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Recent developments in liquid-phase separation techniques for metabolomics Metabolomics is the comprehensive analysis of low molecular weight compounds in biological samples such as cells, body fluids and tissues. Comprehensive profiling of metabolites in complex sample matrices with the current analytical toolbox remains a huge challenge. Over the past few years, liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis–mass spectrometry (CE–MS) have emerged as powerful complementary analytical techniques in the field of metabolomics. This Review provides an update of the most recent developments in LC–MS and CE–MS for metabolomics. Concerning LC–MS, attention is paid to developments in column technology and miniaturized systems, while strategies are discussed to improve the reproducibility and the concentration sensitivity of CE–MS for metabolomics studies. Novel interfacing techniques for coupling CE to MS are also considered. Representative examples illustrate the potential of the recent developments in LC–MS and CE–MS for metabolomics. Finally, some conclusions and perspectives are provided. The metabolome is the complete set of endogenous low molecular weight metabolites present in a given biological sample, such as for example, cells, tissues and body fluids [1,2]. The comprehensive or global analysis of these metabolites in biological samples is known as metabolomics or metabonomics (these terms are often used interchangeably in the literature). Metabolomics provides a direct functional read-out of the physiological status of an organism and is, in principle, ideally suited to describe someone’s health status [3]. According to the most recent version of the Human Metabolome Database [4], the human metabolome is comprised of more than 40,000 metabolites covering various classes of compounds, such as lipids, amino acids, organic acids, steroids, nucleotides and carbohydrates. It should be noted that this number includes both endogenous and exogenous compounds (the latter originating from nutrients, microbiota, drugs and other sources) [4]. It is evident that multiple complementary analytical techniques should be used in conjunction in order to profile as many metabolites as possible in a given biological sample. For example, by using a combination of five analytical approaches, more than 4000 chemically diverse metabolites have been identified in human serum at concentration levels spanning more than nine orders of magnitude [5,6]. Over the past few years, major advancements have been made in analytical techniques such as NMR spectroscopy and MS coupled to GC, LC or CE for metabolomics studies [7]. At present, most metabolomics studies are performed with
NMR, LC–MS and GC–MS [7]. NMR is a powerful analytical platform for relatively fast and reproducible metabolic profiling of body fluids such as serum and urine. However, the sensitivity of NMR is limited, and as a result low-abundant metabolites may not be detected [8]. GC–MS is considered a robust technique for the profiling of various metabolite classes with a high resolution and sensitivity [9]. However, this approach is not suited for the profiling of nonvolatile, thermally labile and/or highly polar compounds, and as such, derivatization is often required to yield volatile and thermostable analytes. A major fraction of the metabolome is composed of polar and nonvolatile metabolites [10]; therefore, liquid-phase separation techniques, such as LC and CE, are very attractive complementary approaches as they can be used for the analysis of a wide range of compounds without using derivatization. Moreover, (reversed-phase) LC and CE are directly compatible with the analysis of aqueous samples without using complicated sample pretreatment procedures. Over the past decade, LC–MS [11,12], and to a rather modest extent CE–MS [13,14], emerged as important analytical techniques for global metabolite profiling studies. In the present paper, an overview of recent developments in LC–MS and CE–MS for metabolomics is given. With regard to LC–MS, attention will be paid to advancements in the various complementary LC modes, such as reversed-phase LC (RP–LC) and HILIC. In this context, the potential of novel chromatographic LC materials, such as
10.4155/BIO.14.51 © 2014 Future Science Ltd
Bioanalysis (2014) 6(7), 1011–1026
Rawi Ramautar*1 & Gerhardus J de Jong2 Analytical Biosciences, LACDR, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands 2 Biomolecular Analysis, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands *Author for correspondence: Fax: +31 30 253 5180 [email protected] 1
ISSN 1757-6180
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Key Terms Metabolomics:
Comprehensive analysis of low molecular weight (endogenous) metabolites present in a biological sample.
Core-shell particles: New
generation of silica particles with an outer porous layer developed for LC offering high chromatographic efficiency.
core-shell and sub-2 µm particles, for metabolic profiling is discussed. Concerning CE–MS, special attention will be devoted to capillary coatings and MS interfacing techniques. Developments in MS technology for metabolomics have been recently reviewed [15,16] and will not be covered in this paper. Aspects related to sample pretreatment for metabolomics have also been reviewed recently [17,18]. Developments in LC column technology LC is comprised of different separation modes, such as RP-LC, normal-phase LC, ion-exhange LC and HILIC. The choice of a particular LC mode depends on the biological question that should be addressed with the metabolomics approach and on the sample matrix of interest. RP-LC
The first LC–MS-based metabolomics study was performed with RP-LC–MS using a C18 column (100× 2.1 mm internal diameter [I.D.]) with a particle size of 3.5 µm [19]. At present, most LC–MS-based metabolomics studies are still carried out with RP-LC columns based on C18 material [7,12]. RP-LC–MS offers many advantages such as: a high sensitivity; improved separation power (i.e., state-of-the-art MS technologies provide an excellent mass accuracy and resolution); the availability of many RP-LC column chemistries for method development; a wide selectivity for the analysis of various metabolite classes; and, it is highly compatible with the analysis of biological samples. For RP-LC separations a gradient is often used, which starts at a high aqueous content (95–100% water, containing 0.1% formic acid), and ramps up to a high organic content (95–100% acetonitrile or methanol, containing 0.1% formic acid) [20]. RP-LC–MS-based metabolomics studies are generally performed with ESI in the positive ionization mode as it can be effectively used for the ionization of a wide range of metabolite classes. For global metabolic profiling studies both the positive and the negative ionization mode are used as they provide complementary information [20]. Negative ion mode is often used for metabolites that are difficult to ionize in positive ion mode, such as acidic metabolites (organic acids) and certain neutral compounds. A volatile basic additive, such as ammonium acetate, is often added to the LC mobile phase in order to improve the chromatographic separation and ionization efficiency for acidic metabolites. 1012
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Global or nontargeted profiling of metabolites in complex mixtures requires highly efficient separations and, therefore, the latest metabolomics studies are increasingly based on columns packed with sub-2 µm porous particles used for UHPLC analyses [12,20–24]. Compared with conventional RP-LC columns packed with 5 or 3.5 µm porous particles, the use of sub-2 µm porous particles offers an improved peak capacity and decreased analysis times. For example, Evans et al. developed a UHPLC–MS method using a C18 column packed with sub-2 µm porous particles for nontargeted metabolic profiling of human plasma [25]. This approach provided a higher resolution and the analysis time was reduced approximately threefold, that is, from 32 min on the conventional HPLC–MS method to 12 min on UHPLC–MS. Two injections were performed – one for basic and the other for acidic metabolites, which improved the number and variety of small compounds measured (i.e., metabolic coverage). Thus far, various groups have demonstrated the strong potential of RP-UHPLC–MS for global metabolic profiling of biological samples, and validated protocols for the analysis of urine, serum and tissues are now available [20,26]. Recently, a new generation of superficially porous or core-shell silica particles was developed for RP-LC [27–29]. In comparison to the sub-2 µm fully-porous hybrid silica particles used for UHPLC, the use of sub-2 µm coreshell material can provide a similar separation performance, but at a lower operational pressure. Sanchez et al. recently evaluated the performance of RP-LC columns packed with 1.0–1.4 µm core-shell particles, including the commercially released Kinetex 1.3 µm C18, for metabolic profiling of human urine [29]. This work illustrated that very low minimum plate heights of 2.2 µm could be obtained using columns packed with 1.3 µm particles, corresponding to a plate count of over 450,000 plates/m. Application of the Kinetex 1.3 µm C18 column (50 mm × 2.1 mm I.D.) to metabolic profiling of human urine resulted in the separation of many components and 128 peaks were detected within just 5 min (Figure 1). According to the authors, the use of a 75 mm or a 100 mm long column made with the smallest core-shell particles should resolve many more components in urine samples. Overall, coreshell particles provide the possibility to obtain highly efficient separations on standard HPLC equipment. These columns seem to have very future science group
Recent developments in liquid-phase separation techniques for metabolomics
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Figure 1. Metabolic profile of human urine. (A) Obtained with LC–UV using a Kinetex 1.3 µm C18 column with dimensions 50 mm × 2.1 mm internal diameter. Upper trace shows the details of the urinary metabolic profile starting at 2 min. Mobile phase A: acetonitrile/water/trifluoroacetic acid 5/95/0.1; mobile phase B: acetonitrile/water/trifluoroacetic acid 95/5/0.1; start at 100% A; hold for 1 min; raise to 50% B in 10 min; step to 100% B and hold for 0.5 min; re-equilibrate for 1 min; flow rate: 0.5 ml/min; sample injection: 20 µl. (B) The lower trace is a zoom out (full chromatogram) of the upper trace. Reproduced from [29] © Elsevier (2013).
attractive characteristics for relatively fast metabolic profiling of complex mixtures, however, the robustness for the analysis of biological samples needs to be demonstrated. A wide range of physiologically relevant metabolite classes can be analyzed by RP-LC systems, however, highly polar and charged metabolites are difficult to retain and often coelute with the void volume. Ion-pairing agents such as tributylamine or hexylamine can be added to the mobile phase of RP-LC in order to improve the analysis for polar compounds [30,31]. However, the use of ion-pair agents in RP-LC–MS may result in severe ion suppression and may contaminate the ion source [32]. An alternative approach is to use reversed-phase pentafluorophenylpropyl (PFPP) columns, in which a pentafluorophenyl ring structure is future science group
attached to the silica over a propyl chain, thereby exhibiting both reversed- and normal-phase retention [33]. For example, Yang et al. developed a RP-LC–MS method using a PFPP column for the profiling of metabolites in bacterial extracts [34]. Highly polar compounds displayed good chromatographic retention on the PFPP column and it was shown that isomers/isobars (e.g., isoleucine/leucine, methylsuccinic acid/ethylmalonic acid) and metabolites of similar structure (e.g., malate/fumarate) were better resolved on the PFPP than on a HILIC column. Another way to increase retention of polar compounds in RP-LC–MS is to use chemical derivatization. For example, Guo et al. developed an approach for the labeling of metabolites containing a primary amine, secondary amine or phenolic hydroxyl group(s) [35]. This www.future-science.com
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Key Term Aqueous normal-phase LC:
LC based on silica hydride-based stationary phases, where hydrophilic partitioning occurs via a gradient-elution program over a wide water-content range.
dansylation labeling improved the chromatographic retention of polar and charged metabolites, which were normally not retained on the RP-LC column, and it also offered one to three orders of magnitude ESI signal enhancement. In general, not all compounds are amenable to chemical derivatization and, therefore, this approach is especially suited for targeted, quantitative RP-LC–MS-based metabolomics studies [36–38]. Complementary
LC stationary phases At present, various complementary LC separation mechanisms are available for the analysis of (highly) polar and/or charged metabolites, such as HILIC, aqueous normal-phase LC (ANP-LC) and ion-exhange LC. HILIC can be regarded as a variant of normal-phase LC as the separation is performed on a polar stationary phase [39–41]. In contrast to normal-phase LC, HILIC uses a polar stationary phase in combination with aqueous organic eluents (typically 3–50% water), which are well suited for efficient ESI-MS analyses. In HILIC, retention of analytes occurs via hydrophilic partitioning of polar compounds between an organicsolvent rich mobile phase and an aqueous layer formed on the stationary phase [42]. The various HILIC modes available can be divided into neutral, charged and zwitter-ionic stationary phases [42–45]. Gika et al. developed a HILICUHPLC–MS method using an Acquity BEH HILIC column (1.7 µm, 2.1 × 150 mm) for metabolic profiling of urine from male Zucker rats [46]. Typical metabolic profiles obtained for the same set of rat urine samples by HILIC- and RP-UHPLC–MS profiles are shown in Figure 2 . Overall, the HILIC-MS analysis provided a higher signal response for the peaks detected in rat urine, while the RP-LC–MS analysis resulted a higher chromatographic resolution and more compounds were observed in rat urine. The HILIC approach was especially suited for the profiling of highly polar metabolites and as such it provided a complementary view on the composition of the urine samples as compared with the results obtained by RP-UHPLC–MS. In order to profile as many metabolites as possible in a given biological sample, Ivanisevic et al. developed a single extraction-dual LC–MS platform comprised of RP-LC and HILIC in order to allow the global profiling of both hydrophobic and hydrophilic metabolites [47]. An aminopropyl-based column was used for HILIC-MS, which provided a good resolution 1014
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for highly polar compounds. The optimized method appeared to be highly reproducible with minimal drifts in retention time over more than 60 h of analysis. The potential of the approach was illustrated for metabolic profiling of bacterial cells, human cancer cells and human plasma. The combined HILIC/RPLC–MS approach generated over 30,000 molecular features in each sample, with the highest number of unique features detected by RP-LC in positive ESI-MS mode and by HILIC in negative ESI-MS mode. Overall, HILIC-MS emerged as a powerful analytical approach for the analysis of (highly) polar metabolites, however, a rather long re-equilibration time between runs is often required for an acceptable performance [48]. Recently, ANP-LC emerged as an effective LC method for the analysis of (highly) polar metabolites [42]. In these columns, hydride groups substitute 95% of the silanol groups on the surface [49]. The less polar surface minimizes the attraction of the stationary phase for water, allowing the column to function in ANP-LC mode. Callahan et al. developed a ANP-LC–MS method using a diamond hydride stationary phase for the rapid profiling of polar metabolites in various biological samples [50]. For the LC separation, 90% acetonitrile with 0.1% ammonium acetate and 0.1% acetic acid was employed as the organic mobile phase and 100% water with 0.1% ammonium acetate and 0.1% acetic acid (pH 3.4) as the aqueous mobile phase. Approximately 1000 compounds were reproducibly detected in human urine and 400 compounds were observed in xylem fluid from soyabean (Glycine max) plants. This work also demonstrated that both ANP-LC and RP-LC are required for nontargeted metabolic profiling. Chen et al. developed an ANP-LC–MS method for nontargeted metabolic profiling of mice plasma in order to discover systemic changes arising from inactivation of xanthine oxidoreductase, an enzyme that catalyzes the final steps in purine degradation [51]. Figure 3 shows the technical reproducibility of the ANP-LC–MS method for nontargeted metabolic profiling of plasma (25-fold diluted), which was determined for 56 repeated analyses. 374 metabolites could be reproducibly detected in the plasma samples. Figure 3 also illustrates that good chromatographic peaks were obtained for various metabolite classes on the ANP-LC column. The metabolomics data obtained corroborated the predicted derangements in purine metabolism and also revealed unexpected perturbations in metabolism of pyrimidines, future science group
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Figure 2. Comparison of HILIC and reversed-phase-UPLC–MS-based metabolic profiles of rat urine. (A) HILIC and (B) the reversed phase (RP) analysis of urine from (fa/fa) obese Zucker rat of 10 weeks of age are shown. (C) HILIC and (D) the RP metabolic profiles of urine from a lean same age Zucker rat are shown. An extracted ion mass (m/z 154.517) in the fa/fa rat urine shows the difference at the retention time and the intensity between the RP and the HILIC mode. Reproduced from [46] © John Wiley & Sons (2008).
nicotinamides, trypto phan, phospholipids, Krebs and urea cycles. Zhang et al. evaluated for the first time the performance of RP-LC–MS, ANP-LC–MS, and two zwitter-ionic HILIC-MS systems, for metabolic profiling of urine [42]. It was shown that the zwitter-ionic HILIC columns provided the most optimal chromatographic performance and the future science group
greatest metabolic coverage of polar compounds in urine. Kloos et al. performed a study in which three porous HILIC columns (cyano, amino and diol) and three RP-LC columns (core-shell C18, XB-C18 and pentafluorophenyl) were evaluated for relatively fast metabolic profiling [52]. The performance of these columns was assessed using a representative metabolite mixture (comprising www.future-science.com
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Figure 3. Reproducibility of nontargeted metabolic profiling of plasma by aqueous normal phase-LC–MS. (A) Overlay of chromatograms acquired for 56 repeated analyses of a single plasma sample. (B) Profile plot overlay of normalized extracted ion intensities for 374 distinct metabolites, quantified as a function of injection number for the 56 repeated plasma analyses depicted in A. Results depict flat run-to-run variation (mean CV = 6.51%) in the levels of the 374 metabolites with repeated analysis. (C) Peak overlay of 56 repeated assessments of extracted ion counts for detection of some typical plasma metabolites, demonstrating reproducibility of quantification. Reproduced from [51] © PLoS One (2012).
54 compounds) and pooled human urine samples. Figure 4 shows that the amino- and diolHILIC columns provided the most suitable analysis for the metabolite mixture, whereas poor retention for most of the analytes was obtained on the RPLC columns. When applied to human urine samples, the diol-HILIC column provided the most optimal results, that is, the highest number of detectable molecular features, in both ESI(+) and ESI(-) mode at pH 7. Therefore, the diol-HILIC column combined 1016
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with polarity switching in ESI-MS may be considered an attractive tool for fast urinary metabolic profiling as it can lead to a 50% reduction of analysis time. Miniaturized
LC systems Until now, most LC–MS-based metabolomics studies have been performed with RP-LC systems using 2.1 mm I.D. columns [48]. The sensitivity of ESI-MS can be improved by using LC columns with smaller internal diameters. Nano-LC–MS future science group
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Figure 4. Extracted ion chromatograms for all analytes of the metabolite test mixture. Obtained with: (A) pentafluorophenyl-reversed-phase(RP)-LC; (B) amino-HILIC, (C) XB-C18-RPLC; (D) diol-HILIC; (E) C18-RPLC; and (F) cyano-HILIC. The colored bars indicate the type of compounds eluting in the given time window. Each color represents a different class of metabolites. Please see colour figure at www.future-science.com/doi/full/10.4155/BIO.14.51 Reproduced from [52] © Elsevier (2013).
has a higher sensitivity if only small sample volumes are available and this approach is routinely applied in the field of proteomics. In the field of metabolomics, the use of nano-LC–MS would be an attractive approach for in-depth metabolic profiling studies and for the analysis of volume-limited biological samples. Still, the application of nano-LC–MS in metabolomics is very limited. In proteomics, RP-nano-LC–MS is a commonly used approach, however, unlike peptides, (highly) polar and charged metabolites are poorly retained on RP-LC columns. Therefore, Myint et al. evaluated various future science group
stationary phase and mobile phase compositions for nano-LC–MS analysis of cationic metabolites [53]. It was found that polar cationic metabolites were strongly bound to mixed-functional RP with cation exchange mode resin. Different types of coating for the internal wall of the capillary columns were studied to improve peak shapes and to reduce wall effects for highly polar metabolites, and in this regard most optimal results were obtained with the hydrophilic coated TCWAX capillary. The method was used for the analysis of human cerebrospinal fluid (CSF) and more than 100 peaks were detected from 10 µl www.future-science.com
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Ramautar & de Jong of CSF. The same group has also developed a nano-LC–MS method for the comprehensive analysis of anionic metabolites in biological samples [54]. A polyamine-bonded polymerbased apHera™ NH2 column was used for the analysis of anionic polar compounds, such as organic acids and sugar phosphates. However, distorted peak shapes were obtained for multiply phosphorylated or carboxylated compounds. The addition of the metal chelating reagent EDTA to the sample significantly improved peak shapes of multiply charged anionic compounds. LODs of some polar anionic metabolites in full-scan mode ranged from 0.19 to 2.81 pmol. The method was applied to the analysis of Hela cells, mouse brain extracts, plasma and CSF, and revealed that phosphorylated metabolites were abundant in HeLa cells and brain, while plasma and CSF mostly contained organic acids. Recently, Kiefer et al. developed a nanoscale ion-pair RP-LC–MS method for the profiling of anionic compounds in cell extracts [55]. Anionic metabolites were separated on a 100 µm I.D. C18 column using tributylamine as the ionpairing reagent and methanol as the mobile phase. A basic pH (9.4) of the mobile phase was required to obtain adequate retention and efficient chromatographic peaks (Figure 5) at a low concentration of tributylamine (1.7 mM). LODs determined for 54 metabolite standards were in the upper attomole range, and compared with conventional ion-pair RP-LC–MS and HILICMS methods using 2 mm I.D. columns the LODs were in general 100 times lower with the nanoscale ion-pair RP-LC–MS approach. The method was applied to metabolic profiling of cell extracts from the methylotroph model organism Methylobacterium extorquens AM1. So far, microfluidic or chip-based LC–MS systems have been primarily used for bioanalytical applications often focused on the analysis of a few analytes [56,57]. Microfluidic separations are usually performed with microchip CE systems as they are relatively easy to implement, whereas downscaling of LC remains a challenging process [57,58]. CE Separation optimization CE emerged as a highly efficient and attractive separation technique for the profiling of polar and charged metabolites in biological samples [59–64]. In comparison to RP-LC and HILIC, CE can provide complementary metabolomics information as recently demonstrated for the
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analysis of colon cancer cells, K562 leukemia cells and mouse urine [65–67]. Capillary zone electrophoresis, normally referred to as CE, is the main CE separation mode used in CE–MS for metabolomics [60]. In this mode, the capillary is filled with a volatile background electrolyte (BGE) only and analytes are separated according to differences in their electrophoretic mobility. For CE–MS-based metabolomics, ESI is the most widely used ionization technique. As ESI is prone to analyte signal suppression, the BGE should not contain nonvolatile constituents and/or surfactants. Moreover, nonvolatile constituents may cause source contamination and high background signals. Therefore, volatile BGEs, such as formic acid, acetic acid and/or ammonium acetate, are typically used for CE–MS-based metabolomics studies. In general, low-pH (~2) and high-pH (~9) BGE conditions in combination with ESI(+) and ESI(-) are used for the analysis of cationic and anionic metabolites, respectively [68–70]. Soga and co-workers developed the first CE–MS approach for global metabolic profiling of bacterial extracts [14,71]. In this study, distinct CE–MS methods were developed for cationic and anionic metabolites, that is, cationic metabolites were analyzed with a bare fusedsilica capillary using 1 M formic acid (pH 1.8) as BGE, whereas anionic metabolites were analyzed with a cationic polymer-coated capillary using 50 mM ammonium acetate (pH 8.5) as BGE. This approach allowed the analysis of more than 1600 metabolites in bacterial extracts. The CE–MS method for cationic metabolites developed by Soga and co-workers is now used by other research groups [69,72–74]. A stable CE–MS method is of pivotal importance for metabolomics studies where multiple biological samples have to be profiled and compared. In CE, adsorption of matrix components and/or analytes to the capillary wall may cause changes in the electro-osmotic flow that, in turn, can lead to irreproducible migration times. This aspect is especially critical when bare fused-silica capillaries are employed [75]. In such cases, it is important to employ an extensive washing step between successive analyses to allow the ana lysis of biological samples with minimal sample pretreatment. Another way to improve migration time reproducibility in CE is by coating the inner wall of fused-silica capillaries with polymers, as has been demonstrated for various CE–MS-based metabolomics studies [71,75,76]. In this context, charged capillary coatings are future science group
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Figure 5. Extracted ion chromatograms of selected metabolites detected in the cell extract of Methylobacterium extorquens AM1 obtained with nanoscale ion-pair reversed-phase-LC–MS using a 100 µm internal diameter C18 column with tributylamine as ion-pairing reagent. Metabolites were extracted with cold acidified acetonitrile. Injected amount corresponds to 50 ng of biomass cell dry weight. HB: Hydroxybutyryl; PEP: Phospoenolpyruvate; tR: Retention time. Reproduced with permission from [55] © American Chemical Society (2011).
very attractive as they can be used for changing the direction and magnitude of the electro-osmotic flow in CE–MS using normal or reversed CE polarity, providing CE systems future science group
for anions and cations [60,69]. The potential of these coated capillaries was recently assessed for metabolic profiling of rat urine [69]. In this study, fused-silica capillaries were coated with a www.future-science.com
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Recent developments in liquid-phase separation techniques for metabolomics Metabolomics is the comprehensive analysis of low molecular weight compounds in biological samples such as cells, body fluids and tissues. Comprehensive profiling of metabolites in complex sample matrices with the current analytical toolbox remains a huge challenge. Over the past few years, liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis–mass spectrometry (CE–MS) have emerged as powerful complementary analytical techniques in the field of metabolomics. This Review provides an update of the most recent developments in LC–MS and CE–MS for metabolomics. Concerning LC–MS, attention is paid to developments in column technology and miniaturized systems, while strategies are discussed to improve the reproducibility and the concentration sensitivity of CE–MS for metabolomics studies. Novel interfacing techniques for coupling CE to MS are also considered. Representative examples illustrate the potential of the recent developments in LC–MS and CE–MS for metabolomics. Finally, some conclusions and perspectives are provided. The metabolome is the complete set of endogenous low molecular weight metabolites present in a given biological sample, such as for example, cells, tissues and body fluids [1,2]. The comprehensive or global analysis of these metabolites in biological samples is known as metabolomics or metabonomics (these terms are often used interchangeably in the literature). Metabolomics provides a direct functional read-out of the physiological status of an organism and is, in principle, ideally suited to describe someone’s health status [3]. According to the most recent version of the Human Metabolome Database [4], the human metabolome is comprised of more than 40,000 metabolites covering various classes of compounds, such as lipids, amino acids, organic acids, steroids, nucleotides and carbohydrates. It should be noted that this number includes both endogenous and exogenous compounds (the latter originating from nutrients, microbiota, drugs and other sources) [4]. It is evident that multiple complementary analytical techniques should be used in conjunction in order to profile as many metabolites as possible in a given biological sample. For example, by using a combination of five analytical approaches, more than 4000 chemically diverse metabolites have been identified in human serum at concentration levels spanning more than nine orders of magnitude [5,6]. Over the past few years, major advancements have been made in analytical techniques such as NMR spectroscopy and MS coupled to GC, LC or CE for metabolomics studies [7]. At present, most metabolomics studies are performed with
NMR, LC–MS and GC–MS [7]. NMR is a powerful analytical platform for relatively fast and reproducible metabolic profiling of body fluids such as serum and urine. However, the sensitivity of NMR is limited, and as a result low-abundant metabolites may not be detected [8]. GC–MS is considered a robust technique for the profiling of various metabolite classes with a high resolution and sensitivity [9]. However, this approach is not suited for the profiling of nonvolatile, thermally labile and/or highly polar compounds, and as such, derivatization is often required to yield volatile and thermostable analytes. A major fraction of the metabolome is composed of polar and nonvolatile metabolites [10]; therefore, liquid-phase separation techniques, such as LC and CE, are very attractive complementary approaches as they can be used for the analysis of a wide range of compounds without using derivatization. Moreover, (reversed-phase) LC and CE are directly compatible with the analysis of aqueous samples without using complicated sample pretreatment procedures. Over the past decade, LC–MS [11,12], and to a rather modest extent CE–MS [13,14], emerged as important analytical techniques for global metabolite profiling studies. In the present paper, an overview of recent developments in LC–MS and CE–MS for metabolomics is given. With regard to LC–MS, attention will be paid to advancements in the various complementary LC modes, such as reversed-phase LC (RP–LC) and HILIC. In this context, the potential of novel chromatographic LC materials, such as
10.4155/BIO.14.51 © 2014 Future Science Ltd
Bioanalysis (2014) 6(7), 1011–1026
Rawi Ramautar*1 & Gerhardus J de Jong2 Analytical Biosciences, LACDR, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands 2 Biomolecular Analysis, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands *Author for correspondence: Fax: +31 30 253 5180 [email protected] 1
ISSN 1757-6180
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Key Terms Metabolomics:
Comprehensive analysis of low molecular weight (endogenous) metabolites present in a biological sample.
Core-shell particles: New
generation of silica particles with an outer porous layer developed for LC offering high chromatographic efficiency.
core-shell and sub-2 µm particles, for metabolic profiling is discussed. Concerning CE–MS, special attention will be devoted to capillary coatings and MS interfacing techniques. Developments in MS technology for metabolomics have been recently reviewed [15,16] and will not be covered in this paper. Aspects related to sample pretreatment for metabolomics have also been reviewed recently [17,18]. Developments in LC column technology LC is comprised of different separation modes, such as RP-LC, normal-phase LC, ion-exhange LC and HILIC. The choice of a particular LC mode depends on the biological question that should be addressed with the metabolomics approach and on the sample matrix of interest. RP-LC
The first LC–MS-based metabolomics study was performed with RP-LC–MS using a C18 column (100× 2.1 mm internal diameter [I.D.]) with a particle size of 3.5 µm [19]. At present, most LC–MS-based metabolomics studies are still carried out with RP-LC columns based on C18 material [7,12]. RP-LC–MS offers many advantages such as: a high sensitivity; improved separation power (i.e., state-of-the-art MS technologies provide an excellent mass accuracy and resolution); the availability of many RP-LC column chemistries for method development; a wide selectivity for the analysis of various metabolite classes; and, it is highly compatible with the analysis of biological samples. For RP-LC separations a gradient is often used, which starts at a high aqueous content (95–100% water, containing 0.1% formic acid), and ramps up to a high organic content (95–100% acetonitrile or methanol, containing 0.1% formic acid) [20]. RP-LC–MS-based metabolomics studies are generally performed with ESI in the positive ionization mode as it can be effectively used for the ionization of a wide range of metabolite classes. For global metabolic profiling studies both the positive and the negative ionization mode are used as they provide complementary information [20]. Negative ion mode is often used for metabolites that are difficult to ionize in positive ion mode, such as acidic metabolites (organic acids) and certain neutral compounds. A volatile basic additive, such as ammonium acetate, is often added to the LC mobile phase in order to improve the chromatographic separation and ionization efficiency for acidic metabolites. 1012
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Global or nontargeted profiling of metabolites in complex mixtures requires highly efficient separations and, therefore, the latest metabolomics studies are increasingly based on columns packed with sub-2 µm porous particles used for UHPLC analyses [12,20–24]. Compared with conventional RP-LC columns packed with 5 or 3.5 µm porous particles, the use of sub-2 µm porous particles offers an improved peak capacity and decreased analysis times. For example, Evans et al. developed a UHPLC–MS method using a C18 column packed with sub-2 µm porous particles for nontargeted metabolic profiling of human plasma [25]. This approach provided a higher resolution and the analysis time was reduced approximately threefold, that is, from 32 min on the conventional HPLC–MS method to 12 min on UHPLC–MS. Two injections were performed – one for basic and the other for acidic metabolites, which improved the number and variety of small compounds measured (i.e., metabolic coverage). Thus far, various groups have demonstrated the strong potential of RP-UHPLC–MS for global metabolic profiling of biological samples, and validated protocols for the analysis of urine, serum and tissues are now available [20,26]. Recently, a new generation of superficially porous or core-shell silica particles was developed for RP-LC [27–29]. In comparison to the sub-2 µm fully-porous hybrid silica particles used for UHPLC, the use of sub-2 µm coreshell material can provide a similar separation performance, but at a lower operational pressure. Sanchez et al. recently evaluated the performance of RP-LC columns packed with 1.0–1.4 µm core-shell particles, including the commercially released Kinetex 1.3 µm C18, for metabolic profiling of human urine [29]. This work illustrated that very low minimum plate heights of 2.2 µm could be obtained using columns packed with 1.3 µm particles, corresponding to a plate count of over 450,000 plates/m. Application of the Kinetex 1.3 µm C18 column (50 mm × 2.1 mm I.D.) to metabolic profiling of human urine resulted in the separation of many components and 128 peaks were detected within just 5 min (Figure 1). According to the authors, the use of a 75 mm or a 100 mm long column made with the smallest core-shell particles should resolve many more components in urine samples. Overall, coreshell particles provide the possibility to obtain highly efficient separations on standard HPLC equipment. These columns seem to have very future science group
Recent developments in liquid-phase separation techniques for metabolomics
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attractive characteristics for relatively fast metabolic profiling of complex mixtures, however, the robustness for the analysis of biological samples needs to be demonstrated. A wide range of physiologically relevant metabolite classes can be analyzed by RP-LC systems, however, highly polar and charged metabolites are difficult to retain and often coelute with the void volume. Ion-pairing agents such as tributylamine or hexylamine can be added to the mobile phase of RP-LC in order to improve the analysis for polar compounds [30,31]. However, the use of ion-pair agents in RP-LC–MS may result in severe ion suppression and may contaminate the ion source [32]. An alternative approach is to use reversed-phase pentafluorophenylpropyl (PFPP) columns, in which a pentafluorophenyl ring structure is future science group
attached to the silica over a propyl chain, thereby exhibiting both reversed- and normal-phase retention [33]. For example, Yang et al. developed a RP-LC–MS method using a PFPP column for the profiling of metabolites in bacterial extracts [34]. Highly polar compounds displayed good chromatographic retention on the PFPP column and it was shown that isomers/isobars (e.g., isoleucine/leucine, methylsuccinic acid/ethylmalonic acid) and metabolites of similar structure (e.g., malate/fumarate) were better resolved on the PFPP than on a HILIC column. Another way to increase retention of polar compounds in RP-LC–MS is to use chemical derivatization. For example, Guo et al. developed an approach for the labeling of metabolites containing a primary amine, secondary amine or phenolic hydroxyl group(s) [35]. This www.future-science.com
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Key Term Aqueous normal-phase LC:
LC based on silica hydride-based stationary phases, where hydrophilic partitioning occurs via a gradient-elution program over a wide water-content range.
dansylation labeling improved the chromatographic retention of polar and charged metabolites, which were normally not retained on the RP-LC column, and it also offered one to three orders of magnitude ESI signal enhancement. In general, not all compounds are amenable to chemical derivatization and, therefore, this approach is especially suited for targeted, quantitative RP-LC–MS-based metabolomics studies [36–38]. Complementary
LC stationary phases At present, various complementary LC separation mechanisms are available for the analysis of (highly) polar and/or charged metabolites, such as HILIC, aqueous normal-phase LC (ANP-LC) and ion-exhange LC. HILIC can be regarded as a variant of normal-phase LC as the separation is performed on a polar stationary phase [39–41]. In contrast to normal-phase LC, HILIC uses a polar stationary phase in combination with aqueous organic eluents (typically 3–50% water), which are well suited for efficient ESI-MS analyses. In HILIC, retention of analytes occurs via hydrophilic partitioning of polar compounds between an organicsolvent rich mobile phase and an aqueous layer formed on the stationary phase [42]. The various HILIC modes available can be divided into neutral, charged and zwitter-ionic stationary phases [42–45]. Gika et al. developed a HILICUHPLC–MS method using an Acquity BEH HILIC column (1.7 µm, 2.1 × 150 mm) for metabolic profiling of urine from male Zucker rats [46]. Typical metabolic profiles obtained for the same set of rat urine samples by HILIC- and RP-UHPLC–MS profiles are shown in Figure 2 . Overall, the HILIC-MS analysis provided a higher signal response for the peaks detected in rat urine, while the RP-LC–MS analysis resulted a higher chromatographic resolution and more compounds were observed in rat urine. The HILIC approach was especially suited for the profiling of highly polar metabolites and as such it provided a complementary view on the composition of the urine samples as compared with the results obtained by RP-UHPLC–MS. In order to profile as many metabolites as possible in a given biological sample, Ivanisevic et al. developed a single extraction-dual LC–MS platform comprised of RP-LC and HILIC in order to allow the global profiling of both hydrophobic and hydrophilic metabolites [47]. An aminopropyl-based column was used for HILIC-MS, which provided a good resolution 1014
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for highly polar compounds. The optimized method appeared to be highly reproducible with minimal drifts in retention time over more than 60 h of analysis. The potential of the approach was illustrated for metabolic profiling of bacterial cells, human cancer cells and human plasma. The combined HILIC/RPLC–MS approach generated over 30,000 molecular features in each sample, with the highest number of unique features detected by RP-LC in positive ESI-MS mode and by HILIC in negative ESI-MS mode. Overall, HILIC-MS emerged as a powerful analytical approach for the analysis of (highly) polar metabolites, however, a rather long re-equilibration time between runs is often required for an acceptable performance [48]. Recently, ANP-LC emerged as an effective LC method for the analysis of (highly) polar metabolites [42]. In these columns, hydride groups substitute 95% of the silanol groups on the surface [49]. The less polar surface minimizes the attraction of the stationary phase for water, allowing the column to function in ANP-LC mode. Callahan et al. developed a ANP-LC–MS method using a diamond hydride stationary phase for the rapid profiling of polar metabolites in various biological samples [50]. For the LC separation, 90% acetonitrile with 0.1% ammonium acetate and 0.1% acetic acid was employed as the organic mobile phase and 100% water with 0.1% ammonium acetate and 0.1% acetic acid (pH 3.4) as the aqueous mobile phase. Approximately 1000 compounds were reproducibly detected in human urine and 400 compounds were observed in xylem fluid from soyabean (Glycine max) plants. This work also demonstrated that both ANP-LC and RP-LC are required for nontargeted metabolic profiling. Chen et al. developed an ANP-LC–MS method for nontargeted metabolic profiling of mice plasma in order to discover systemic changes arising from inactivation of xanthine oxidoreductase, an enzyme that catalyzes the final steps in purine degradation [51]. Figure 3 shows the technical reproducibility of the ANP-LC–MS method for nontargeted metabolic profiling of plasma (25-fold diluted), which was determined for 56 repeated analyses. 374 metabolites could be reproducibly detected in the plasma samples. Figure 3 also illustrates that good chromatographic peaks were obtained for various metabolite classes on the ANP-LC column. The metabolomics data obtained corroborated the predicted derangements in purine metabolism and also revealed unexpected perturbations in metabolism of pyrimidines, future science group
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nicotinamides, trypto phan, phospholipids, Krebs and urea cycles. Zhang et al. evaluated for the first time the performance of RP-LC–MS, ANP-LC–MS, and two zwitter-ionic HILIC-MS systems, for metabolic profiling of urine [42]. It was shown that the zwitter-ionic HILIC columns provided the most optimal chromatographic performance and the future science group
greatest metabolic coverage of polar compounds in urine. Kloos et al. performed a study in which three porous HILIC columns (cyano, amino and diol) and three RP-LC columns (core-shell C18, XB-C18 and pentafluorophenyl) were evaluated for relatively fast metabolic profiling [52]. The performance of these columns was assessed using a representative metabolite mixture (comprising www.future-science.com
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Figure 3. Reproducibility of nontargeted metabolic profiling of plasma by aqueous normal phase-LC–MS. (A) Overlay of chromatograms acquired for 56 repeated analyses of a single plasma sample. (B) Profile plot overlay of normalized extracted ion intensities for 374 distinct metabolites, quantified as a function of injection number for the 56 repeated plasma analyses depicted in A. Results depict flat run-to-run variation (mean CV = 6.51%) in the levels of the 374 metabolites with repeated analysis. (C) Peak overlay of 56 repeated assessments of extracted ion counts for detection of some typical plasma metabolites, demonstrating reproducibility of quantification. Reproduced from [51] © PLoS One (2012).
54 compounds) and pooled human urine samples. Figure 4 shows that the amino- and diolHILIC columns provided the most suitable analysis for the metabolite mixture, whereas poor retention for most of the analytes was obtained on the RPLC columns. When applied to human urine samples, the diol-HILIC column provided the most optimal results, that is, the highest number of detectable molecular features, in both ESI(+) and ESI(-) mode at pH 7. Therefore, the diol-HILIC column combined 1016
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with polarity switching in ESI-MS may be considered an attractive tool for fast urinary metabolic profiling as it can lead to a 50% reduction of analysis time. Miniaturized
LC systems Until now, most LC–MS-based metabolomics studies have been performed with RP-LC systems using 2.1 mm I.D. columns [48]. The sensitivity of ESI-MS can be improved by using LC columns with smaller internal diameters. Nano-LC–MS future science group
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Figure 4. Extracted ion chromatograms for all analytes of the metabolite test mixture. Obtained with: (A) pentafluorophenyl-reversed-phase(RP)-LC; (B) amino-HILIC, (C) XB-C18-RPLC; (D) diol-HILIC; (E) C18-RPLC; and (F) cyano-HILIC. The colored bars indicate the type of compounds eluting in the given time window. Each color represents a different class of metabolites. Please see colour figure at www.future-science.com/doi/full/10.4155/BIO.14.51 Reproduced from [52] © Elsevier (2013).
has a higher sensitivity if only small sample volumes are available and this approach is routinely applied in the field of proteomics. In the field of metabolomics, the use of nano-LC–MS would be an attractive approach for in-depth metabolic profiling studies and for the analysis of volume-limited biological samples. Still, the application of nano-LC–MS in metabolomics is very limited. In proteomics, RP-nano-LC–MS is a commonly used approach, however, unlike peptides, (highly) polar and charged metabolites are poorly retained on RP-LC columns. Therefore, Myint et al. evaluated various future science group
stationary phase and mobile phase compositions for nano-LC–MS analysis of cationic metabolites [53]. It was found that polar cationic metabolites were strongly bound to mixed-functional RP with cation exchange mode resin. Different types of coating for the internal wall of the capillary columns were studied to improve peak shapes and to reduce wall effects for highly polar metabolites, and in this regard most optimal results were obtained with the hydrophilic coated TCWAX capillary. The method was used for the analysis of human cerebrospinal fluid (CSF) and more than 100 peaks were detected from 10 µl www.future-science.com
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Ramautar & de Jong of CSF. The same group has also developed a nano-LC–MS method for the comprehensive analysis of anionic metabolites in biological samples [54]. A polyamine-bonded polymerbased apHera™ NH2 column was used for the analysis of anionic polar compounds, such as organic acids and sugar phosphates. However, distorted peak shapes were obtained for multiply phosphorylated or carboxylated compounds. The addition of the metal chelating reagent EDTA to the sample significantly improved peak shapes of multiply charged anionic compounds. LODs of some polar anionic metabolites in full-scan mode ranged from 0.19 to 2.81 pmol. The method was applied to the analysis of Hela cells, mouse brain extracts, plasma and CSF, and revealed that phosphorylated metabolites were abundant in HeLa cells and brain, while plasma and CSF mostly contained organic acids. Recently, Kiefer et al. developed a nanoscale ion-pair RP-LC–MS method for the profiling of anionic compounds in cell extracts [55]. Anionic metabolites were separated on a 100 µm I.D. C18 column using tributylamine as the ionpairing reagent and methanol as the mobile phase. A basic pH (9.4) of the mobile phase was required to obtain adequate retention and efficient chromatographic peaks (Figure 5) at a low concentration of tributylamine (1.7 mM). LODs determined for 54 metabolite standards were in the upper attomole range, and compared with conventional ion-pair RP-LC–MS and HILICMS methods using 2 mm I.D. columns the LODs were in general 100 times lower with the nanoscale ion-pair RP-LC–MS approach. The method was applied to metabolic profiling of cell extracts from the methylotroph model organism Methylobacterium extorquens AM1. So far, microfluidic or chip-based LC–MS systems have been primarily used for bioanalytical applications often focused on the analysis of a few analytes [56,57]. Microfluidic separations are usually performed with microchip CE systems as they are relatively easy to implement, whereas downscaling of LC remains a challenging process [57,58]. CE Separation optimization CE emerged as a highly efficient and attractive separation technique for the profiling of polar and charged metabolites in biological samples [59–64]. In comparison to RP-LC and HILIC, CE can provide complementary metabolomics information as recently demonstrated for the
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analysis of colon cancer cells, K562 leukemia cells and mouse urine [65–67]. Capillary zone electrophoresis, normally referred to as CE, is the main CE separation mode used in CE–MS for metabolomics [60]. In this mode, the capillary is filled with a volatile background electrolyte (BGE) only and analytes are separated according to differences in their electrophoretic mobility. For CE–MS-based metabolomics, ESI is the most widely used ionization technique. As ESI is prone to analyte signal suppression, the BGE should not contain nonvolatile constituents and/or surfactants. Moreover, nonvolatile constituents may cause source contamination and high background signals. Therefore, volatile BGEs, such as formic acid, acetic acid and/or ammonium acetate, are typically used for CE–MS-based metabolomics studies. In general, low-pH (~2) and high-pH (~9) BGE conditions in combination with ESI(+) and ESI(-) are used for the analysis of cationic and anionic metabolites, respectively [68–70]. Soga and co-workers developed the first CE–MS approach for global metabolic profiling of bacterial extracts [14,71]. In this study, distinct CE–MS methods were developed for cationic and anionic metabolites, that is, cationic metabolites were analyzed with a bare fusedsilica capillary using 1 M formic acid (pH 1.8) as BGE, whereas anionic metabolites were analyzed with a cationic polymer-coated capillary using 50 mM ammonium acetate (pH 8.5) as BGE. This approach allowed the analysis of more than 1600 metabolites in bacterial extracts. The CE–MS method for cationic metabolites developed by Soga and co-workers is now used by other research groups [69,72–74]. A stable CE–MS method is of pivotal importance for metabolomics studies where multiple biological samples have to be profiled and compared. In CE, adsorption of matrix components and/or analytes to the capillary wall may cause changes in the electro-osmotic flow that, in turn, can lead to irreproducible migration times. This aspect is especially critical when bare fused-silica capillaries are employed [75]. In such cases, it is important to employ an extensive washing step between successive analyses to allow the ana lysis of biological samples with minimal sample pretreatment. Another way to improve migration time reproducibility in CE is by coating the inner wall of fused-silica capillaries with polymers, as has been demonstrated for various CE–MS-based metabolomics studies [71,75,76]. In this context, charged capillary coatings are future science group
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ATP tR 20.2
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Figure 5. Extracted ion chromatograms of selected metabolites detected in the cell extract of Methylobacterium extorquens AM1 obtained with nanoscale ion-pair reversed-phase-LC–MS using a 100 µm internal diameter C18 column with tributylamine as ion-pairing reagent. Metabolites were extracted with cold acidified acetonitrile. Injected amount corresponds to 50 ng of biomass cell dry weight. HB: Hydroxybutyryl; PEP: Phospoenolpyruvate; tR: Retention time. Reproduced with permission from [55] © American Chemical Society (2011).
very attractive as they can be used for changing the direction and magnitude of the electro-osmotic flow in CE–MS using normal or reversed CE polarity, providing CE systems future science group
for anions and cations [60,69]. The potential of these coated capillaries was recently assessed for metabolic profiling of rat urine [69]. In this study, fused-silica capillaries were coated with a www.future-science.com
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Ramautar & de Jong bilayer of polybrene (PB) and polyvinyl sulfonate, or with a triple layer of PB, dextran sulfate and PB. The coated capillaries were evaluated at low and high pH conditions, thereby providing separation conditions for basic and acidic compounds. The use of the coated capillaries resulted in a good migration-time repeatability (RSD
