Challenges in bioanalytical laboratories include the development of fast LCeMS methods able to separate closely related compounds, such as analytes and metabolites, from endogenous components (Spaggiari et al., 2014).
Because UHPLC greatly enhances the separation throughput and resolution, peaks as narrow as (or less than) 1 s can be obtained. This induces practical issues for bioanalytical applications using MS because sufficient data points (e.g.,>15 points per peak) are essential to ensure reliable quantitation.
Several critical applications can be found in the literature. For example,Petsalo et al. (2008)published a UHPLCetandem mass spectrometry (MS/MS) procedure for analyzing nine drugs and their respective metabolites in urine, using a 4 min gradient. An ESI source operating sequentially in positive and negative polarity modes was employed, and dwell times (DT) of 20e30 ms were applied
for each SRM transition. Because peak widths of 4 s were experimentally obtained, only six points were acquired to define peaks, which could limit performance, particularly at the lower limit of quantitation. To accommodate the small UHPLC peak widths, DT can be reduced when many SRM transitions have to be monitored (Schappler et al., 2009), but this can result in sensitivity loss. An alternative approach consists of using various time windows during the acquisition, as proposed by Berg et al. (2009). Finally, to circumvent DT reduction, Li et al. (2008) suggested a useful “peak parking” strategy, which consisted of reducing the flow rate during peak elution, and thus extending the MS acquisition window for quantitative bioanalytical assays. However, the latter strategy is only suitable when a limited number of targeted analytes are analyzed.
In addition, it has been demonstrated in various studies that a significant reduction of matrix effects was brought about by UHPLC technology, compared to regular HPLC. As an example,Chambers et al.
(2007) proved that polymeric mixed-mode solid-phase extraction (SPE), combined with UHPLC technology and appropriate mobile phase pH, provided significant benefits for reducing matrix effects from plasma matrix components, and improving the ruggedness and sensitivity of bioanalytical methods.
Considering the analysis time reduction offered by UHPLC technology, the sample preparation procedure becomes the limiting step in terms of total analysis time. Numerous UHPLC bioanalytical applications still involve traditional sample preparation procedures, which drastically increase the total analysis time. A few authors have suggested solutions to this issue, while maintaining sufficient sample preparation selectivity. To date, solideliquid extraction (SLE) and SPE based on a 96-well plate format were used prior to UHPLCeMS/MS bioanalysis, allowing for selective, sensitive, and, above all, high-throughput analyses (Licea-Perez et al., 2007; Yadav et al., 2008).
6.2 HIGH RESOLUTION DRUG METABOLISM BY ULTRAHIGH-PRESSURE LIQUID CHROMATOGRAPHY e MASS SPECTROMETRY USING QUADRUPOLE TIME-OF- FLIGHT MASS SPECTROMETER ANALYZERS
In drug metabolism experiments, there are also many challenges because of the complex nature of biological matrices and the large diversity of produced metabolites. To identify unknown metabolites, high chromatographic resolution and mass accuracy for fragmentation patterns are key requirements.
UHPLC with high resolution analyzers such as QqTOF are particularly useful to fulfill both tasks in a high-throughput environment.
Castro-Perez et al. (2005)were the first to report the use of UHPLC technology with a QqTOF analyzer in the early drug discovery process. This study emphasized improved resolution, in terms of chromatographic and mass spectral quality, and the associated gain in sensitivity afforded by the UHPLCeQqTOF/MS system. These features were explained by the combination of reduced peak width and low ion suppression due to the enhanced resolution of metabolites and endogenous compounds.
Walles et al. have investigated the benefits and drawbacks of three UHPLCeQqTOF/MS methods for fast metabolite identification using alternative MS/MS experiments (MSE) (Walles et al., 2007;
Plumb et al., 2006; Crockford et al., 2008). The high efficiency attributable to UHPLC was the key to the successful identification of isobaric metabolites. In fact, they could not be distinguished with the accurate mass of QqTOF, as they had an identical elemental composition and often similar MS/MS fragmentation patterns. It can be noted that the time spent for structure elucidation created additional bottlenecks and becomes the limiting step when UHPLCeQqTOF experiments are performed.
6.3 ULTRAHIGH-PRESSURE LIQUID CHROMATOGRAPHY e MASS SPECTROMETRY FOR MULTIRESIDUE SCREENING
Multiresidue screening techniques are generally developed to quickly assess the presence of con- taminants in a complex sample. Thus, the developed method should be able to detect as many com- ponents as possible in a single analytical run. In this context, UHPLC coupled with tandem MS or TOF/MS remains the gold standard. Multiresidue screening methods with UHPLC technology have been applied to a wide variety of analytes and matrices, including: (1) doping agents (Thorngren et al., 2008; Badoud et al., 2009, 2010; Nicoli et al., 2016) and veterinary drugs (Kaufmann et al., 2007) in biological matrices; (2) drugs (Kasprzyk-Hordern et al., 2008a,b), pesticides (Gervais et al., 2008), and herbicides (Pastor Montoro et al., 2007) in environmental matrices; and (3) veterinary drugs (Stolker et al., 2008), drugs (Cai et al., 2008), and pesticides (Romero-Gonzalez et al., 2008; Taylor et al., 2008;
Garrido Frenich et al., 2008) in food samples.
Because of the high number of investigated compounds, conventional HPLC runs can be relatively long, particularly to avoid peak coelution leading to matrix effects. It is indeed important to attain sufficient chromatographic resolution, to minimize coelution of compounds with closem/zratios and similar fragmentation pathways. As shown inFig. 1.11, for the separation of 103 doping agents in a
0 50 100 150 200 250 300 350 400 450 500
0 1 2 3 4 5 6
Time [min]
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ESI positive ESI negative IS 1 positive IS 2 positive IS negative
LC
MS
m/z180.10 ±0.02Da m/z179.11 ±0.02Da 1
Time [min]
3 2
Intensity [%]
FIGURE 1.11
Separation of 103 doping agents in urine sample according tom/zandtRvalues. Data from electrospray ionization (ESI) positive and negative mode are plotted together. The three I.S arecircled with a continuous line. A zone (dashed line) is magnified to show the selectivity of coupling ultrahigh-pressure liquid chromatography to the quadrupole time-of-flight mass spectrometer. In the magnified zone, the compounds (1) methylephedrine, (2) 3,4- methylenedioxyamphetamine, and (3) nikethamide are separated as a function of time, intensity, andm/z.
Adapted from Badoud, F., Grata, E., Perrenoud, L., Avois, L., Saugy, M., Rudaz, S., Veuthey, J.L., 2009. Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatographyequadrupole time-of-flight mass spectrometry: I. Screening analysis.
J. Chromatogr. A 1216, 4423e4433, with permission.
urine sample, gradient conditions were selected to elute analytes presenting a wide polarity range, and formic acid is generally the preferred additive in the mobile phase. To screen compounds by UHPLC, 50e100 mm column lengths were used with gradient times ranging between 5 and 15 min, followed by a reequilibration time of 2e4 min. The column length should be selected in agreement with the gradient time because the longest column did not always provide the highest peak capacity in UHPLC.
Indeed, a 50 mm column was found to be optimal for gradient times shorter than 7 min, whereas a 100 mm column was only beneficial for longer gradients. Finally, the 2.1 mm I.D. column was often preferred, to limit extra-column band broadening contributions. Only Kasprzyk-Hordern et al.
(2008a,b)reported the successful screening of about 50 pharmaceuticals in wastewater using a 1 mm I.D. column. Even if the consumption of mobile phase and analyte was drastically reduced, peaks were notably broader and distorted with a 1 mm I.D. column, as expected from the influence of external volume contributions.
From the above papers, an approximate increase in throughput by three- to fivefold was observed in UHPLC compared to conventional HPLC methods. In addition to the analysis time decrease, an equivalent or higher chromatographic resolution was reported (Pastor Montoro et al., 2007; Petrovic et al., 2006; Farre et al., 2008). Such improvements were attributed not only to an increase in peak capacity but also to column selectivity changes. Because of different column chemistries, strict comparisons were not always possible.
6.4 ULTRAHIGH-PRESSURE LIQUID CHROMATOGRAPHY e MASS SPECTROMETRY IN METABOLOMICS
Due to the inherent complexity of biological samples and because metabolites can be found at low concentrations, there is a need for analytical systems providing high resolution and increased sensitivity. For this reason, the value of UHPLCeTOF/MS and QqTOF/MS platforms has been demonstrated in a number of studies. The strong reduction of analysis time provided by UHPLC versus HPLC opens up the possibility of high-throughput screening for metabolomic fingerprinting. On the other hand, a longer UHPLC run can be employed to increase the amount of information, essentially for metabolomic profiling. The UHPLC platform has been applied for the global metabolic profiling of (1) human and animal biological fluids, including rat urine (Gika et al., 2008b,c; Plumb et al., 2005), human urine (Gika et al., 2008a,b,c;Guy et al., 2008; Wong et al., 2008), and human serum (Dunn et al., 2008), as well as (2) plant extracts, such asArabidopsis thaliana(Grata et al., 2008; Glauser et al., 2008) andPanaxherbs (Xie et al., 2008a,b; Dan et al., 2008).
Wilson et al. have used UHPLC for the profiling of rat and mouse urine since 2005 (Gika et al., 2008a,b,c; Plumb et al., 2005). Initially, biological fluids were analyzed on a 50 mm column packed with 1.7mm particles in combination with TOF/MS. In terms of chromatography, the average peak widths were around 1 s, thus generating a peak capacity of 60 for UHPLC runs of only 1 min. With the additional TOF/MS information, a total of 1000 features (i.e., signals observed with specificm/zand retention times that can be considered as a variable for data treatment) were determined in rat urine.
This number was equivalent, or even better than, that achieved on conventional HPLC instrumentation, but with a 10-fold reduction in analysis time. The study performed byNordstrom et al. (2006)on the quantitative analysis of endogenous and exogenous metabolites in human serum confirmed these results. Indeed, UHPLC provided 20% more detected components in comparison with HPLC. Finally, it was demonstrated that UHPLC displayed some additional advantages over HPLC, such as better
retention time repeatability and signal-to-noise ratios. Finally, two interesting approaches, namely, the application of elevated temperature in UHPLC (up to 180C) and the use of HILIC columns packed with sub-2mm particles, were proposed by Wilson et al. to further extend the applicability of UHPLC in metabolomics (Gika et al., 2008b,c).
Because of the complexity and chemical diversity of metabolites present in natural plant extracts, metabolomics is also gaining interest in the field of phytochemistry. The use of UHPLCeTOF/MS as well as QqTOF/MS for untargeted metabolic profiling has been reported by two research groups. Jia et al. reported the profiling of several medicinalPanaxherbs (Xie et al., 2008a,b; Dan et al., 2008), while Wolfender et al. evaluated a UHPLCeTOF/MS platform for the analysis of a model plant,A.
thaliana(Grata et al., 2008; Glauser et al., 2008). As shown inFig. 1.12, they proposed a useful and sensitive multistep strategy for the detection, isolation, and identification of stress-induced metabolites
time
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Fast UHPLC-TOF/MS
High resolution UHPLC-TOF/MS
Micro Fractionation μg scale
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CapNMR gCOSY
H-10 H-9
H-11
H-12 H-8
1
FIGURE 1.12
Plant metabolomics based on a four-step strategy: fingerprinting, profiling, isolation, and identification of stress biomarkers.UHPLCeTOF/MS, ultrahigh-pressure liquid chromatographyetime-of-flight mass spectrometry;cap NMR, cap nuclear magnetic resonance spectroscopy.
Adapted from Guillarme, D., Ruta, J., Rudaz, S., Veuthey, J.-L., 2010a. New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches. Anal. Bioanal. Chem. 397, 1069e1082; Guillarme, D., Schappler, J., Rudaz, S., Veuthey, J.L., 2010b. Coupling ultra-high pressure liquid chromatography with mass spectrometry. Trends Anal.
Chem. 29, 15e27, with permission.
inA. thalianaafter leaf wounding, which mimicked herbivore attack (Grata et al., 2008; Glauser et al., 2008). In the first step, a rapid screening gradient was carried out by UHPLCeTOF/MS using a short column of 50 mm. This metabolite fingerprinting was performed on numerous plant specimens to evaluate the intrasample variability and achieve adequate pool formation (Grata et al., 2008). The second step consisted of high resolution metabolite profiling of selected pool samples using a UHPLC column of 150 mm. Gradient conditions used in the metabolomic fingerprinting were adequately transferred to the new column geometry, and analysis times were increased up to 100 min. This profiling allowed confirmation of the presence of different stress-related compounds. The high peak capacity afforded by long columns packed with sub-2mm was indeed essential to obtain a complete deconvolution of the biomarkers, and resolution of numerous closely related isomers (Grata et al., 2008). The last step of the process was the complete structural determination of minor biomarkers in plants using LCeMS triggered preparative isolation. For this purpose, the UHPLC separation obtained during the metabolic profiling was transferred to semipreparative conditions, using a 19 mm I.D.
column packed with 5mm particles of the same chemistry. Based on the use of a capillary-nuclear magnetic resonance probe, 1D and 2D spectra of good quality were obtained at themg level, allow- ing unambiguous structural elucidation of the isolated wound-biomarkers (including known signaling molecules, as well as original oxylipins and jasmonates) (Glauser et al., 2008). This generic analytical platform can be used to screen various other plant extracts.
7. CONCLUSION/PERSPECTIVES
As shown in this chapter, UHPLC is a powerful technology, which is quite easy to implement. With this strategy, it is possible to increase drastically the throughput, while maintaining equivalent per- formance and/or to increase the resolution within an acceptable analysis time. However, to take full advantage of this platform, it is important to keep in mind that the performance of UHPLC is based not only on the column packing but also on the quality of the chromatographic system. For this reason, UHPLC experiments should be performed on a system compatible with ultrahigh pressure, which possesses reduced system and dwell volumes.
The coupling of UHPLC with MS appears to be the ultimate approach, in terms of sensitivity, selectivity, and peak assignment for the determination of analytes at low concentrations in complex matrices. This strategy has become very popular and has now been applied in numerous fields of application, such as bioanalysis, drug metabolism, multiresidue screening, and metabolomics. How- ever, it is recommended to work with an MS device of the latest generation (quadrupole-based and TOF instruments are the most appropriate) that possesses a sufficient data acquisition rate.
In the future, if 2.1 mm I.D. column becomes the standard dimension for UHPLC, there is certainly not much interest in reducing even more the particle size of the support. Indeed, the backpressure generated by such packing would be detrimental for the chromatographic separation because of the axial and longitudinal temperature gradients within the column (frictional heating effects). To limit these negative effects, the solution would be to work with reduced I.D. (500mme1 mm) columns. But in this case, the current chromatographic system needs to be strongly improved in terms of extra- column and dwell volumes. From our point of view, the use of small particles, in conjunction with ultrahigh pressure and elevated temperature, up to 90C (HT-UHPLC), is certainly more promising both not only for throughput and resolution but also for selectivity and peak shape.
Finally, monolithic columns as well as columns packed with sub-3mm SPP should be neglected as they could represent some good alternatives to columns packed with fully porous sub-2mm particles.