Coupling ultra-high-pressure liquid chromatography with mass spectrometry

Trends in Analytical Chemistry, Vol. 29, No. 1, 2010

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Coupling ultra-high-pressure liquid chromatography with mass spectrometry Davy Guillarme, Julie Schappler, Serge Rudaz, Jean-Luc Veuthey In recent years, different approaches have been taken to improve chromatographic performance in terms of analysis time and/or resolution. The use of columns packed with sub-2lm particles in ultra-high-pressure liquid chromatography (UHPLC) has become a technique of choice in many laboratories. Furthermore, for the analysis of complex matrices (e.g., biological fluids, plant extracts, and food and environmental samples), coupling UHPLC with mass spectrometry (MS) or tandem MS (MS2) provides a powerful analytical tool. This review describes major advances in the field of UHPLC-MS and UHPLC-MS2. We strongly emphasize the possibility of speeding up bioanalysis, drug metabolism, and multi-residue screening assays, while maintaining qualitative and quantitative performance equivalent to HPLC-MS and HPLC-MS2. We also report the possibility of gaining additional information in metabolomics, using high-resolution UHPLC with a time-of-flight analyzer. The studies summarized are discussed in this review in terms of throughput increases and resolution enhancements afforded by UHPLC. In addition, we highlight the impact of UHPLC conditions on MS detection capabilities (e.g., acquisition rate, limits of detection and matrix effects). ª 2009 Elsevier Ltd. All rights reserved. Abbreviations: DT, Dwell time; ESI, Electrospray ionization; FWHM, Full width at half maximum; HILIC, Hydrophilic interaction liquid chromatography; HLB, Hydrophilic-lipophilic balanced; IP, Identification point; LLE, Liquid-liquid extraction; LLOQ, Lower limit of quantitation; MCX, Medium cation exchange; ME, Matrix effect; MRL, Maximum residue limit; NMR, Nuclear magnetic resonance; PP, Protein precipitation; QqTOF, Quadrupole/time of flight; RP, Reversed phase; RSD, Relative standard deviation; SPE, Solid-phase extraction; SIM, Selected ion monitoring; SRM, Selected reaction monitoring; TOF, Time of flight; UHPLC, Ultra-high-pressure liquid chromatography; UPLC, Ultra-performance liquid chromatography Keywords: Bioanalysis; Drug metabolism; Mass spectrometry (MS); Metabolomics; Multi-residue screening; Time of flight; Ultra-high-pressure liquid chromatography (UHPLC); Ultra-performance liquid chromatography (UPLC); UHPLC-MS; UPLC-MS

1. Introduction Davy Guillarme*, Julie Schappler, Serge Rudaz, Jean-Luc Veuthey School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard dÕYvoy 20, 1211 Geneva 4, Switzerland

*

Corresponding author. Tel.: +41 22 379 64 77; Fax: +41 22 379 68 08; E-mail: [email protected]

Over the past decade, advances in conventional HPLC have speeded up separation and/or increased peak capacity (i.e. the number of peaks that can be separated in a given time window). Several approaches toward improvement have been described in the literature [1,2], e.g., using: (i) high temperatures (up to 200C) to decrease mobile-phase viscosity and polarity; (ii) monolithic supports instead of columns packed with silica particles; and, (iii) small particles.

0165-9936/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.09.008

From these, columns packed with sub2-lm particles in ultra-high pressure conditions (UHPLC) have emerged in a powerful approach, particularly because of the ability to transfer existing HPLC conditions directly. In addition, the reduction of particle size down to sub-2 lm (compared to 5-lm particles) allows either speeding up of the analytical process by a factor of 9 while maintaining similar efficiencies or a theoretical nine-fold increase in efficiency for a similar run time [3]. Fig. 1 demonstrates the growing interest in UHPLC, with around 600 papers published since 2003. The combination of UHPLC with an MS detector appears to be a suitable approach

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Figure 1. Number of papers published each year in the field of UHPLC and UHPLC-MS, since 2003. Before this date, only a few papers were published (by JorgensonÕs and LeeÕs groups). Black bars were obtained with keywords ‘‘UPLC’’ and ‘‘UHPLC’’, while white bars were obtained with an additional filter (keyword ‘‘MS’’). Source: Scifinder scholar 2007 search of the Chemical Abstracts database 2003–09. Date of information gathering: May 2009.

that fulfills key requirements in terms of sensitivity, selectivity, and peak-assignment certainty for the rapid determination of analytes at low concentrations in complex matrices. However, due to the very narrow peaks produced by UHPLC (commonly 1–3 s), coupling with MS devices can be critical. For this reason, specific quadrupole-based instruments that possess improved acquisition rates were commercialized for UHPLC hyphenation. With this new generation of analyzers, full-scan acquisition was enhanced up to 10,000 m/z/s and DT reduced to 5 ms for SIM and SRM modes [4]. Aside from quadrupole-based analyzers, TOF instruments are also well adapted to record and store data over a broad mass range without compromising sensitivity. Lee et al. [5] first investigated the coupling of a TOF instrument with a home-built UHPLC system able to withstand pressures up to 3600 bar. They demonstrated high-speed separations (ca. 1 min) of combinatorial chemistry samples, pharmaceutical compounds, and herbicides. Later, a significant proportion of the published works involving commercial UHPLC-MS systems were carried out with TOF or QqTOF instruments due to their elevated acquisition rate over a broad mass range. Fig. 1 shows that MS (or MS2) is the detector of choice for more than 60% of separations carried out by UHPLC. To date, only two reviews have been published regarding the benefits, the limitations and the applications of UHPLC [2,3], while there has been no extensive discussion on its coupling with MS. In this article, we report on the current state of the art of UHPLC-MS and UHPLCMS2, highlighting the throughput increase and/or the resolution improvement afforded by UHPLC technology and its impact on MS-detection capabilities. For this purpose, we considered three promising fields of applications: 16

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bioanalysis and drug metabolism to perform highthroughput separations with tandem MS in the SRM mode and QqTOF analyzers (more than 50% of the reported applications); (ii) rapid multi-residue screening in environmental, biological, and food matrices, using either quadrupole-based instruments in SRM mode or TOF analyzers (10% of the published papers); and, (iii) metabolomics to gain maximal information from a single run, taking advantage of the very high chromatographic resolution of UHPLC combined with the exact-mass measurement of TOF-MS (about 20% of the UHPLC-MS publications). As sample preparation is a non-negligible part of the whole analytical process, irrespective of the application and the analyzer, we critically discuss it throughout the manuscript in terms of required time and observed MEs.

2. UHPLC-MS for bioanalysis and drug metabolism Bioanalytical methods used for the quantitative determination of drugs and their metabolites in biological fluids play a significant role in evaluating and interpreting bioavailability, bioequivalence, pharmacokinetics, and toxicokinetics. Challenges in bioanalytical laboratories include development of fast LC-MS methods able to separate closely related compounds (e.g., analytes and metabolites) from endogenous components. The final objective consists of releasing a bioanalytical method that meets the rigorous criteria set by authorities in terms of selectivity, accuracy (trueness and precision) and linearity [6], so suitable sample preparation and chromatography are required to ensure robust, accurate bioanalytical methods. Concerning selectivity, and as described in the regular guidelines for bioanalytical method validation proposed by the US Food and Drug Administration (FDA), assessment and quantitation of MEs are important issues. Because UHPLC enhances chromatographic resolution overall, co-elution is reduced, and that, in turn, leads to a decrease of ion suppression, improving MS sensitivity and reliability. However, since UHPLC greatly enhances separation throughput and resolution, base peaks as narrow as (or less than) 1 s can be obtained. This creates practical issues for bioanalytical applications using MS, as sufficient data points (e.g., >15 points per peak) are essential to ensure reliable quantitation. In drug-metabolism experiments, there are also many challenges due to the complex nature of biological matrices and the diversity of metabolites produced. Reactive or toxic metabolites have to be detected and identified as early as possible in the drug-discovery process to reduce compound attrition in late-phase development. In order to eliminate false positives and to determine unexpected metabolites, high chromatographic

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resolution and mass accuracy on fragmentation patterns are key requirements. UHPLC with high-resolution analyzers (e.g., QqTOF) is particularly suitable to fulfill both tasks in a high-throughput environment. 2.1. Sample preparation and matrix effects Highly selective sample preparation is an important step for minimizing MEs and ionization alterations that could occur with ESI-MS detection. Chambers et al. suggested a systematic, comprehensive strategy to reduce MEs in bioanalytical LC-MS and LC-MS2 assays [7]. Experimental factors were controlled to ensure valid comparison of results, and several criteria (e.g., analyte recovery and MEs) were used to compare treatments of plasma samples (e.g., PP) and sample-preparation methods (e.g., LLE and SPE). One of their major conclusions was that polymeric mixed-mode SPE, combined with appropriate mobile-phase pH and UHPLC technology provided significant advantages for reducing MEs from plasma-matrix components and improving ruggedness and sensitivity of bioanalytical methods. They also emphasized that a statistically-significant decrease in MEs was brought about by UHPLC, when compared with traditional HPLC. Another study [8] also supported these conclusions. Despite these observations, one-third of the reported applications in UHPLC-MS bioanalysis were carried out with LLE (one-third with SPE and one-third with only PP). Considering the reduction in analysis time offered by UHPLC technology, sample preparation has become the limiting step in terms of total analysis time. Numerous publications still involve traditional sample-preparation procedures, which dramatically increase total analysis time. A few authors have suggested solutions, while maintaining sufficient selectivity in sample preparation. To date, only LLE based on a 96-well plate format has been used prior to UHPLC-MS and UHPLC-MS2 bioanalysis, allowing for selective, sensitive and highthroughput analyses. For example, Licea-Perez et al. used a semi-automated LLE system for the determination of oral contraceptives in human plasma [9], while Yadav et al. used a completely automated LLE system for the quantitation of methoxsalen in human plasma [10]. Licea-Perez et al. [9] reportedly improved throughput by a factor of three compared to conventional HPLC, allowing sample run from four 96-well plates in an overnight sequence [ca. 300 samples in addition to blanks, calibration standards, and quality controls (QCs)]. This strategy is particularly appropriate to support pharmacokinetic studies, in which large numbers of samples generated from clinical studies have to be extracted and analyzed. Yadav et al. [10] exhibited a run time of 1.5 min, and the automated LLE procedure lowered the time needed for sample preparation three-fold compared to a manual procedure. The validated UHPLC-MS and UHPLC-MS2

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method established an LLOQ (ca. 1 ng/mL) sufficient to conduct a bioequivalence study of methoxsalen in healthy human volunteers after oral administration of 10 mg. Apart from these two attempts, few studies have evaluated the possibility of speeding up sample preparation using automated well-plate formats. 2.2. Rapid bioanalysis by UHPLC-MS2 using quadrupole analyzers When coupling UHPLC technology with quadrupole analyzers, there are often insufficient numbers of data points across the narrow UHPLC peaks. At least 15 points are usually required to define an LC peak for accurate, reliable quantitation. Several critical examples can be found in the literature. For example, Petsalo et al. recently published an UHPLC-MS and UHPLC-MS2 method for analyzing nine drugs and their respective metabolites in urine, with a 4-min gradient [11]. After reaction with a CYP450 enzymatic system, an ESI source was operated simultaneously in positive- and negative-polarity modes, and DTs of 20–30 ms were selected for each SRM transition. Average peak widths of 4 s were obtained experimentally, so only 6 points were acquired to define peaks, and that could limit performance, particularly at concentrations near the LLOQ. To accommodate the small UHPLC peak widths, it is straightforward to reduce DTs when many SRM transitions have to be monitored [4]. On the one hand, this can enhance the number of acquired data points and positively affect measurement precision. On the other hand, this can result in loss of sensitivity as the signal-tonoise ratio is approximately proportional to the square root of DT. For instance, low DTs were used in a study performed by Churchwell et al. to achieve 15 data points per peak [12]. Good quantitative performance was obtained using the fastest possible scanning rate (i.e. 5 ms), while sensitivity remained sufficient because of the elevated UHPLC efficiency and the high selectivity afforded by the MS2 instrument. Another option involves using various time windows during the acquisition, as recently suggested by Berg et al. [14], who presented a validated UHPLC-MS and UHPLC-MS2 method for the determination of eight opiates, cocaine, and metabolites in urine. Because six different deuterated internal standards were added and at least two SRM transitions per analyte were acquired, the acquisition time was divided into four different time windows. Consequently, the analyses were performed in less than 4 min with a DT of 20 ms, maintaining a sufficient data points across the peaks for quantitation, as shown in Fig. 2. LLOQs down to 8 ng/mL in urine were obtained with RSD values <10%. To circumvent DT reduction, Li et al. suggested a clever ‘‘peak-parking’’ strategy, which involved reducing the flow rate during peak elution and thus extending the useful MS-acquisition window for quantitative bioanahttp://www.elsevier.com/locate/trac

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Figure 2. Time-window strategy for enhanced performance by UHPLC-MS and UHPLC-MS2 for bioanalytical studies. Chromatograms obtained by monitoring SRM transitions of selected opiates, cocaine, and metabolites at 0.10 lM in positive electrospray ionization mode (Adapted from [14], with permission from Elsevier).

lytical assays [13]. The high-throughput advantage of UHPLC was maintained since there was no significant increase of the total analysis time. This approach was successfully applied for lansoprazole in human plasma 18

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with a 100 ms DT, affording an LLOQ of 1 pg/mL with an analysis time of 30 s. Fig. 3(A) shows the chromatogram of lansoprazole without ‘‘peak parking’’, and Fig. 3(B) that obtained with ‘‘peak parking’’. Fig. 3(C)

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Figure 3. ‘‘Peak parking’’ strategy for enhanced performance by UHPLC-MS and UHPLC-MS2 in bioanalytical assays. Lansoprazole chromatograms obtained under (A) regular UHPLC conditions and (B) ‘‘peak parking’’ UHPLC conditions obtained by (C) reducing the UHPLC flow rate during peak elution (Adapted from [13], with permission from Wiley).

shows that the duration of the ‘‘peak-parking’’ window was set at three-fold the base peak width, which was increased by 50% (from 2.8 s to 4.2 s), allowing more MS data points (from 14 to 21). Consequently, the peakarea RSD at the LLOQ was lowered to 13% with this strategy (in contrast to 38% without ‘‘peak parking’’). However, it is worthwhile noting that this strategy could be applied only to a limited number of targeted analytes. Finally, relatively simple analyses dealing with only a few compounds can be carried out with high DTs, generating high sensitivities without compromising peak definition. For instance, Pedraglio et al. developed a UHPLC-MS and UHPLC-MS2 method for the analysis of a pre-clinical antitumor candidate [15]. A DT of 200 ms was used for SRM transitions of the drug candidate and its internal standard. This methodology showed an LLOQ down to 0.1 ng/mL in plasma with RSD <12%, allowing quantitation of the 24-h samples. Results demonstrated a different elimination phase, significantly affecting the evaluation of the pharmacokinetic parameters and oral bioavailability.

2.3. High-resolution drug metabolism by UHPLC-MS using QqTOF analyzers In metabolite screening, numerous metabolites have to be detected in a single run, so long chromatographic separations were initially implemented to avoid co-elution, taking into account issues such as sensitivity loss from ion suppression and the need to resolve structural isomers (e.g., hydroxyl metabolites) [16,17]. Castro-Perez et al. were the first to report the use of UHPLC technology with a QqTOF analyzer in the early drug-discovery process, providing high-quality data within very short time frames [18]. The power of UHPLC compared to conventional HPLC was illustrated by the analysis of the in vitro metabolism of dextromethorphan, which undergoes dealkylation in two positions, giving three major phase I metabolites. By UHPLC, both demethylated metabolites were clearly resolved, and the di-demethylated metabolite was easily detected in less than 3 min, while a new peak was also observed. Thanks to the QqTOF-MS analyzer, the new peak was identified as a dealkylated glucuronide conjugate. This study http://www.elsevier.com/locate/trac

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emphasized the improved resolution, in terms of chromatographic and mass-spectral quality, and the concomitant gain in sensitivity afforded by the UHPLCQqTOF-MS system. These features were explained by the combination of reduced peak width (and consequent increase in analyte concentration) and low ion suppression that may result from the co-elution of metabolites and endogenous compounds. Finally, the authors successfully applied their methodology to the analysis of a wider library of compounds that were extensively hydroxylated, forming numerous metabolites of varying abundance. Others showed the ability of UHPLC-QqTOF-MS in drug-metabolism studies. Walles et al. investigated the benefits and the drawbacks of three methods for fast metabolite identification using alternative MS experiments (e.g., MSE) [19–21]. Columns packed with sub-2lm particles coupled to QqTOF-MS were compared for the high-throughput determination of major metabolites of compounds with high, medium, and low metabolic turnover, in a 384-well-plate format. The high efficiency attributable to UHPLC was the key to the successful identification of isobaric metabolites, which could not be distinguished with accurate-mass data generated by QqTOF, as they had identical elemental composition and often identical MS2-fragmentation patterns. Although accurate mass did not clearly assist in the identification of isobaric metabolites, it eliminated false-positive isobaric interferences, based on their mass defects (>5 ppm). Finally, the time spent for structure elucidation created additional bottlenecks. Although analytical run times were below 3 min, processing a whole 384-well plate took over 20 h, unless automated algorithms were used (then total analysis time was reduced to 7 h).

3. UHPLC-MS for multi-residue screening Multi-residue screenings are generally developed for rapidly assessing the presence of contaminants in a complex sample. The method developed for multi-component screening should be able to detect as many pollutants as possible in a single analytical run, so RP-LC coupled with MS2 remains the gold standard, as recommended in regulatory texts (e.g., SANCO/10852000) that establish MRLs and the number of required IPs. In recent years, there have been some important improvements in this field. First, the possibility of replacing conventional HPLC with UHPLC was evaluated to improve analysis throughput and reduce response time. Second, the availability of TOF analyzers, providing accurate-mass measurements, represented a promising alternative to triple-quadrupole analyzers, particularly for screening purposes. A detailed discussion on MS analyzers would be outside the scope of the present re20

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view, but some additional information can be found elsewhere [22,23]. Multi-residue screenings by UHPLCMS2 and UHPLC-TOF-MS have been applied to a wide variety of analytes and matrices, including: (i) doping agents [24,25] and veterinary drugs [26] in biological matrices; (ii) drugs [27], pesticides [28] and herbicides [29] in environmental matrices; and, (iii) veterinary drugs [30], drugs [31] and pesticides [32–34] in food samples. Because all of these applications involve a similar assay format, emphasis is put on environmental matrices, where there has been the greatest impact, while continuous growth is observed in other fields of application. 3.1. Sample-preparation procedures Sample preparation is one of the most critical steps of the whole analytical process in multi-component analysis, because compounds often possess different physicochemical properties. As the number of target analytes can vary between dozens and more than hundreds [26,30], simultaneous extraction of multiple compounds from a complex matrix should be generic. This usually involves a compromise in selecting experimental conditions for maximal recovery of individual analytes. SPE is largely used prior to UHPLC-MS, except in the determination of doping agents or veterinary drugs in urine, where a simple dilution (i.e. dilute and shoot approach) was considered [24–26]. However, this approach could not be applied to determine very low levels of residues because of sensitivity issues. Regarding environmental matrices, the use of SPE prior to UHPLC-MS has been reported for only HLB RPs (e.g., Oasis HLB) or polymeric mixed-mode sorbents (e.g., Oasis MCX) [27–29]. These supports were selected because they allow simultaneous extraction of acidic, neutral and basic analytes from water. In addition, the extract could be concentrated, enabling detection of analytes at very low concentration levels. Extraction recoveries after SPE were systematically determined for various types of analyte in wastewater and surface water and comprised 70–110% for both pesticides [28] and herbicides [29], after extraction on HLB support. However, recoveries were more critical for pharmaceuticals with HLB or MCX supports, particularly for the simultaneous extraction of acidic and basic drugs [27,35,36]. As mentioned earlier for bioanalytical applications, SPE was carried out only in the conventional cartridge format for multi-residue screening, and no attempt was made to reduce the time needed for sample preparation. 3.2. UHPLC conditions Due to the large number of compounds investigated, conventional LC runs can be relatively long, particularly to avoid peak co-elution leading to MEs. It is indeed important to attain sufficient chromatographic

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resolution, to minimize co-elution of compounds with close m/z ratios and similar fragmentation pathways, and to decrease ionization alterations. For multi-component screening, gradient conditions have been systematically selected to elute analytes over a wide polarity range. Formic acid was generally added to the mobile phase, just as it is commonly employed in LC-MS. Only Barcelo et al. reported the use of suitable buffer solutions with a controlled pH value (e.g., acetate pH 4.8) for proper tuning of chromatographic selectivity [36,37]. To screen compounds by UHPLC, 50–100-mm column lengths were used with gradient times of 5–15 min, followed by a re-equilibration time of 2–4 min. The selection of column length was made in line with the gradient duration, since the longest column did not systematically provide the highest peak capacity in UHPLC. Indeed, a 50-mm column has been shown to be optimal for gradient times shorter than 7 min, while a 100 mm column only proved to be beneficial for longer gradients [38]. Finally, the 2.1-mm I.D. column was often preferred to limit extra-column band broadening contributions. Only Kasprzyk-Hordern et al. [27,35] reported the successful screening of more than 50 pharmaceuticals in wastewater using a 1-mm I.D. column. Despite reduced mobile-phase and analyte consumptions, the peaks were significantly broadened and distorted with a 1-mm I.D. column, as expected from theory, due to the influence of external volume contributions. In conclusion, an approximate reduction of 3–5-fold in analysis time has been observed in UHPLC compared to conventional HPLC methods. In addition, an equivalent or higher chromatographic resolution has also been reported [29,36,37]. Such improvement could be attributed to increased peak capacity, but also to the change in column selectivity. Indeed, comparisons were often not made using strictly the same column chemistries. 3.3. Triple-quadrupole vs. TOF analyzers Because of the huge number of analytes to be analyzed in multi-residue screening, MS instruments must possess high acquisition rates. In this context, only two types of MS analyzer have been employed for multi-residue screening in UHPLC, namely MS2 operating in the SRM mode (70% of the papers) and TOF-MS (around 30% of the papers). Triple-quadrupole instruments are particularly well suited for targeted analysis and provide excellent sensitivity and selectivity. Indeed, modern MS2 instruments can attain high signal-to-noise ratio even with short SRM DT (i.e. as low as 5 ms), but a compromise needs to be found between sensitivity and acquisition rate, since the number of MS2 transitions in a single SRM segment is limited. When analyzing over 20 compounds, the MS2 method should be segmented into various time-schedule win-

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dows containing different SRM channels and time intervals. This approach is particularly useful in multiresidue screening because of the elevated number of analytes. As example, a SRM window method with a DT of 15 ms was considered for the UHPLC analysis of 26 pesticides in wastewater (Fig. 4) [28] or 53 pesticides in fruits and vegetables [34], while it was reduced to 5 ms for the determination of 130 prohibited substances in urine [24]. The main limitation of this procedure came from elution-time variability. Indeed, when analyzing hundreds of analytes, time windows became very thin and analytes sometimes move from the originally-defined window, requiring some adjustments. Another procedure was reported to obtain a good compromise between speed and sensitivity, taking into account the achievable DT of the MS2 instrument. It involved several successive injections of the unknown sample, while the number of target analytes followed in the SRM method was different and limited to 10–20 for each run. This allowed the maintenance of a sufficient number of data points across a peak for quantitation (ca. 10–15 points per peak). This time-consuming strategy has been implemented {e.g., in the determination of 48 drugs in wastewater (DT = 20 ms) [39] or 90 pesticides in fruit juices (DT = 15 ms) [32]}. TOF instruments provide an attractive alternative, since they can provide elevated mass resolution (>10,000 FWHM) and accuracy over a broad mass range. In addition, TOF instruments are suitable for both targeted and non-targeted determination. In multi-residue screening, response times of 0.2–0.3 s (equivalent to 3–5 spectra per s) were considered for the determination of more than 100 veterinary drugs in various matrices [26,30]. To attain a sufficient level of selectivity, a mass window of ±5–20 amu was selected to extract target analytes from the full spectrum. In addition to its potential high acquisition rate, TOF could: (i) help structural elucidation of non-targeted compounds based on accurate-mass measurements and isotopic patterns; and, (ii) follow an unlimited number of compounds, with no restriction of acquisition rate. However, an important limitation of TOF-MS, compared to MS2, is the lower sensitivity [26], as well as the elevated cost. An in-depth discussion on the advantages and the limitations of both analyzers in UHPLC multicomponent analysis can be found elsewhere [33]. Finally, hybrid QqTOF-MS instruments combine the advantages of both technologies, offering great potential for the screening, the confirmation, and above all the structural elucidation of unknown compounds in complex matrices due to valuable fragmentation information. At the moment, only Barcelo et al. have reported the use of such instrumentation with UHPLC for fast, powerful multi-residue determination [36,37]. http://www.elsevier.com/locate/trac

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3.4. Matrix effects and quantitative performance: HPLC-MS vs. UHPLC-MS The determination of MEs is an important but tedious task in multi-residue screening because of the number of compounds to be determined and the possible matrix variability. Due to the narrow peaks and high resolution afforded by UHPLC, analyte co-elution is restricted, reducing ionization alterations. An interesting study [40] showed that the MEs for 9 basic drugs in surface water were minor by UHPLC compared to conventional HPLC using identical analytical conditions. This was confirmed by other studies involving the determination of 24 pesticides in water samples [28] or 29 pharmaceuticals in wastewater [36]. For a larger number of compounds (>30), signal suppression in ESI-MS was often observed for a few compounds [27], decreasing sensitivity but maintaining acceptable performance. Quantitative performances (i.e. trueness and precision), as well as LLOQs, were found equivalent (or of the 22

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same order of magnitude) in both HPLC and UHPLC, and the LLOQs were in agreement with MRLs set in regulatory texts. Indeed, with an SPE-UHPLC-MS2 method, LLOQs obtained for the determination of contaminants in wastewater and surface water were around 0.1–1 ng/L for pharmaceuticals [27,35,37,39] and 5–10 ng/L for herbicides and pesticides [28,29].

4. UHPLC-MS in metabolomics The metabolome refers to the whole collection of lowmolecular-weight compounds (i.e. <1000 g/mol) (e.g., metabolic intermediates, secondary metabolites, and other signaling molecules) contained within a biological sample [41,42]. A metabolomic experiment is a multistep process (i.e. sample collection and preparation, separation, detection, and data mining) for comprehensive identification and quantitation of all metabolites in a

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complex biological system [43,44]. Due to the complexity of metabolomic samples and because metabolites can be found at very low concentrations, there is a great need for analytical systems that provide high resolution and increased sensitivity. For this reason, the value of UHPLC-TOF-MS and UHPLC-QqTOF-MS platforms has been demonstrated in a number of studies, by comparing the number of signals detected in UHPLC vs. conventional HPLC. Such analytical tools have been applied for the global metabolic fingerprinting and profiling of: (i) human and animal biological fluids, including rat urine [45–47], human urine [48–50], and human serum [47,51]; and, (ii) plant extracts (e.g., Arabidopsis thaliana [52,53] and Panax herbs [54–56]). In this review, we particularly emphasize separation and detection conditions used in metabolomics. Sample preparation and data mining (i.e. pattern-recognition methodology using multivariate statistical analysis) remain of prime importance [57], but are outside the scope of the present review. 4.1. Human and animal tissues The strong reduction in analysis time afforded by UHPLC, compared to conventional HPLC, opens up the possibility of high-throughput screening for metabolomic fingerprinting. Alternatively, a longer UHPLC run can be envisaged to increase the amount of information, which is essential for metabolomic profiling. Wilson and co-workers have applied this technique to rat and mouse urine since 2005 [45–48]. Initially, biological fluids were analyzed on a 50-mm column packed with 1.7-lm particles using TOF-MS. From a chromatographic point of view, the average peak widths were around 1 s, generating a peak capacity of 60 for UHPLC runs of 1 min. With the additional TOF-MS information, a total of 1000 features (i.e. signals observed with specific m/z and RT that differ from the noise and which can be considered as a variable for data treatment) were determined in rat urine. This number was equivalent to or better than that achieved on a conventional HPLC material, but with a 10-fold reduction in analysis time. The proposed UHPLC-MS methodology presented some obvious advantages, and was applied to normal and obese Zucker rodents to provide rapid discrimination between age, strain, gender, and diurnal variation of rats and mice. Further examples on various rat, mouse or human plasmas and urines confirmed the potential of UHPLC-MS for metabolomics, with improvement of sensitivity, peak capacity, and analysis time as main objectives [46–51]. For these works, the UHPLC column length was always between 50 and 100-mm, with a 2.1-mm I.D. Generally, a step elution gradient for 10 min at 400 lL/min provided a good compromise for urinary metabolite profiling [50], while a 30 min run time offered 30% more features [49].

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The study performed by Nordstrom et al. on the quantitative analysis of endogenous and exogenous metabolites in human serum confirmed these results [58]. Indeed, UHPLC provided 20% more detected components compared to HPLC. Finally, it was demonstrated that UHPLC displayed some additional benefits over HPLC, such as better retention-time (RT) reproducibility and signal-to-noise ratios. Regarding detection, TOF-MS or QqTOF-MS analyzers remain the gold standards to gain maximal information from a single run using scan times of 0.1 s (for UHPLC separations <5 min) to 0.3 s (when tgrad > 5 min). An LTQ-Orbitrap device was also evaluated in conjunction with UHPLC for the successful metabolomic profiling of serum [51]. With this analytical platform, mass resolution was as high as 30,000 FWHM and mass accuracy better than 2 ppm, allowing a potential assessment of disease biomarkers in complex samples. Despite these evident benefits, some problems related to quantitative determination (i.e. trueness and precision) were observed when analyzing hundreds of metabolites contained within a complex biological sample. This topic was extensively discussed in several recent publications [48,59], and reported RT drifts and mass shifts in UHPLC-TOF-MS were acceptable. For example, over the course of 600 injections of a pooled QC sample over a 5-day period, the drift in RT was only 0.03 min and the mass shift <4 ppm [50]. These results were confirmed in another study [49], where RT and mass shifts were estimated at 0.03 min and <5 ppm, respectively. Finally, it was also found that the first few injections on the UHPLC system were not representative and should be discarded [48], so, based upon the assessment of QC samples, UHPLC-MS provides a powerful, repeatable method for the global metabolomic analysis of biological samples [48,59]. Finally, two interesting strategies, namely application of elevated temperature in UHPLC and use of HILIC material packed with sub-2lm particles, were proposed to extend further the range of UHPLC in metabolomics. For the sake of comparison, a similar sample was investigated (the urine of normal and obese Zucker rats). First, the possibility of using UHPLC at an elevated temperature was evaluated as a means of improving chromatographic performance and obtaining alternative selectivity for complex samples [47]. Indeed, the application of high temperatures in UHPLC was recently shown to permit delivery of mobile phase at higher flow rates, thereby reducing analysis times, or to increase column length and obtain higher resolutions [60]. Wilson et al. investigated thermal gradients (up to 180C) using only pure water as the eluent for the global-metabolite profiling of plasma and urine, with two 100-mm columns connected in series and a 21-mingradient run [47]. The columns used in their study did not show any loss of performance after >250 injections http://www.elsevier.com/locate/trac

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of urine under elevated temperature conditions. The results demonstrated the potential of such an approach, with an increased number of ions detected when a thermal gradient was applied (around 2000 features), as shown in Figs. 5A and 5B. However, the metabolomic profiling of plasma samples was not satisfactory, because the eluotropic strength of water was not sufficient, even at 180C. Second, the use of HILIC columns packed with sub2lm particles as a complementary technique for the retention of polar compounds in urine-metabolite profiling was also recently reported by Wilson et al. [46]. As shown in Figs. 5C and 5D, some obvious differences were observed for the profiles obtained in HILIC-UHPLC (95– 50% ACN in 12 min) vs. RP-UHPLC (5-100% ACN in 11 min). However, the elution order was essentially reversed between both modes, with some increase in retention and a change in selectivity for certain classes of very polar analytes. In addition, the signal intensity was

improved in HILIC mode (ca. four-fold), because of the higher organic content of the solvent, which was particularly beneficial to the ESI process [4]. However, the number of features tracked in HILIC was lower than in the RP dataset (i.e. 2098 vs. 3284), but the markers for sample-type differentiation differed, depending on the technique. This demonstrated the complementary nature of both separation modes, providing improved metabolome coverage. Until now, the main limitation of this approach remained the lack of retention for most acidic metabolites. 4.2. Plant extracts Because of the complexity and the chemical diversity of metabolites present in natural products, metabolomics is also gaining interest in phytochemistry. The use of UHPLC-TOF-MS and QqTOF-MS for large-scale, untargeted metabolic profiling has been recently reported by two research groups. Jia and co-workers reported the

Figure 5. Typical urine profile of Zucker rat in various analytical conditions. (A) Conventional UHPLC gradient under isothermal conditions (58C), (B) Isobaric elevated temperature UHPLC analysis, with thermal and flow-rate gradients, (C) Metabolite profiling in RP-UHPLC mode, and (D) Metabolite profiling in HILIC-UHPLC mode (Figs. 5A and 5B adapted from [47], with permission from Elsevier. Figs. 5C and 5D adapted from [46], with permission from Wiley).

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profiling of several medicinal Panax herbs [52–54], while Wolfender and co-workers evaluated an UHPLC-TOF-MS platform for the analysis of model plant Arabidopsis thaliana [55,56]. The possibility of differentiating five Panax herbs using a UHPLC-QqTOF-MS system and principal-component analysis (PCA) was demonstrated [53]. A comparison was made between the performance of UHPLC using a 100 x 2.1-mm I.D. column and a 20-min gradient and conventional HPLC on a 250 x 4.6-mm I.D. column and an 80-min gradient. By UHPLC, 25 chemical markers (i.e. saponins) were identified within the 20-min runtime, while only 11 markers were recognized by HPLC with four-fold longer analysis time. However, this result was strongly attributed to the detector technology. Indeed, a powerful QqTOF-MS was used in UHPLC while a single-quadrupole instrument was used in HPLC. Finally, due to the high mass accuracy and resolution of the QqTOF-MS analyzer and the RT repeatability of UHPLC, it was possible to identify bioactive compounds responsible for variations between Panax herbs, with the help of available reference standards [52,53]. The metabolite

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profiling of various parts of Panax notoginseng was also carried out using UHPLC-QqTOF-MS, and clear discrimination obtained between the composition of flower buds, roots, and rhizomes [54]. The saponins responsible for such differences were identified with the help of QqTOF-MS. Finally, the proposed strategy was applied to discriminate between slight variations within the same plant species due to different geographical locations, cultivations and collection times [53]. As shown in Fig. 6, Wolfender and co-workers proposed a powerful, sensitive, multi-step strategy for detection, isolation, and identification of stress-induced metabolites in Arabidopsis thaliana after leaf wounding, which mimicked herbivore attack [55,56]. In the first step, a rapid-screening gradient (i.e. 7 min) was carried out by UHPLC-TOF-MS using a 50 x 1-mm I.D. column. This metabolite fingerprinting was performed on numerous plant specimens to evaluate the intra-sample variability and to achieve adequate pool formation [55]. The second step involved high-resolution metabolite profiling of selected pool samples using UHPLC columns of 150 to 300 x 2.1-mm I.D. Gradient conditions used in

Figure 6. Plant metabolomics based on a four-step strategy: fingerprinting, profiling, isolation, and identification of stress biomarkers (From [55] and [56], by courtesy of the authors).

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the metabolomic fingerprinting were directly transferred to the new column geometry, and analysis times were extended up to 100 min and 300 min. This profiling allowed confirmation of the presence of different stressrelated compounds. The high peak capacity afforded by long columns packed with sub-2lm particles was found essential to obtain a complete deconvolution of the biomarkers and resolution of various closely-related isomers [55]. The final step of the process was the complete structural determination of minor biomarkers in plants using LC-MS-triggered preparative isolation. For this purpose, the UHPLC separation obtained during the metabolic profiling was directly transferred to semi-preparative conditions, using a 19-mm I.D. column packed with 5-lm particles of the same material. Based on the use of a capillary-NMR probe, 1D and 2D spectra of good quality were obtained at the lg level, allowing unambiguous structural elucidation of the wound biomarkers isolated (including known signaling molecules, as well as original oxylipins and jasmonates) [56]. Since the proposed approach is generic, this analytical platform could be used to screen various other plant extracts without further reoptimization.

5. Conclusions This review highlights the obvious benefits of columns packed with sub-2lm particles (UHPLC) in terms of throughput and resolution. These features are particularly useful in bioanalysis, for the large screening of molecules and in the metabolomic field. Regarding the coupling of UHPLC with MS, modern triple-quadrupole instruments operating in the SRM mode were preferred for targeted analysis (e.g., bioanalysis, drug metabolism, and multi-component screening), while TOF-MS analyzers were particularly useful for non-targeted analysis (e.g. metabolomics, drug metabolism and multi-component screening). Because UHPLC technology has significantly reduced run times without reducing chromatographic resolution, the main bottleneck has moved to sample preparation and/or data processing, particularly in complex applications, such as metabolomics. At present, the major limitation of UHPLC is the price of dedicated equipment and consumables. However, the cost should decrease with general use of the technique in analytical laboratories. In the near future, the combination of UHPLC with higher mobile-phase temperatures will surely become a useful tool to increase analysis throughput further and to improve the chromatographic resolution of more complex samples, while keeping within acceptable analysis time. Another attractive outlook concerns the recent development and commercialization of HILIC columns packed with sub-2lm particles. Indeed, such columns present some evident advantages for bioana26

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Trends [55] E. Grata, J. Boccard, D. Guillarme, G. Glauser, P.A. Carrupt, E.E. Farmer, J.L. Wolfender, S. Rudaz, J. Chromatogr., B 871 (2008) 261. [56] G. Glauser, D. Guillarme, E. Grata, J. Boccard, A. Thiocone, P.A. Carrupt, J.L. Veuthey, S. Rudaz, J.L. Wolfender, J. Chromatogr., A 1180 (2008) 90. [57] M. Katajamaa, M. Oresic, J. Chromatogr., A 1158 (2007) 318. [58] A. Nordstrom, G. OÕMaille, C. Qin, G. Siuzdak, Anal. Chem. 78 (2006) 3289. [59] E. Zelena, W.B. Dunn, D. Broadhurst, S. Francis-McIntyre, K.M. Carroll, P. Begley, S. OÕHagan, J.D. Knowles, A. Halsall, I.D. Wilson, D.B. Kell, Anal. Chem. 81 (2009) 1357. [60] S. Heinisch, J.L. Rocca, J. Chromatogr., A 1216 (2009) 642. [61] P.N.M. Demacker, A.M. Beijers, H. van Daal, J.P. Donnelly, N.M.A. Blijlevens, J.M.W. den Ouweland, J. Chromatogr., B 877 (2009) 387. Davy Guillarme gained his PhD in analytical chemistry from the University of Lyon (France) in 2004. He is now Maıˆtre Assistant of the School of Pharmaceutical Sciences, University of Geneva, University of Lausanne (Switzerland). His interests include the development of new approaches to perform fast and ultra-fast separations in LC and the possibility of hyphenating these techniques with alternative detection modes. Julie Schappler gained her PhD in pharmaceutical sciences from the University of Geneva (Switzerland) in 2007. She is now Maıˆtre Assistant of the School of Pharmaceutical Sciences, University of Geneva, University of Lausanne (Switzerland). Her interests involve LC and CE hyphenations to various detection modes, and particularly focus on the technological issues for MS coupling. Her research includes analysis of drugs and metabolites in biological matrices, as well as biomolecules analysis. Serge Rudaz gained his PhD in pharmacy from the University of Geneva (Switzerland) in 1997. He is now senior lecturer of the School of Pharmaceutical Sciences, University of Geneva, University of Lausanne (Switzerland). His research is focused on sample preparation, CE, LC and MS. He is also interested in the development and use of methods for mathematical and statistical analysis of data produced from chemical instrumentation (design of experiments, method validation, and data mining) including metabolomic approaches. Jean-Luc Veuthey obtained his PhD in analytical chemistry from the University of Geneva (Switzerland) in 1987. He is now full professor of the School of Pharmaceutical Sciences, University of Geneva, University of Lausanne (Switzerland). His interests include the development of LC and CE hyphenated to several detection modes for the analysis of drugs and metabolites. Sample preparation and validation of the procedures are also particularly studied in his laboratory.

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