Zic philic

Manufactured by Merck Group

Sourced in Germany, United Kingdom

ZIC-pHILIC is a liquid chromatography column designed for the separation and analysis of polar and ionizable compounds. It utilizes zwitterionic stationary phase technology to provide efficient separation of a wide range of polar analytes.

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ZIC-pHILIC 2.1-mm i.d × 150 mm column SeQuant ZIC-pHILIC polymeric column SeQuant Zic-pHilic ZIC-pHILIC stationary phase SeQuant ZIC-pHILIC 2.1 × 150 mm (5 μm Sequant Zic-pHILIC 2.1 × 100 mm column SeQuant® ZIC®-pHILIC 5 µm 2.1 mm × 100 mm column ZIC-pHILIC guard column SeQuant ZIC-pHILIC column SeQuant ZIC-pHILIC LC column

12 protocols using zic philic

Targeted Metabolomics Analysis Using HILIC-LCMS

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The analyses were performed using separation on 150 x 4.6 mm ZIC-pHILIC (Merck) on UltiMate 3000 RSLC (Thermo Scientific) followed by mass detection on an Orbitrap Exactive mass spectrometer (Thermo Fisher) at Glasgow Polyomics. Analyses were performed in positive and negative polarity switching mode, using 10 μl injection volume and a flow rate of 300 μl/min. The samples were run alongside 249 authentic standards at 10 μM each. The data were processed and analyzed using mzMatch software [57 (link)], IDEOM [58 (link)], mzMatchISO [59 (link)], and MetaboAnalyst [60 (link)]. All the MS analyses were performed in 4 replicates, means of which are indicated. For the studies involving U-¹³C-glucose labelling, non-labelled samples were run in parallel and the natural labelling subtracted. Metabolites identified based on match with respective standards are indicated, the rest is predicted based on mass and retention time. Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (10.1093/nar/gks1004. PubMed PMID: 23109552) with the identifier MTBLS491.

Kovářová J., Pountain A.W., Wildridge D., Weidt S., Bringaud F., Burchmore R.J., Achcar F, & Barrett M.P. (2018). Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana. PLoS Pathogens, 14(3), e1006953.

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Metabolite profiling by LC-MS

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LC–MS-based metabolite profiling was performed on an Accela system coupled to an Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK) operating with electrospray ionisation (ESI) running in the negative (ESI−) and positive (ESI+) modes as previously described in (Kim et al. 2015 (link)). Briefly, the spray voltage was 4500 V (ESI+) and 3500 V (ESI−), capillary voltage was 40 V (ESI+) and 30 V (ESI−), tube lens voltage was 70 V for the both modes and skimmer voltage was 20 V (ESI+) and 18 V (ESI−). The temperature for capillary and probe was maintained at 275 °C and 150 °C, respectively. Chromatographic separation was carried out using ZIC-pHILIC (4.6 × 150 mm and 5 μm particle size, Merck Sequant). The mobile phase composed of 20 mM ammonium carbonate in water (solvent A) and 100% acetonitrile (solvent B). Metabolites were separated according to a linear gradient as following: 0–15 min (20% A), 15–17 min (95% A), and 17–24 min (20% A) at 300 µl/min flow rate. The injection volume of 10 µl and the column was kept at 45 °C.

Abuawad A., Mbadugha C., Ghaemmaghami A.M, & Kim D.H. (2020). Metabolic characterisation of THP-1 macrophage polarisation using LC–MS-based metabolite profiling. Metabolomics, 16(3), 33.

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HPLC Analysis of Biomolecules

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HPLC measurements were performed on a Shimadzu (Kyoto, Japan) HPLC system consisting of two LC-10ADvp pumps, a SIL-10ADvp auto-injector, an SPD-M10AVvp diode array detector and a CTO-10ASvp oven at 25 °C and a SCL-10Avp controller. A 5 μm, 150 x 4.6 mm ZIC-pHiLIC (Merck, Darmstadt, Germany) column was employed. Injection volume and flow rate were 1 μL and 0.50 mL min -1 , respectively.
Aqueous buffers used in the preparation of mobile phases were directly prepared from ammonium acetate at different concentrations in order to provide a total concentration of 5 mM after mixing with the organic solvent.

Subirats X., Abraham M.H, & Rosés M. (2019). Characterization of hydrophilic interaction liquid chromatography retention by a linear free energy relationship. Comparison to reversed- and normal-phase retentions. Analytica chimica acta, 1092.

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EDTA-Plasma Metabolomics Analysis

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Available EDTA-plasma samples from a non-selected sub-cohort of patients in the low and the high Na⁺ intake groups, stored in Padua biobank from the time of screening, were extracted with chloroform/methanol/water (1:3:1 v/v) and stored at −80°C until analysis by LC-MS. Briefly, samples were eluted on a hydrophilic interaction LC column (ZIC-pHILIC, Merck) and analysed on a Thermo Q-Exactive (Thermo Scientific), operated in polarity switching mode to record both positive and negative ionization mode data for each sample. A pooled sample, prepared by combining an aliquot from each individual sample, was injected every 5th injection to confirm the stability of the analysis. The raw MS data were processed in a non-targeted way, using a pipeline consisting of XCMS (peak picking) and MZMatch (grouping and filtering).^26–30 (link)

Rossitto G., Maiolino G., Lerco S., Ceolotto G., Blackburn G., Mary S., Antonelli G., Berton C., Bisogni V., Cesari M., Seccia T.M., Lenzini L., Pinato A., Montezano A., Touyz R.M., Petrie M.C., Daly R., Welsh P., Plebani M., Rossi G.P, & Delles C. (2020). High sodium intake, glomerular hyperfiltration, and protein catabolism in patients with essential hypertension. Cardiovascular Research, 117(5), 1372-1381.

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Metabolite Footprinting by LC-MS

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For metabolite footprinting of spent culture media, LC-MS was performed on an Accela system coupled to an Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK). Spectral data was acquired in full scan ion mode (m/z 70-1400, resolution 50 000) in both positive and negative electrospray ionisation modes. The probe temperature and capillary temperature were maintained at 150 and 275 °C, respectively. The calibration mass range was extended to cover small metabolites by inclusion of low-mass contaminants with the standard Thermo calibration mixture masses (below m/z 1400), C 2 H 6 NO 2 for positive ion electrospray ionisation (PIESI) mode (m/z 76.0393) and C 3 H 5 O 3 for negative ion electrospray ionisation (NIESI) mode (m/z 89.0244). Chromatographic separation was carried out using a ZIC-pHILIC (150 mm × 4.6 mm, 5 µm column, Merck Sequant) maintained at 45 °C and a flow rate of 300 µL min -1 as previously described in ref. 26. Briefly, the mobile phase consisted of (A) 20 mM ammonium carbonate in water, (B) 100% acetonitrile which were eluted with a linear gradient from 80% B to 5% B over 15 min, followed by a 2 min linear gradient from 5% B to 80% B, and 7 min re-equilibration with 80% B. The injection volume was 10 µL and samples were maintained at 4 °C.

Surrati A., Linforth R., Fisk I.D., Sottile V, & Kim D.H. (2016). Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footprinting. The Analyst, 141(12).

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Hydrophilic Interaction Liquid Chromatography

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The ZIC^®-pHILIC (Merck Sequant, Watford, UK) column (4.6 mm × 150 mm, particle size 5 µm) was used for chromatographic separation which was performed on an Accela system (Thermo Fisher Scientific, Hemel Hempstead, UK). Mobile phases were 20 mM ammonium carbonate in water (phase A, pH 9.1) and acetonitrile (phase B). A 24 min-per-sample gradient system was used, with a flow rate of 300 µL/min, at a column temperature of 45 °C. With a starting gradient of 20% A, increasing to 95% A over 15 min, an equilibration followed to give the 24 min run time per sample. Samples were maintained at 4 °C and an injection volume of 10 µL was used.

Wellham P.A., Hafeez A., Gregori A., Brock M., Kim D.H., Chandler D, & de Moor C.H. (2021). Culture Degeneration Reduces Sex-Related Gene Expression, Alters Metabolite Production and Reduces Insect Pathogenic Response in Cordyceps militaris. Microorganisms, 9(8), 1559.

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UHPLC-MS Analysis of Metabolites

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Liquid chromatography-mass spectrometry analysis was carried out using an Ultimate 3000 ultra high performance liquid chromatography and a Thermo Q-Exactive orbitrap mass spectrometer (MS) interfaced with a heated electrospray ionization source. The mass spectrometer was operated in positive and negative ionization modes. The full scan and data-dependent MSⁿ scan were collected at a resolution of 70,000 and 35,000, respectively. The heated capillary was held at 275°C, and the HESI probe held at 350°C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit. The MS data acquisition was performed in a range of 59–880 m/z. Chromatographic separation was achieved using Sequant ZIC-pHILIC column (2.1 × 150 mm, 5 μm, EMD Millipore) with a guard column (ZIC-pHILIC, 2.1 × 20 mm, 5 μm, EMD Millipore). Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide; buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.150 ml/min as follows: 0–20 min, linear gradient from 80% to 20% B; 20–21 min, hold at 20% B min; 21–22 min, linear gradient to 80% B; 22–28 min, re-equilibrate at 80% B. The injection volume was 5 μL.

Deng P., Valentino T., Flythe M.D., Moseley H.N., Leachman J.R., Morris A.J, & Hennig B. (2021). Untargeted stable-isotope probing of the gut microbiota metabolome using 13C-labeled dietary fibers. Journal of proteome research, 20(5), 2904-2913.

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LC-MS Metabolite Profiling Protocol

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For both the intracellular and extracellular metabolite profiling, LC-MS was performed on an Accela system coupled to an Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK) as previously described [28, 29] . Briefly, capillary voltage was 40 V (ESI+) and 30 V (ESI-), the spray voltage was 4.5 kV (ESI+) and 3.5 kV (ESI-), skimmer voltage was 20 V (ESI+) and 18 V (ESI-) and tube lens voltage was 70 V for the both modes. The temperature for capillary and probe was maintained at 275°C and 150°C, respectively. Intracellular and extracellular metabolites were separated on ZIC-pHILIC (4.6 × 150 mm and 5 μm particle size, Merck Sequant, Watford, UK). Chromatographic separation was carried out according to a linear gradient as following: 0-15 min (20% A -20 mM ammonium carbonate in water; 80% B -acetonitrile), 15-17 min (95% A), and 17-24 min (20% A) at 300 μl/min flow rate. The injection volume of 10 μl and the column was kept at 45°C.

Surrati A., Evseev S., Jourdan F., Kim D.H, & Sottile V. (2021). Osteogenic Response of Human Mesenchymal Stem Cells Analysed Using Combined Intracellular and Extracellular Metabolomic Monitoring. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 55(3).

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UPLC-QTOF-MS Metabolomics Profiling

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Single injections of 3 μl of the aqueous extract were made on a Waters Acquity UPLC system in discrete, randomized blocks. The pooled QC was injected every after every 10 injections. Separation was performed using a ZIC‐pHilic (5 μM, 2.0 × 150 mm; EMD Millipore), using a gradient from solvent A (acetonitrile) to solvent B (water, 10 mM Ammomium Bicarbonate, pH 9.6). Flow rate was 0.27 ml/min unless noted otherwise, and the column was held at 50°C. The gradient is as follows: time (t) = 0 min, 10% A; t = 1.5 min, 10% A; t = 8.5 min, 38% A; t = 11 min, 60% A; t = 11.5 min, 100% A, 0.2 ml/min flow; t = 16.5 min, 100% A; t = 17 min, 10% A; t = 18 min, 10% A, 0.6 ml/min flow; t = 22 min 10% A; t = 22.5 min, 10% A, 0.27 ml/min flow; t = 23 min, 10% A, end of equilibration. The column eluent was infused into a Xevo G2 Q‐TOF‐MS (Waters Corporation) with an electrospray source in negative ionization mode, scanning 50–1,200 m/z at 0.2 s per scan, alternating between MS (6 V collision energy) and MSE mode (15–30 V ramp). Calibration was performed using sodium formate with 1 ppm mass accuracy. The capillary voltage was held at 2,200 V, source temperature at 150°C, and N desolvation temperature at 350°C with a flow rate of 800 L/hr.

Sheflin A.M., Chiniquy D., Yuan C., Goren E., Kumar I., Braud M., Brutnell T., Eveland A.L., Tringe S., Liu P., Kresovich S., Marsh E.L., Schachtman D.P, & Prenni J.E. (2019). Metabolomics of sorghum roots during nitrogen stress reveals compromised metabolic capacity for salicylic acid biosynthesis. Plant Direct, 3(3), e00122.

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Metabolite Profiling by HPLC-MS

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Samples and a mixture of 240 standards (in three mixes) were separated by HPLC on a Dionex Ultimate 3000 RSLC system (Thermo Fisher Scientific) using a ZIC-pHILIC (Merck) column and then analyzed on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) using the method of Rattigan et al. (93 (link)), including fragmentation of the pooled samples.

Zoltner M., Campagnaro G.D., Taleva G., Burrell A., Cerone M., Leung K.F., Achcar F., Horn D., Vaughan S., Gadelha C., Zíková A., Barrett M.P., de Koning H.P, & Field M.C. (2020). Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes. The Journal of Biological Chemistry, 295(24), 8331-8347.

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