Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling

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1 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling Introduction KEY WORDS Metabolomics, nano LC ESI-MS, Thermo LTQ orbitrap XL FTMS, chip based separation, microfabrication, plant metabolites GOAL Demonstrate the gain in analytical performance and increase in detection sensitivity that can be achieved for the separation of plant metabolite extracts by using micro a Pillar Array Column (μpac ) Metabolic profiling studies have become indispensable in a wide range of research fields; including biomarker and drug discovery, toxicology, foodomics and plant sciences [1-4]. The goal is to acquire profound insights into the biochemistry of an organism at a certain moment by identifying endo- and exogenous metabolites present in extracts of biological tissue or biofluids. Given its tremendous complexity and the dynamic concentration range in which certain metabolites can be present, there is a growing demand for powerful analytical techniques with increased sensitivity to unravel the composition of the metabolome even further. The continuously evolving fields of liquid chromatography and mass spectrometry (LC-MS) have become vital tools in these studies. Ever more sensitive mass spectrometers and new or improved LC techniques allow analytical scientists to retrieve more information from minute sampling amounts. In absence of molecular amplification techniques (in contrast with PCR for genomics), successful metabolomics profiling is essentially dependent on detection sensitivity. A serious leap forward concerning detection sensitivity has been made with the introduction of nanoflow-hplc-nanoesi-ms, where flow rates down to several hundred nanoliters per minute enable the use of nanospray emitters with reduced internal diameters and increased ionization efficiency [5-6]. Up until recently, practically all nanoflow driven LC-MS research was focused on proteomic analyses [7-8]. However, technological advances in robustness and stationary phase availability of nanoflow LC-MS have also drawn the attention of other analytical research fields, including metabolomics with a growing number of publications [9-11]. Whereas current state-of-the-art UPLC columns offer a viable solution for high-throughput analyses, µpac nano LC columns can provide unprecedented separation efficiency for complex analyses where long gradient times are applied to achieve maximal separation. Proprietary microfluidic design and lithographic manufacturing techniques ensure minimal peak dispersion [12] and the high permeability allows to increase the separation path length without the drawbacks of legacy LC column technology, where the build-up of back pressure restricts the use of longer columns [See the inset on page 1]. In this application note, plant extract samples are used to compare the analytical performance of a µpac nano LC column to state-of-the-art UPLC columns. The combination of superior separation performance and increased detection sensitivity will enable analytical scientists to identify even more components in a single metabolic profiling run.

2 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 2 EXPERIMENTAL EXPERIMENTAL Nano LC conditions UPLC conditions LC system 126 Infinity Nanoflow LC Column 2 cm C18 μpac Mobile phase A; Water (1%) with,1% (v/v) FA B; Acetonitrile/water (8/2) with,1% (v/v) FA Flow rate μl/min Gradient profiles 1-5% B in 3, 6, 12, 18, 3 and 42 min gradient Temperature 3 C Sample poplar bark metabolite extract, Arabidopsis thaliana leaf metabolite extract Injection 4 nl injection UPLC system Accela autosampler coupled to an Accela pump Column type A: C18 (15 cm x 2.1 mm x 1.7 μm) type B: C18 (15 cm x 2.1 mm x 1.8 μm) Mobile phase A; Water (1%) with,1% (v/v) FA B; Acetonitrile/water (8/2) with,1% (v/v) FA Flow rate 3 μl/min Gradient profiles 1-5% B in 3, 6, 12 and 18 min gradient Temperature 3 C Sample poplar bark metabolite extract Injection 5 μl injection Nano-ESI-MS conditions ESI-MS conditions MS system LTQFT; lineair ion trap mass spectrometer connected with an FT-ICR mass spectrometer Acquisition mode full MS scan (m/z 2-1) Ionization mode nano ESI (Advion coupler) positive mode Source voltage 2. kv (Advion coupler) Capillary temperature 2 C MS system LTQFT; lineair ion trap mass spectrometer connected with an FT-ICR mass spectrometer Acquisition mode full MS scan (m/z 2-1) Ionization mode electropsray ionization (ESI) positive mode Source voltage 5. kv Capillary temperature 275 C Sheath gas 4 (/) Auxiliary gas 2 (/) Sample preparation Poplar bark tissue (1 mg) was extra extracted with 1 ml Methanol. The supernatant was freeze-dried and redissolved in 6 ml milliq water/cyclohexane (1/1, v/v). Arabidopsis tissue (1 mg) was extra extracted with 1 ml Methanol. The supernatant was freeze-dried and redissolved in 6 ml milliq water/cyclohexane (1/1, v/v).

3 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 3 Results and Discussion In order to compare the analytical performance of state-of-the-art UPLC columns to µpac nano LC column technology, a defined set of increasing gradient times is used to separate poplar bark metabolite extracts on all columns. UPLC columns are operated with a Thermo Accela UPLC system coupled to a Thermo Orbitrap-XL FTMS mass spectrometer (UPLC-ESI-MS), where 5 µl a sample is injected and subsequently separated at a flow rate of 3 µl/min. Alternatively, only 4 nl of sample is injected onto the µpac nano LC column at a flow rate of 1 µl/min using an Agilent 126 Infinity Nanoflow LC system. An Advion TriVersa Nanomate was used to couple the µpac nano LC column to a Thermo Orbitrap-XL FTMS mass spectrometer. Figure 1: Representative base peak chromatograms of a poplar bark metabolite extract separated on (a) a 2 cm PharmaFluidics μpac nano LC column, (b) a state-of-the-art 15 mm x 2.1 mm x 1.7 μm UPLC column, referred to as type A; (c) another state-of-the-art 15 mm x 2.1 mm x 1.8 μm UPLC column, referred to as type B (see experimental section for the gradient condition, gradient time was set at 12 min). RT: NL: 3.E6 Base Peak F: FTMS + 8 p NSI Full ms [1.-1.] MS 6 µpac C18 (2 mm, PharmaFluidics) RT: NL: 3.E6 Base Peak F: FTMS + p 8 ESI Full ms [1.-1.] MS 6 UPLC type A C18 (15 mm x 2.1 mm x 1.7 µm) RT: NL: 3.E6 Base Peak F: FTMS + p 8 ESI Full ms [1.-1.] MS 6 UPLC type B C18 (15 mm x 2.1 mm x 1.8 µm) Base peak chromatograms obtained for 12 min gradient separations are shown in figure 1. The intensity scale has been normalized to 3. x 1E6 to compare detection sensitivity and elution profiles. Despite the presence of four obviously overloaded peaks in all chromatograms, some clear conclusions can be drawn. Whereas only a fraction of the sample amount that is used for the UPLC separations is injected onto µpac nano LC column (1/125), similar elution profiles with increased detection response can be observed. 3% more distinct features are displayed in the chromatogram corresponding to µpac nano LC column, shown in figure 1a. Differences in stationary phase morphology also affect the retention. Peak capacity (n C ), where average peak width as well as the retention that is generated is taken into account, will therefore be used as a parameter for proper comparison of analytical performance. Peak capacity is calculated using equation (1). With t i the time at which the last component

4 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 4 of the sample elutes and t the column void volume time. Peak widths have been defined at half maximum () and have been converted to peak width at 4σ using equation (2). (1) (2) Three m/z values corresponding to compounds that elute over the entire retention are selected to compare chromatographic performance and detection sensitivity. Extracted ion chromatograms for each compound are shown in figure 2. Chromatograms obtained on a µpac nano LC column are displayed in blue, while those obtained on UPLC column type A and type B are displayed in orange. Measured retention time (RT), peak width () and signal intensity values are listed in the tables next to the chromatograms. Figure 2: Comparison of extracted ion chromatograms obtained from 12 min gradient poplar bark separations. M/z value (a), m/z value (b) and m/z value range of the elution. A 2 cm PharmaFluidics μpac nano LC column (blue) is compared to types, type A (orange) and type B (orange). Corresponding elution times (RT), peak widths () and signal intensities are shown to the left. RT: RT: NL: 1.51E6 m/z= F: FTMS + p NSI Full ms [1.-1.] MS PAC C18 (2 mm, PharmaFluidics) NL: 7.96E5 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type A C18 (15 mm x 2.1 mm x 1.7 m) NL: 9.53E5 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type B C18 (15 mm x 2.1 mm x 1.8 m) NL: 1.75E6 m/z= F: FTMS + p NSI Full ms [1.-1.] MS PAC C18 (2 mm, PharmaFluidics) NL: 6.87E5 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type A C18 (15 mm x 2.1 mm x 1.7 m) NL: 7.44E5 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type B C18 (15 mm x 2.1 mm x 1.8 m) PharmaFluidics µpac nano LC C18 (2 cm) UPLC Type A C18 (15 cm x 2.1 mm x 1.7 µm) UPLC Type B C18 (15 cm x 2.1 mm x 1.8 µm) PharmaFluidics µpac nano LC C18 (2 cm) UPLC Type A C18 (15 cm x 2.1 mm x 1.7 µm) UPLC Type B C18 (15 cm x 2.1 mm x 1.8 µm) RT Intensity [/] E E E+5 RT Intensity [/] E E E+5 RT: NL: 2.13E6 m/z= F: FTMS + p NSI Full ms [1.-1.] MS PAC C18 (2 mm, PharmaFluidics) NL: 1.34E6 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type A C18 (15 mm x 2.1 mm x 1.7 m) NL: 1.28E6 m/z= F: FTMS + p ESI Full ms [1.-1.] MS UPLC type B C18 (15 mm x 2.1 mm x 1.8 m) PharmaFluidics µpac nano LC C18 (2 cm) UPLC Type A C18 (15 cm x 2.1 mm x 1.7 µm) UPLC Type B C18 (15 cm x 2.1 mm x 1.8 µm) RT Intensity [/] E E E+6 Smaller and higher peaks are observed for all selected m/z values when comparing µpac nano LC columns to UPLC column type A and B. Narrower peak widths can be attributed to the high degree of order that is present in µpac nano LC columns ensuring minimal peak dispersion. Additionally, compounds elute more concentrated from columns with reduced peak dispersion, hereby positively affecting detection sensitivity. Increased ionization efficiency typically observed for nanospray emitters will amplify the increase in detection sensitivity even further. peak widths () based on the three m/z values shown in figure 2 have been determined for a range of gradient times and are listed in table 1. The corresponding retention that is generated for each separation is shown next to the average peak width. For short gradient times (< 6 min), no significant difference in peak width nor in retention is observed. When longer gradient times (> 6 min) are applied, the potential of

5 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 5 Table 1: peak width () and retention obtained for the separation of a poplar bark metabolite extract at increasing gradient times. M/z value , m/z value and m/z value have been used to calculate the average peak width. Column void volume time (t) has been subtracted to define the retention. PharmaFluidics µpac nano LC C18 (2 cm) UPLC Type A C18 (15 cm x 2.1 mm x 1.7 µm) UPLC Type B C18 (15 cm x 2.1 mm x 1.8 µm) 3 min 6 min 12 min 18 min µpac nano LC technology becomes apparent. By increasing gradient time from 3 to 18 min, average peak width is only affected by a factor of 1.8 on a 2 cm long µpac nano LC column, whereas an average increase by a factor of 2.7 is observed on both UPLC column types. The exceptional separation path length of the µpac nano LC column (2 cm) ensures that minimal peak dispersion can be maintained, even for very long gradient times which are needed to achieve optimal separation of highly complex samples. The back-pressure build-up associated with sub 2 µm UPLC packed bed column formats restricts the use of longer columns on the employed instrument and therefore limits the gain in chromatographic resolution that can be achieved for long gradient times. This is clearly demonstrated when the operating pressures of both formats are compared. To operate 15 cm long UPLC columns at their optimal flow rate (3 µl/min), pressures ranging from 6 to 7 bar have to be generated. In contrast, only 3 bar is needed to operate a 2 cm long µpac nano LC column at a flow rate of 1 µl/min The superior analytical performance of µpac nano LC columns for long gradient times is demonstrated in figure 3 where peak capacity (n C ) is plotted as a function of gradient time. A considerable gain in peak capacity (by a factor up to 1.5) can be realized when gradient times are extended beyond 12 min. However, it has to be taken into account that the µpac nano LC columns are not operated at their optimal flow rate and that even more striking differences in peak capacity can be accomplished when they are operated at lower flow rates. In addition, reducing the flow rate will increase ionization efficiency and allow for even more sensitive analysis. Figure 3: Comparison of peak capacity (nc) for 2 commercially available UPLC column types (15 cm x 2.1 mm, sub-2 μm particles) type A (orange), type B (orange) and a 2 cm long μpac column (blue). Sample: poplar bark metabolite extract. Peak capacity is calculated according to Equations (1) and (2) Peak capacity n C [/] MICRO PILLAR ARRAY COLUMN (µpac ) PACKED BED UPLC COLUMNS Gradient time

6 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 6 Figure 4: Left: Base peak chromatograms obtained from 18 min gradient Arabidopsis thaliana leaf metabolite extract separations on a 2 cm PharmaFluidics μpac nano LC column at flow rates of (a).9, (b).6 and (c).3 μl/min. Right: Corresponding extracted ion chromatograms obtained for m/z value RT: NL: 3.E6 Base Peak F: FTMS + p NSI Full ms 8 [1.-1.] MS PAC C18 (2 cm, 6 PharmaFluidics).9 l-min RT: NL: 3.E6 8 Base Peak F: FTMS + p NSI Full ms 6 [1.-1.] MS PAC C18 (2 cm, PharmaFluidics).6 l-min RT: NL: 3.E6 Base Peak F: FTMS + p 8 NSI Full ms [1.-1.] MS PAC C18 (2 cm, 6 PharmaFluidics).3 l-min RT: NL: 3.E5 m/z= F: FTMS + p NSI Full ms 8 [1.-1.] MS PAC C18 (2 cm, 6 PharmaFluidics).9 l-min RT: NL: 3.E5 m/z= F: FTMS + p NSI Full ms [1.-1.] MS PAC C18 (2 cm, 6 PharmaFluidics).6 l-min RT: NL: 3.E5 8 m/z= F: FTMS + p NSI Full ms [1.-1.] MS PAC 6 C18 (2 cm, PharmaFluidics).3 l-min To investigate the effect of flow rate on analytical performance and detection sensitivity, 4 nl of an Arabidopsis thaliana leaf metabolite extract was injected onto a 2 cm long of µpac nano LC column and separated using linear gradients of 18, 3 and 42 min at different flow rates (.9,.6 and.3 µl/min). Base peak chromatograms obtained for 18 min gradient separations are shown to the left in figure 4. Aside from having a pronounced effect on the elution profile, with the column void time t shifting to longer analysis times, reducing the flow rate also has a significant impact on the ionization efficiency. Substantially more features are present in the bottom chromatogram, where the flow rate has been reduced down to.3 µl/min. Corresponding extracted ion chromatograms for a compound with m/z value are displayed next to the base peak chromatograms to demonstrate the increase in detection sensitivity. Measured retention time (RT), peak width () and signal intensity values for this compound are compared in table 1. By reducing the flow rate from.9 to.3 µl/min, detection sensitivity is increased by a factor of 1.7. Table 2: Elution times (RT), peak widths () and signal intensities obtained for m/z value in 18 min Arabidopsis thaliana leaf metabolite extract separations. µpac nano LC C18 (2 cm) Flow rate [µl/min] RT Intensity [/] E E E+5 To quantify the effect of reduced flow rates in terms of analytical performance, six m/z values corresponding to compounds that elute over the entire elution have been selected and have been monitored in all separations. The base peak chromatogram obtained for a 42 min gradient separation operated at a flow rate of.3 µl/min is shown in figure 5a. Superimposed extracted ion chromatograms corresponding to the monitored m/z values are shown underneath in figure 5b. peak widths () based on m/z values shown in figure 5b have been determined for all gradient time and flow rate conditions and are listed next to the corresponding retention in table 3. Both retention and average peak width are affected by the flow rate, resulting in a larger retention for gradient separations at lower flow rates with slightly increased average peak widths. However, the increase in retention is more pronounced, resulting in higher peak capacities for low flow rates.

7 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 7 Figure 5: (a) Base peak chromatogram obtained from a 42 min gradient Arabidopsis thaliana leaf metabolite extract separation on a 2 cm PharmaFluidics μpac nano LC column at a flow rate of.3 μl/min. (b) Overlay of extracted ion chromatograms obtained for 6 m/z values. RT 42.1: m/z value 48.93; RT 61.81: m/z value ; RT 94.63: m/z value ; RT : m/z value ; RT : m/z value ; RT : m/z value RT: RT: Table 3: peak width () and retention obtained for the separation of a Arabidopsis thaliana leaf metabolite extract at increasing gradient times. M/z value 48.93, , , , and have been used to calculate the average peak width. Column void volume time (t) has been subtracted to define the retention. PharmaFluidics µpac nano LC C18 (2 cm) Flow rate [µl/min] 18 min 3 min 42 min Peak capacities calculated for the Arabidopsis thaliana leaf metabolite extract separation at different flow rates are shown in figure 6. A comparison has been made with peak capacities that have been determined earlier for gradient separations of poplar bark metabolite extracts on µpac as well as on both UPLC columns. By operating µpac nano LC columns at their optimal flow rate of.3 µl/min, a substantial increase in peak capacity can be made for very long gradient time separations where the relative contribution of column void time to total analysis time becomes of little significance Figure 6: Peak capacity (n c ) obtained on a 2 cm long μpac column for long gradient times and at different flow rates. Sample: Arabidopsis thaliana leafmetabolite extract. Peak capacity is calculated according to Equations (1) and (2). Flow rate.9 μl/min;.6 μl/min;.3 μ l/min. Peak capacities obtained for separation of polar bark metabolite extract on UPLC columns are shown in orange Peak capacity n C [/] PACKED BED UPLC COLUMNS MICRO PILLAR ARRAY COLUMN (µpac ) Gradient time

8 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 8 Conclusions This application note highlights the added value that can be generated with PharmaFluidics micro Pillar Array Column technology (µpac ) in the field of metabolic profiling studies: µpac nano LC columns offer the advantage that samples with high complexity can be separated at long gradient times without running into the separation efficiency limitations typically observed on packed bed HPLC or UHPLC columns. In addition to this superior analytical performance, a serious increase in detection sensitivity can be observed by combining columns with minimal peak dispersion and flow rates in the range of several hundreds of nanoliter per minute. When comparing µpac nano LC columns to conventional UPLC columns, an increase in peak capacity up to 1.5 times is observed for gradient separations of bark poplar metabolite extract samples when run under conditions that generate comparable void times. In addition, detection sensitivity is enhanced by a factor of 2. The advantages of µpac nano LC columns become even more striking when these columns are operated at their optimal flow rate, with peak capacity values up to 2 times those obtained on state-of-the-art UPLC columns and a supplementary increase in detection sensitivity by nearly a factor of 2. References [1] J.L. Griffin, J.P. Schockcor, Metabolic profiles of cancer cells, Nat. Rev. Cancer 4 (24) [2] D.S. Wishart, Metabolomics: applications to food science and nutrition research, Trends Food Sci Technol. 19 (28) [3] J. Bundy, M. P. Davey, M.R. Viant, Environmental metabolomics: a critical review and future perspectives, Metabolomics 5 (29) 3-21 [4] K. Morreel, Y. Saeys, O. Dima, F. Lu, Y. Van depeer, R. Vanholme, J. Ralph, B. Vanholme, W. Boerjan, Systematic Structural Characterization of Metabolites in Arabidopsisvia CandidateSubstrate-ProductPairNetworks, Plant Cell 26 (214) [5] M. Wilm, M. Mann. Analytical properties of the nanoelectrospray ion source. Anal. Chem 68 (1996) 1-8 [6] R. Juraschek, T. Dulcks, M. Karas, Nanoelectrospray-more than just a minimized-flow electrospray ionization source, J. Am. Soc. Mass Spectrom. 1 (1999) 3-38 [7] L. D. Vu, E. Stes, M. Van Bel, H. Nelissen, D. Maddelein, D. Inzé, F. Coppens, L. Martens, K. Gevaert, I. De Smet, Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves, J. Proteome Res. 15 (216) [8] R. Aebersold, M. Mann, Mass-spectrometric exploration of proteome structure and function, Nature 537 (216) [9] J. Ding, C. M. Sorensen, Q. Zhang, H. Jiang, N. Jaitly, E. A. Livesay, Y. Shen, R. D. Smith, T. O. Metz, Capillary LC Coupled with High-Mass Measurement Accuracy Mass Spectrometry for Metabolic Profiling, Anal. Chem. 79 (27) [1] A. David, A. Abdul-Sada, A. Lange, C.R. Tyler, E.M. Hill, A new approach for plasma (xenp)metabolomics based on solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionization mass spectrometry, J. Chromatogr A 1365 (214) 72-82

9 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 9 [11] A. J. Chetwynd, A. David, E.M. Hill, A. Abdul-Sada, Evaluation of analytical performance and reliability of direct nanolc-nanoesi-high resolution mass spectrometry for profiling the (xeno)metabolome, J. Mass Spectrom. 49 (214) [12] W. De Malsche, J. Op De Beeck, S. De Bruyne, H. Gardeniers, G. Desmet, Realization of 1 16 Theoretical Plates in Liquid Chromatography Using Very Long Pillar Array Columns, Anal. Chem. 84 (212) Acknowledgements We would like to acknowledge Kris Moreel and Wout Boerjan from the VIB Department of Plant Systems Biology (Ghent University, Belgium) for their analytical expertise and for providing the samples used for the work presented in this application note.

10 Analytical performance of micro Pillar Array Columns (μpac ) for metabolic profiling 1 µpac driven separations Better by Design Conventionally LC columns are fabricated by stacking (packed beds) or depositing (monoliths) material into a capillary. PharmaFluidics µpac technology (micro Pillar Array Column) is unique in its kind as it is built upon the precise micromachining of designed chromatographic separation beds into silicon. This approach brings along three crucial and unique characteristics: Perfect Order. µpac beds are designed with a high degree of order, eliminating heterogeneous flow paths otherwise present in conventional columns (so called Eddy dispersion). Flow through µpac columns adds very little dispersion to the overall separation. As a result, peaks remain sharper and sensitivity is increased. High Permeability. µpac s operate at moderate pressures, typically lower than 3 bar. Separation channels with exceptional length (5 cm to 2 cm) are therefore possible. These are folded onto a small footprint by a interconnecting concatenating bed segments. Solid Backbone. The micromachined backbone of the separation bed forms a rigid structure that is not influenced by pressure. There are no obstructions by touching surfaces, and there is no risk for perturbations by pressure fluctuations. Technologiepark-Zwijnaarde 3 B-952 Ghent (Zwijnaarde) Belgium info@pharmafluidics.com