram, where one separates complex mixtures in singleand multiple-dimensional separations.5 As many intracellular metabolites are not volatile, gas chromatography is not suitable for the comprehensive analysis of these metabolites without derivatization. Liquid chromatography (LC) and capillary electrophoresis (CE) are rational choices. To date, the liquid-phase multidimensional separation systems include LC-LC, CE-CE, and LC-CE, most of which were applied to proteome analysis, as reviewed by Issaq and Wang et al.6,7 The LC-CE multidimensional separation system has two modes, on-line and off-line, each of which has its advantages and limitations. Lemmo et al. reported on-line coupling microcolumn size exclusion chromatography/ reversed-phase micro-LC with capillary zone electrophoresis (CZE) to resolve protein mixtures8 and fluorescein 5-isothiocyanate derivatized amino acids and amine samples.9 On-line mode is fast and high throughput. But it requires that the second (2) Soga, T.; Ueno, Y.; Naraoka, H.; Ohashi, Y.; Tomita, M.; Nishioka, T. Anal. Chem. 2002, 74, 2233-2239. (3) Terabe, S.; Markuszewski, M. J.; Inoue, N.; Otsuka, K.; Nishioka, T. Pure Appl. Chem. 2001, 73, 1563-1572. (4) Giddings, J. C. J. High Resolut. Chromatogr. 1987, 10, 319-323. (5) Felinger, A. Data Analysis and Signal Processing in Chromatography; Elsevier: Amsterdam, 1998. (6) Issaq, H. J. Electrophoresis 2001, 22, 3629-3638. (7) Wang, H.; Hanash, S. J. Chromatogr., B 2003, 787, 11-18. (8) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (9) Hooker, T. F.; Jorgenson, J. W. Anal. Chem. 1997, 69, 4134-4142.

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dimension (CE) must be much faster than the first dimension (LC). A number of papers on off-line combination of LC and CE have been published in the past.10-22 Off-line mode has the following advantages: simplicity and ease of performance, no limitation on column type and size, and commercial availability of instrument. However, it is time-consuming because many runs are required to analyze the fractions of effluent from LC by CE using a single capillary. Issaq et al.17 and He et al.20 employed an off-line combining RPLC with multiplex-CE method to separate protein digests and cancer cell extracts, which shortened the entire CE analysis time. Although Issaq’s research group reported two-dimensional mapping of cancer cell extracts by LC-CE with UV absorbance detection,20 the experimental conditions used mainly focused on analyzing proteins, not intracellular metabolites, and peaks were not identified. Peptides, proteins, or their digests are hydrophilic and have similar characteristics, which makes the development of a LC-CE method less difficult. CE suffers from low concentration sensitivity due to a minute sample volume and limited optical path length for on-capillary UV photometric detection, which is the most widely used detection mode. To enhance the concentration sensitivity in CE, several online sample preconcentration techniques, such as stacking,23-26 dynamic pH junction,27,28 and sweeping,29-32 have been reported. Using different on-line preconcentration techniques, from ten to more than several hundred thousand-fold improvements in sensitivity is obtained. An advantage of dynamic pH junction and sweeping is that sample matrix can contain relatively high concentrations of electrolytes because low conductivity is not required for the sample matrix. In this paper, dynamic pH junction and sweeping were utilized as concentration techniques in CZE and micellar electrokinetic chromatography (MEKC), respectively. (10) Janssen, P. S. L.; Van Nispen, J. W.; Van Zeeland, M. J. M.; Melgers, P. A. T. A. J. Chromatogr., A 1989, 470, 171-183. (11) Frenz, J.; Wu, S.; Hancock, W. S. J. Chromatogr., A 1989, 480, 379-391. (12) Castagnola, M.; Cassiana, L.; Rabino, R.; Rossetti, D. V. J. Chromatogr., A 1991, 572, 51-58. (13) Rudnick, S. E.; Hilser, V. J.; Worosila, G. D. J. Chromatogr., A 1994, 672, 219-229. (14) Boss, H. J.; Watson, D. B.; Rush, R. S. Electrophoresis 1998, 19, 26542664. (15) Voelter, W.; Schutz, J.; Tsitsiloni, O. E.; Weiler, A.; Grubler, G.; Paulus, G.; Stoeva, S.; Lehmann, R. J. Chromatogr., A 1998, 807, 135-149. (16) Issaq, H. J.; Chan, K. C.; Janini, G. M.; Muschik, G. M. Electrophresis 1999, 20, 1533-1537. (17) Issaq, H. J.; Chan, K. C.; Liu, C.; Li, Q. Electrophoresis 2001, 22, 11331135. (18) Shen, Y.; Berger, S. J.; Smith, R. D. J. Chromatogr., A 2001, 914, 257-264. (19) Sanz-Nebot, V.; Benavente, F.; Barbosa, J. J. Chromatogr., A 2002, 950, 99-111. (20) He, Y.; Yeung, E. S.; Chan, K. C.; Issaq, H. J. J. Chromatogr., A 2002, 979, 81-89. (21) Bergh, G. V. D.; Clerens, S.; Vandesande, F.; Arckens, L. Electrophoresis 2003, 24, 1471-1481. (22) Udiavar, S.; Apffel, A.; Cakel, J.; Swedberg, S.; Hancock, W. S.; Pungor, E., Jr. Anal. Chem. 1998, 70, 3572-3578. (23) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, P. E. M. J. Chromatogr. 1979, 169, 1-10. (24) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (25) Liu, Z.; Sam, P.; Sirimanne, S. R.; McClure, P. C.; Grainger, J.; Patterson, D. G. J. Chromatogr., A 1994, 673, 125-132. (26) Shihabi, Z. K. Electrophoresis 2000, 21, 2872-2878. (27) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (28) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729-1735. (29) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (30) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (31) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-1687. (32) So, T. S. K.; Jia, L.; Huie, C. W. Electrophoresis 2001, 22, 2159-2166.

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The determination of specific or a group of compounds in Bacillus subtilis metabolites has been reported in the past.2,33-37 Soga et al. developed several CE-MS methods for analysis of anionic intermediates in the B. subtilis cell.2,34,35 Dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection was applied to analyze trace amounts of flavin derivatives in B. subtilis at picomolar concentrations.33 Pyridine and adenine nucleotide metabolites in B. subtilis cell extract were determined by sweeping borate complexation CE.36 Using 2,6-pyridinedicarboxylic acid as a carrier electrolyte and with electrokinetic injection mode, carboxylic acids metabolites from the tricarboxylic acid cycle in the B. subtilis cell were analyzed by CE with UV detection.37 For better understanding of the cellular process in relation to the genome and proteome and providing direct information on metabolic phenotypes, it is necessary to analyze the intracellular metabolite profile. With the trend in analytical chemistry toward miniaturization, microseparation systems have become hot research fields in separation sciences. Micro-LC offers a useful tool for fast, economic analysis because of the very small inner diameter of separation columns, low flow rates, and low injection volumes compared with conventional LC. To meet the requirement for a micro-LC column, one type of special column, i.e., a monolithic column, has been developed recently, which can overcome some inherent limitations possessed by conventional commercial packed columns, such as low permeability and large volume of mobile phase.38-45 The major problems confronting separation scientists in the metabolome analysis are limited separation efficiency and detection sensitivity. In this paper, micro-LC and CE, which operated on different separation mechanisms, were combined to enhance the separation efficiency. Two on-line sample preconcentration techniques, dynamic pH junction and sweeping, were utilized to interface reversed-phase micro-LC with CZE and MEKC to improve the detection sensitivity. The classification of major metabolites in a cell according to the chemical structures was listed in our previous work.3 Several classes of important metabolite standards (including purine and pyrimidine bases, ribonucleosides, nucleotides, aromatic acids, flavin and folate derivatives, and steroids) were chosen as model compounds to optimize the micro-LC and CE conditions. There(33) Britz-McKibbin, P.; Markuszewski, M. J.; Iyanagi, T.; Matsuda, K.; Nishioka, T.; Terabe, S. Anal. Biochem. 2003, 313, 89-96. (34) Soga, T.; Ueno, Y.; Naraoka, H.; Matsuda, K.; Tomita, M.; Nishioka, T. Anal. Chem. 2002, 74, 6224-6229. (35) Soga, T.; Ohashi, Y.; Ueno, Y.; Naraoka, H.; Tomita, M.; Nishioka, T. J. Proteome Res. 2003, 2, 488-494. (36) Markuszewski, M. J.; Britz-McKibbin, P.; Terabe, S.; Matsuda, K.; Nishioka, T. J. Chromatogr., A 2003, 989, 293-301. (37) Markuszewski, M. J.; Otsuka, K.; Terabe, S.; Matsuda, K.; Nishioka, T. J. Chromatogr., A 2003, 1010, 113-121. (38) Svec, F.; Frechet, J. M. J. Anal. Chem. 1992, 64, 820-822. (39) Fields, S. M. Anal. Chem. 1996, 68, 2709-2712. (40) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (41) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 73, 420A-429 A. (42) Tanaka, N.; Kobayashi, H. J. Chromatogr. Libr. 2001, 62, 165-181. (43) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hosoya, K.; Ikegami, T. J. Chromatogr., A 2002, 965, 35-49. (44) Zou, H.; Huang, X.; Ye, M.; Luo, Q. J. Chromatogr., A 2002, 954, 5-32. (45) Legido-Quigley, C.; Marlin, N. D.; Melin, V.; Manz, A.; Smith, N. W. Electrophoresis 2003, 24, 917-944.

after, the two separation techniques were hyphenated. B. subtilis cell extract was first separated based on their hydrophobicity using a monolithic silica-ODS column and a laboratory-assembled microLC apparatus operated in the gradient elution mode. The earlyeluting fractions were separated by dynamic pH junction CZE based on their charge-to-size ratios, while the late-eluting fractions were separated by sweeping MEKC based on their hydrophobicity. The middle fractions were analyzed using both modes of CE. Some important metabolites in the B. subtilis cell were identified. EXPERIMENTAL SECTION Apparatus and Procedure. The micro-LC system was set up in our laboratory, which consisted of a microflow pump (GL Sciences MP681, Tokyo, Japan), a UV detector (Jasco CE 970, Tokyo, Japan), and a microbore HPLC injection valve (Valco VICI). Data were acquired by a data acquisition board (National Instrument) at a sampling rate of 50 Hz and a program written in Labview 6.0. A monolithic silica-ODS column (250 × 0.2 mm i.d.) was obtained from GL Sciences. The volume of the sample loop was 160 nL. Gradient elution was performed with methanol-30 mM phosphate buffer (pH 3.0) at a flow rate of 2 µL/min. The gradient started at 5% methanol, which was maintained isocratically for the first 5 min; thereafter, the methanol concentration was raised linearly to 95% in 15 min and kept at 95% for 10 min. Detection wavelength was set at 210 nm. All the CE experiments were performed on a Beckman P/ACE MDQ capillary electrophoresis system (Fullerton, CA) equipped with a photodiode array UV detector. Uncoated capillaries (Polymicro Technologies, Phoenix, AZ) with 75-µm i.d., 60.5-cm total length (50-cm effective length) and 50-µm i.d., 60.5-cm total length (50-cm effective length) were used in dynamic pH junction CZE and sweeping MEKC, respectively. New capillaries were first rinsed with 1.0 M NaOH (30 min), followed by methanol (30 min), deionized water (60 min), and finally with the background electrolyte (60 min) by applying pressure (140 kPa). For conditioning, each separation was preceded by a 2-min rinse with 0.1 M NaOH, followed by a 3-min rinse with the background electrolyte. The temperature of the capillary cartridge was maintained at 20 °C. All injections were made hydrodynamically with a pressure of 3.5 kPa from 3 to 720 s. In dynamic pH junction CZE, the applied voltage was set at 18 kV. In sweeping MEKC, the applied voltage was set at -25 kV. The UV wavelength range was set from 190 to 300 nm for detection. A sample solution was injected into the micro-LC system, and the fractions of effluent from the column were collected using 150-µL vials every minute (2 µL/vial). After collection, the vials were placed in a microcentrifugal vacuum concentrator MV-100 and dried at room temperature under vacuum. Then the sample in each vial was reconstituted with 10 µL of 50 mM phosphoric acid or 75 mM sodium phosphate (pH 6.0) before CE analysis. Reagents and Samples. Steroid standards, which included estrone (ES), 17-β-estradiol (ED), estriol (ET), testosterone (T), 4-androstene-3,17-dione (AS), progesterone (PG), 17-R-hydroxyprogesterone (HPG), cortisone (C), and hydrocortisone (HC), were purchased from Nacalai Tesque (Kyoto, Japan). Stock solutions of each steroid were prepared as 1 mg/mL in methanol and stored at 4 °C prior to use. Aromatic acid standards, which included nicotinic acid (NA), benzoic acid (BA), 4-hydroxybenzoic acid (HBA), protocatechuic

acid (PA), trans-cinnamic acid (CA), 4-hydroxyphenylacetic acid (HPAA), 4-hydroxyphenylpyruvic acid (HPPA), 3,4-dihydroxyphenylacetic acid (DHPAA), phenylpyruvic acid (PPA), and anthranilic acid (AA), were purchased from Wako (Osaka, Japan). Stock solutions of each aromatic acid were prepared as 2 mg/mL in 80% (v/v) methanol-water and stored at 4 °C prior to use. Purine bases (adenine, guanine, xanthine, hypoxanthine, caffeine, theophylline, theobromine, uric acid) and pyrimidine bases (cytosine, thymine, uracil) were purchased from Wako. Stock solutions of each analyte were prepared as 1 mg/mL in deionized water and stored at 4 °C prior to use. Ribonucleosides and nucleotides, including adenosine and its mono-, di-, and triphosphates (A, AMP, ADP, ATP), guanosine and its mono-, di-, and triphosphates (G, GMP, GDP, GTP), uridine and its mono-, di-, and triphosphates (U, UMP, UDP, UTP), inosine and its mono- and triphosphates (I, IMP, ITP), and cytidine and its monophosphate (C, CMP), were purchased from Wako. Stock solutions of each analyte were prepared as 2 mg/mL in deionized water and stored at -20 °C prior to use. Two coenzymes, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), were purchased from Sigma (St. Louis, MO). Stock solutions of standards were prepared as 2 mg/mL in deionized water and stored at -20 °C. Riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) were purchased from Wako, Sigma, and TCI Chemicals (Tokyo, Japan), respectively. FMN and FAD stock solutions were prepared as 2 mg/mL in 50% methanol-water. RF stock solutions were prepared as 1 mg/mL in deionized water. The stock solutions were stored at 4 °C. Folic acid (FA) and 5-methyl-5,6,7,8-tetrahydrofolate disodium salt (5methyl THF) were purchased from Sigma. Stock solutions of each standard were prepared as 1 mg/mL in 10 mM potassium dihydrogen phosphate solution. The stock solutions were stored at -20 °C prior to use. In dynamic pH junction CZE, the running buffer consisted of 160 mM borate, whose pH was adjusted by using concentrated NaOH or 160 mM boric acid. In sweeping MEKC, the running buffer was composed of 50 mM sodium dodecyl sulfate (SDS), 50 mM phosphoric acid, and 20% acetonitrile or methanol. Water used for buffer and sample preparations was purified using a Milli-Q water purification system (Millipore). B. subtilis Cell Culture. Bacteria B. subtilis was cultured as described in our previous work.36,37 Briefly, the B. subtilis strain 168 (trpC2, laboratory stock) was cultured in 100 mL of S6-malate medium at 37 °C by shaking. Growth was monitored by measuring optical density at 660 nm. As the number of bacteria increased with the duration of culture, the growth was discontinued when the cell concentration reached ∼4 × 108 living cells/mL. Then 10 mL of culture medium was withdrawn and passed through a 0.45-µm-pore size filter. Residual B. subtilis cells on the filter were washed with deionized water to prevent contamination of S6-malate medium and stored at -80 °C until extraction. Metabolite Extraction. The metabolite extraction procedure was similar to the method described previously with small modification.36,37 Briefly, cells on the filter were plunged into 2 mL of ice-cooled methanol, incubated at room temperature for 10 min, and then placed at -20 °C for 30 min. The methanol solution was transferred to a 15-mL plastic tube. Chloroform (1.6 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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mL) and deionized water (640 µL) were added to the methanol solution and mixed thoroughly for ∼30 s to remove phospholipids liberated from the cell membrane. After 10 min, the solution was centrifuged at 4 °C and 3000 rpm for 25 min. Then the upper layer was withdrawn and centrifugally filtered through a Millipore 5-kDa-cutoff filter to remove proteins and other debris. The filtrate was evaporated using a vacuum centrifuge at room temperature. Prior to analysis, the dried sample was reconstituted in 50 µL of deionized water. RESULTS AND DISCUSSION Micro-LC Conditions. Because of the complexity of B. subtilis cell metabolites, it is better to separate as many compounds as possible by the first-dimensional micro-LC in order to make the separation by the second-dimensional CE much easier. RPLC is one of the most popular modes in LC although its peak capacity is insufficient to resolve all compounds in very complex samples, such as cell metabolite extract. In this paper, a monolithic silica column with inner diameter of 0.2 mm was modified using ODS. So RPLC mode was selected to separate different types of metabolites. As stated by Vissers et al.,46 for micro-LC, the volume of the sample loop is not a critical parameter as long as on-column focusing techniques are applied, but it affects column lifetime and stability. In this paper, the gradient mode, which has an on-column concentration effect, was employed. To maintain the chromatographic efficiency and resolution, a 160-nL sample loop was used. The reproducibilities of the micro-LC system in isocratic and gradient modes were investigated. Six alkylbenzenes (methylbenzene, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene) were used as samples. In isocratic mode, when 80% acetonitrile-water at a flow rate of 2 µL/min was used as mobile phase, the relative standard deviations (RSD) of migration time, peak area, and peak height of the six alkylbenzens were all less than 3% (n ) 4). In gradient mode, when the gradient started at 60% acetonitrile-water and the acetonitrile concentration was raised linearly to 100% in 15 min and kept at 100% for 5 min and the flow rate was 2 µL/min, the RSDs of migration time, peak area, and peak height of the six alkylbenzens were all less than 5% (n ) 4). In the system, cross-capillary UV illumination was used for UV detection. The inner and outer diameters of the capillary are 50 and 362 µm, respectively. In isocratic mode, the baseline was flat. In gradient mode, the baseline drift was encountered since the shape of the detector cell was cylindrical and there existed a refractive index gradient in the axial direction of the capillary resulting from the concentration gradient. By optimizing the pH of the phosphate buffer mobile phase, the type of organic solvents, and gradient program, 30 mM phosphate buffer (pH 3) and methanol were used as mobile phases. The gradient program started at 5% methanol, which was maintained isocratically for the first 5 min; thereafter, the methanol concentration was raised linearly to 95% in 15 min and kept at 95% for 10 min. Figure 1 shows a series of chromatograms depicting the separation of different types of metabolites under the given conditions. The bases, ribonucleosides, and nucleotides are strongly hydrophilic and were almost not retained onto the monolithic silica-ODS (46) Vissers, J. P. C.; de Ru, A. H.; Ursem, M.; Chervet, J. P. J. Chromatogr., A 1996, 746, 1-7.

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column, resulting in their early elution. Aromatic acids, flavin, and folate derivatives all contain carboxyl groups. The pH of the mobile phase affected the dissociation of carboxyl groups, resulting in affecting the hydrophobicity of the compounds having carboxyl groups. When the pH of the phosphate buffer was 3, these compounds were baseline separated under the given gradient program. However, two pairs of steroids (ES and ED, T and HPG) coeluted. When the program was changed such that the concentration of methanol was increased linearly from 40 to 70% in 30 min and kept at 70% for 10 min, the nine steroids were baseline separated. But the gradient program was not suitable for the separation of other types of compounds. The optimum conditions were applied to the separation of an artificial sample containing 54 standard metabolites and B. subtilis cell extract, as depicted in Figure 2A and B, respectively. From Figure 2A, we can see that 25 peaks appeared for the 54-component mixture since some peaks overlapped. For the B. subtilis cell extract, only 18 peaks appeared, as shown in Figure 2B. The few peaks appearing in the chromatogram of the cell extract indicated that the peak capacity of a single LC technique was not sufficient to resolve all the components in complex samples. CE Conditions. To separate fractions of effluent from microLC, CE conditions to separate the standard metabolites needed to be optimized. It is usually not difficult to develop a CE method to separate one class of compounds. Due to the low injection volume (160 nL) in micro-LC, after each fraction was dried and reconstituted to 10 µL, the analytes were diluted 62.5 times (10 µL/0.16 µL). Therefore, it is important to develop a highly sensitive CE method in order to detect the analytes in the fractions. Two on-line sample preconcentration techniques, dynamic pH junction and sweeping, were used to enhance the sensitivity of CE. As the compounds were separated based on their hydrophobicity in LC, the analytes in the fractions eluted at different retention times have different hydrophobicities. For CE, different on-line sample preconcentration techniques are suitable for different compounds. Therefore, different CE on-line preconcentration techniques were employed to improve the sensitivity of analytes in the fractions eluted at different retention times. Sweeping is a preconcentration technique in MEKC, which utilizes the phenomenon that hydrophobic analytes tend to be incorporated into the micelle. Hence, it is very effective for the preconcentration of hydrophobic analytes. As steroids are strongly hydrophobic, sweeping MEKC was selected. At first, the sweeping concentration efficiency of steroids under suppressed EOF was investigated. Organic solvents are often used in MEKC to improve separation performance. To optimize the separation of the steroids, the effect of adding varying amounts of methanol and acetonitrile in the running buffer was investigated. When the running buffer was 50 mM SDS-50 mM H3PO4-20% acetonitrile (pH 2.0) (6.2 mS/cm) and injection time was 10 s (3.5 kPa), the steroids were separated except for HPG and ES. When methanol was substituted for acetonitrile, the separation became poor. The effects of injection time and different types and concentrations of sample matrix were studied. Different concentrations of phosphoric acid, sodium chloride, citric acid, sodium acetate, and sodium dihydrogen phosphate in the sample matrix proved to have different effects on the resolution and concentration efficiency. Among them, phosphoric acid was considered as the best in terms of

Figure 1. Chromatograms of different types of standard metabolites. (A) Bases and ribonucleosides; (B) nucleotides; (C) aromatic acids; (D) flavin derivatives; (E) folate derivatives; (F) steroids components: 1, cytosine; 2, uracil; 3, C; 4, adenine; 5, guanine; 6, hypoxanthine; 7, uric acid; 8, xanthine; 9, U; 10, thymine; 11, i; 12, A; 13, G; 14, theobromine; 15, theophylline; 16, caffeine; 17, ADP; 18, ATP; 19, GDP; 20, GTP; 21, ITP; 22, UDP; 23, UTP; 24, NADP; 25, CMP; 26, UMP; 27, IMP; 28, AMP; 29, GMP; 30, NAD; 31, NA; 32, PA; 33, DHPAA; 34, PPA; 35, HBA; 36, HPAA; 37, AA; 38, HPPA; 39, BA; 40, CA; 41, FAD; 42, FMN; 43, RF; 44, 5-methyl THF; 45, FA; 46, ET; 47, C; 48, HC; 49, AS; 50, ES; 51, ED; 52, T; 53, HPG; 54, PG. The concentration of each base and ribonucleoside is 10 µg/mL, nucleotide 20 µg/mL, aromatic acid 10 µg/mL except for HPPA and PPA (20 µg/mL), flavin and folate derivative 10 µg/mL except for RF (5 µg/mL), and steroid 2 µg/mL. The experimental conditions are shown in the Experimental Section.

separation and concentration efficiency and was used in subsequent studies for optimizing the conductivity of sample matrix. When the steroids were diluted with water, the concentration

effect was very poor because the analytes migrated too fast due to the high electric field in the sample zone and surfactant had not enough concentration and time to pick up the analytes. Similar Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 2. Chromatograms of the artificial sample and B. subtilis cell extract. (A) Artificial sample containing 54 standards, in which the concentration of each steroid is 5 µg/mL, aromatic acid 10 µg/ mL except for HPPA and PPA (20 µg/mL), base, ribonucleoside and nucleotide 10 µg/mL, and flavin and folate derivative 10 µg/mL except for RF (5 µg/mL). (B) B. subtilis cell extract. The experimental conditions are the same as in Figure 1.

concentration efficiency was observed when the concentration of phosphoric acid was in the range from 25 (2.89 mS/cm) to 200 mM (20.9 mS/cm). Sweeping MEKC has no stringent requirement for the conductivity of sample matrix. The 50 mM phosphoric acid was used to investigate the effect of the injection time. The peak height and area increased with injection time. And the peak height had a linear relationship with injection time. But the resolution decreased with increase of the injection time. Figure 3A depicts an electropherogram of the steroids at the injection time of 720 s (3.5 kPa). Under the given conditions, the detection limits of steroids were in the range from 0.4 to 2.0 ng/mL (1.1 × 10-9 to 7.2 × 10-9 M). Next, the sweeping concentration efficiency of steroids at the presence of EOF was investigated. To optimize the separation efficiency of the steroids, the effects of different surfactants, electrolytes, and organic solvents were studied. When a running buffer containing 60 mM sodium cholate, 20 mM CAPS, and 30% 1424 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

methanol (pH 8.9) was used, the steroids were separated. Hence, the above running buffer was used for investigating the effect of injection time. When 100 mM sodium chloride was used as the sample matrix, peak height increased with the increase of the injection time. But the injection time cannot be prolonged too much in the presence of EOF. Beyond 90 s, the peaks broadened seriously. Due to the limitation of injection time, the detection limits of the steroids in the presence of EOF are ∼1 order less than that under suppressed EOF. Based on these results, the lateeluting fractions from micro-LC were reconstituted using 50 mM phosphoric acid and analyzed under suppressed EOF. Dynamic pH junction is an efficient preconcentration technique for the analytes if the difference in pH between the sample matrix and background electrolyte can cause significant changes in their mobilities. Britz-Mckibbin and Chen reported using dynamic pH junction as an on-line preconcentration technique in CZE to enhance the sensitivity of seven purine bases.47 In this paper, dynamic pH junction CZE was utilized to separate purine and pyrimidine bases and their corresponding ribonucleosides since the pH change can cause the changes in their mobilities. The pH of the background electrolyte (160 mM borate) was first optimized. The experimental results showed that, in the pH range from 9.0 to 10.0, these compounds could obtain better separation although they were not all separated yet. The pH of 9.2 was used for further investigation. To improve the separation efficiency, the influences of adding different volumes of methanol or acetonitrile to the background electrolyte were studied. From the experimental results, it was found that when a background electrolyte (pH 9.2) containing 10% methanol was used, the analysis took less than 28 min and the separation was improved although two pairs of compounds, cytosine and caffeine and adenosine and cytidine, still coeluted. When the concentration of methanol was increased to 20%, adenosine and cytidine was separated, but the entire analysis time was increased to ∼55 min, which was almost 2-fold longer than that when 10% methanol was used because EOF decreased with the increase in concentration of methanol in the background electrolyte. Based on these results, a running buffer containing 160 mM borate (pH 9.2) and 10% methanol was used to investigate the focusing effect of dynamic pH junction. The composition of the sample matrix relative to the background electrolyte plays a vital role in the dynamic pH junction on-line sample preconcentration technique in CZE. Optimization of sample matrix composition consisted of the selection of the type of electrolytes (including sodium phosphate and sodium chloride), their concentrations, and the injection length to achieve the highest concentration effect. When the sample matrix contained 75 mM sodium phosphate (pH 6.0) and the injection time was 40 s at 3.5 kPa, the analytes focused better except for adenosine and cytidine. When a running buffer containing 160 mM borate (pH 9.4) devoid of methanol was used, all 16 compounds could be focused although the separation efficiency was not better than that using the running buffer containing methanol. In this paper, as CE acted as the seconddimensional separation technique, the requirement for its separation efficiency is not very stringent. But its sensitivity was of critical importance for the two-dimensional system. Figure 3B shows an electropherogram of the bases and ribonucleosides when 160 mM borate (pH 9.4) was used as a running buffer. Under the given (47) Britz-McKibbin, P.; Chen, D. D. Y. Chromatographia 2003, 57, 87-93.

Figure 3. Electropherograms of 54 standard metabolites. Analyte peak numbering is the same as in Figure 1. (A) Sweeping MEKC for the separation of steroids. Experimental conditions: fused-silica capillary 60.2 cm (effective length 50 cm) × 50 µm i.d.; buffer, 50 mM SDS-50 mM H3PO4-20% acetonitrile (pH 2.0); applied voltage, -25 kV; hydrodynamic injection, 3.5 kPa × 720 s; capillary temperature, 20 °C; detection wavelength, 230 nm; samples solvent, 50 mM phosphoric acid; The concentration of each steroid is 5 µg/mL except for ES (10 µg/mL). (B) Dynamic pH junction CZE for the separation of bases and ribonucleosides. Experimental conditions: fused-silica capillary 60.2 cm (effective length 50 cm) × 75 µm i.d.; buffer, 160 mM borate (pH 9.4); applied voltage, 18 kV; hydrodynamic injection, 3.5 kPa × 40 s; capillary temperature, 20 °C; detection wavelength, 210 nm; Sample solvent, 75 mM phosphorate buffer (pH 6.0); The concentration of each base and ribonucleoside is 1 µg/mL. (C) Dynamic pH junction CZE for the separation of nucleotides. The concentration of each nucleotide is 2 µg/mL except for AMP (1 µg/mL). Other conditions are the same as in Figure 3B. (D) Dynamic pH junction CZE for the separation of aromatic acids. The concentration of each aromatic acid is 2 µg/mL except for DHPAA (1 µg/mL), HPPA, and PPA (4 µg/mL). Other conditions are the same as in (B). (E) Dynamic pH junction CZE for the separation of flavin derivatives. The concentrations of FMN, FAD, and RF are 2, 2, and 1 µg/mL, respectively. Other conditions are the same as in (B). (F) Dynamic pH junction CZE for the separation of folate derivatives. The concentrations of FA and 5-methyl THF are 1 and 1 µg/mL, respectively. Other conditions are the same as in (B).

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Figure 4. Electropherograms of B. subtilis cell extract using sweeping MEKC and dynamic pH junction CZE. (A) Dynamic pH junction CZE; experimental conditions are the same as in Figure 3B. (B) Sweeping MEKC; experimental conditions are the same as in Figure 3A.

conditions, the detection limits of bases and ribonucleosides were in the range from 0.8 to 2.0 ng/mL (0.4 × 10-8 to 1.1 × 10-8 M). Similar experiments were carried out to optimize the running buffer, sample matrix composition, and injection time when separating and concentrating other standards including nucleotides, aromatic acids, flavin, and folate derivatives. Panels C-F of Figure 3 show electropherograms of these compounds under the optimized conditions. From the experimental results, it was found that these compounds showed better separation and concentration under the same experimental conditions used for analyzing bases and ribonucleosides. The detection limits of nucleotides, aromatic acids, flavin, and folate derivatives were in the range from 0.3 to 30 ng/mL (0.2 × 10-8 to 6.5 × 10-8 M) under the optimum conditions. Based on the studies, 75 mM sodium phosphate (pH 6.0) was used to reconstitute the earlyeluting fractions from micro-LC. To compare the peak capacity of a single chromatography or electrophoresis system with that of a two-dimensional separation system, sweeping MEKC and dynamic pH junction CZE were used to analyze the B. subtilis cell extract, as shown in Figure 4A and 1426 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

Figure 5. Electropherograms of the fractions of effluent of artificial sample from micro-LC. Analyte peak numbering is the same as in Figure 1. (A) Dynamic pH junction CZE; experimental conditions are the same as in Figure 3B. (B) Sweeping MEKC; experimental conditions are the same as in Figure 3A.

B, respectively. As inferred, there were not so many peaks appeared. Separation of the Fractions of Effluent of the Artificial Sample and B. subtilis Cell Extract from Micro-LC by CE. After optimization of micro-LC and CE conditions, the two separation techniques were combined to analyze the artificial sample and B. subtilis cell extract. Panels A and B of Figure 5 show electropherograms of fractions of the artificial sample using dynamic pH junction CZE and sweeping MEKC, respectively. The fractions from 5 to 21 min were further analyzed by dynamic pH junction CZE. In 5- and 6-min fractions, there existed 24 compounds, which are strongly hydrophilic and coeluted in RPLC. They were separated by the second dimensional CE except for UMP and GDP. Cytosine and caffeine, adenine, and thymine, A and C, PPA and HPAA, and BA and NA were not separated by CE, as shown in Figure 3B and D. But they were separated by micro-LC, as shown in Figure 1A and C. After combining the two separation systems, these compounds were all separated, as shown in Figure 5A. The fractions from 21 to 24 min, in which strongly hydrophobic steroids existed, were further analyzed by sweeping MEKC. The nine steroids were resolved by MEKC. It should be mentioned that the 21-min fraction was a special fraction as it contained CA and three steroids (ET, HC, C), which have different natures. CA, which contains a carboxyl group, belongs to carboxylic acids and three steroids contain hydroxyl

groups. Therefore, their pKa values are different. Under the microLC conditions used, CA is neutral according to its pKa value of 4.44,48 resulting in its coelution with the three neutral steroids. Under the pH of the buffer used in the dynamic pH junction CZE, CA is ionic due to the dissociation of the carboxyl group, resulting in its separation from the steroids. The three steroids were analyzed by MEKC since MEKC allows the resolution of uncharged molecules. Therefore, the 21-min fraction was analyzed using both dynamic pH junction CZE and sweeping MEKC. Since the two modes of CE demanded different sample matrixes, the artificial sample was injected twice and two sets of fractions were collected. One fraction was reconstituted with 50 mM phosphoric acid and the other with 75 mM sodium phosphate (pH 6.0). When the fraction was analyzed using dynamic pH junction CZE, CA was detected, but the three steroids were not, as shown in Figure 5A. In contrast, when sweeping MEKC was used, the three steroids were detected, but CA was not, as shown in Figure 5B. The four compounds in the fraction were accurately identified by three methods: (1) comparing their migration times with those of standards; (2) adding a pure standard to the fraction so that the peak area of the corresponding compound was increased significantly; (3) comparing the peak spectrum with that of the standard with the same migration time since a photodiode array UV detector was used in the CE system. The four compounds contained in the fraction were indeed resolved by different modes of CE. Compounds with different characteristics existing in one fraction eluted from micro-LC can be analyzed by different modes of CE; that is one of the advantages of off-line combination of LC and CE. The B. subtilis cell extract was tested to determine whether the developed two-dimensional separation system is applicable to real biological samples. Similar experiments were performed to collect two sets of fractions. Panels A and B of Figure 6 show electropherograms of the fractions from micro-LC analyzed further using dynamic pH junction CZE and sweeping MEKC, respectively. As can be seen from Figure 6A, many compounds were detected in 5- and 6-min fractions. Some important metabolites (including NAD, NADP, AMP, ADP, ATP, GMP, GDP, GTP, UMP, UDP, UTP, CMP, IMP, ITP, adenine, guanine, and uracil) in the B. subtilis cell extract were identified by the same three methods as those used for identifying the compounds contained in the 21-min fraction of the artificial sample. There were few peaks that appeared in the fractions from 7 to 24 min when the fractions were analyzed using dynamic pH junction CZE. The fractions from 19 to 24 min were reconstituted using 50 mM phosphoric acid again and analyzed by sweeping MEKC because some metabolites having different characteristics may exist in these fractions. As can be seen from Figure 6B, there existed some hydrophobic compounds in the fractions from 20 to 23 min that were not identified. To embody the two-dimensional separation system, the three-dimensional pictures of the separation of the artificial sample and B. subtilis cell extract by the two-dimensional separation system are given in Figure 7A and B, respectively. As can be seen from Figures 2B and 4, when B. subtilis cell extract was analyzed in one run using a single chromatographic or electrophoretic technique, many peaks overlapped, resulting (48) Lide, D, R., Ed. Handbook of Chemistry and Physics, 82nd ed.; CRC Press: Boca Raton, FL, 2001; pp 8-53.

Figure 6. Electropherograms of the fractions of effluent of B. subtilis cell extract from micro-LC. Analyte peak numbering is the same as in Figure 1. (A) Dynamic pH junction CZE; experimental conditions are the same as in Figure 3B. (B) Sweeping MEKC; experimental conditions are the same as in Figure 3A.

in difficulty in identifying these peaks. When micro-LC and different modes of CE were combined to analyze B. subtilis cell extract, although the peaks that appeared were not as many as we expected, some important metabolites were identified due to the enhancement of peak capacity. One reason much of the twodimensional separation space of the B. subtilis cell extract is empty of peaks is that the concentrations of some metabolites in the micro-LC fractions were too low. Another is that many metabolites in the cell extract lack chromophores or have weak UV absorbance, as noted by Soga et al.35,49 Although a gradient mode having a concentration effect was used in micro-LC, the injection volume was too small. In LC, injection volume is inversely related to separation efficiency. A concentration technique combined with micro-LC, which can permit the increase of injection volume without compromising separation efficiency, might be necessary. To enhance UV detection sensitivity, two on-line preconcentration techniques were used to interface micro-LC and CE, which proved to be successful because some important metabolites were detected and identified. However, the fact that there were not so many peaks appearing suggests that a more sensitive detector is needed. LIF is very sensitive, but it is not suitable for metabolome analysis because most metabolites lack native fluorescence and it is impossible to find a universal fluorescence-labeling reagent (49) http://www.ttck.keio.ac.jp/IAB/microbiology/metabolome/sup/ stab1.pdf.

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fluorescence. Hence, it is a rational choice to replace UV detection in the second-dimensional CE. In this case, CE conditions will be reoptimized to be compatible with the MS detector. CONCLUSIONS A novel two-dimensional separation system was demonstrated to profile B. subtilis cell extract. Micro-RPLC with a monolithic silica-ODS column was used as the first dimension to analyze an artificial sample containing 54 standard metabolites and B. subtilis cell extract. Different modes of CE acted as the second dimension to further resolve fractions from micro-LC. Dynamic pH junction CZE was used to analyze the early-eluting fractions containing hydrophilic and weakly hydrophobic metabolites, while sweeping MEKC was used to analyze the late-eluting fractions containing strongly hydrophobic metabolites. The middle fractions were further resolved using both modes of CE. One of the advantages of the off-line combination of micro-LC and CE is that the system allows the use of different modes of CE to analyze the fractions from micro-LC. Dynamic pH junction and sweeping proved to be useful to interface micro-LC and CE because detection limits from 0.2 to 30 ng/mL for 54 standard metabolites were achieved, resulting in the detection and identification of some important metabolites in B. subtilis cell extract. Based on the results of this study, in the future, an on-line preconcentration technique will be combined with micro-LC, which permits injection of larger sample volume while maintaining separation efficiency. The use of a sensitive and selective MS as the detector in the seconddimensional CE to enable analysis of metabolites having weak or no UV absorbance will also be explored.

Figure 7. Three-dimensional pictures of artificial sample and B. subtilis cell extract by the two-dimensional separation system. (A) Artificial sample; (B) B, subtilis cell extract.

ACKNOWLEDGMENT L.J. is grateful to the Japan Society for the Promotion of Science (JSPS) for supporting her postdoctoral studies. Special thanks are given to Dr. Hideaki Hisamoto and Dr. N. Matsubara for their helpful in-laboratory assistance.

due to the complexity of the metabolome. Mass spectrometry (MS) provides not only excellent sensitivity and selectivity but also structure information of the metabolites. Moreover, it does not require that metabolites have native UV absorbance or

Received for review December 9, 2003.

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AC035039B

September

4,

2003.

Accepted