1290 infinity 2 uplc system

Manufactured by Agilent Technologies

Sourced in United States

The 1290 Infinity II UPLC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical applications. It is capable of delivering precise and accurate solvent flow rates, enabling efficient separation and analysis of complex samples. The system is designed to provide reliable and reproducible results, making it a versatile tool for a wide range of analytical workflows.

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13 protocols using 1290 infinity 2 uplc system

HPLC Analysis of Soluble Sugars in Tomatoes

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Simple soluble sugars were analyzed by HPLC. Briefly, 500 mg of sample was rehydrated with 2 mL of distilled water and homogenized. Tomato samples were extracted three times with 10 mL ethanol (80%), then the mixture was heated at 70 °C for 10 min. After agitation for 20 min and centrifugation (4000× g, 5 min, 15 °C, Beckman Coulter, Brea, CA, USA), the supernatant was filtered through a 0.45 μm membrane before injection in UPLC. Samples were analyzed using a UPLC–1290 System Infinity II (Agilent, Santa Clara, CA, USA) equipped with a refractometer detector. A SHODEX SH1011 column 300 × 8 mm (Tokyo, Japan) was used with an isocratic system of water with H₂SO₄ (0.01%) and a flow rate of 0.7 mL/min. Temperature was set at 30 °C, injection volume at 10 μL, and spectrophotometric detection at 210 and 245 nm. External calibration was established for each standard sugar for concentrations from 0 to 10 g/L.

Boufeldja L., Boudard F., Portet K., Guzman C., Morel S., Berger N., Duchamp O., Dhuique-Mayer C., Dubos C, & Poucheret P. (2023). The Impact of Elevated Atmospheric Carbon Dioxide Exposure on Magic Tomatoes’ Nutrition–Health Properties. International Journal of Molecular Sciences, 24(16), 12815.

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Quantification of Soluble Sugars in Tomatoes

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Simple soluble sugars were analyzed by HPLC. Briefly, 500 mg of sample were rehydrated with 2 mL of distilled water and homogenized. Tomato samples were extracted three times with 10 mL ethanol (80%), then the mixture was heated at 70 °C for 10 min. After agitation for 20 min and centrifugation (4000× g, 5 min, 15 °C, Beckman Coulter, Brea, CA, USA), the supernatant was filtered through a 0.45 μm membrane before injection in UPLC. Samples were analyzed using a UPLC–1290 System Infinity II (Agilent, Santa Clara, CA, USA) equipped with a refractometer detector. A SHODEX SH1011 column 300 × 8 mm (Tokyo, Japan) was used with an isocratic system of water with H₂SO₄ (0.01%) and a flow rate of 0.7 mL/min. Temperature was set at 30 °C, injection volume at 10 μL, and spectrophotometric detection at 210 and 245 nm. External calibration was established for each standard sugar for concentrations from 0 to 10 g/L.

Boufeldja L., Brandt D., Guzman C., Vitou M., Boudard F., Morel S., Servent A., Dhuique-Mayer C., Ollier L., Duchamp O., Portet K., Dubos C, & Poucheret P. (2022). Effect of Elevated Carbon Dioxide Exposure on Nutrition-Health Properties of Micro-Tom Tomatoes. Molecules, 27(11), 3592.

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Quantification of Bacterially Produced Cholic Acid

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Bacterially produced Ch-S was obtained by culturing P. merdae ATCC 43184 (WT) and P. merdae Δsult in the presence of 500 μM Ch for 168 hours alongside media controls (n = 3) and P. merdae WT with no additional Ch. A 1.25 mL sample was removed from each culture, acidified to pH 1 with HCl (Sigma), and extracted twice with ethyl acetate (Sigma). The organic phase was collected and evaporated to dryness using a turbovap (Biotage). Dried extracts were resuspended in 100 μL of HPLC-grade methanol (Sigma) and quantified by running 1:10 dilutions on the UPLC-MS (Agilent Technologies 1290 Infinity II UPLC system coupled online to an Agilent Technologies 6120 Quadrupole LC/MS spectrometry in negative electrospray mode) alongside a standard curve according to the above method. Quantified samples were used for immune cell migration assays (see below).

Helgason S., Petursson G., Gudmundsson S, & Sigurdsson J.A. (2000). Prevalence of postherpetic neuralgia after a first episode of herpes zoster: prospective study with long term follow up. BMJ : British Medical Journal, 321(7264), 794.

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Quantitative Glycerol-d8 and Propanediol-d8 Analysis

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Detailed methods are provided in Supplementary Methods. Briefly, using deuterated glycerol-d8 [Sigma. 447498] and 6 (±)-1,2-propanediol-d8 [CDN, D-1656]) as an internal standard, samples were analyzed by LCMS/MS (Agilent 1290 Infinity II UPLC system with Agilent Zorbax Eclipse Plus C18 column; Santa Clara, CA). Eluted compounds were further separated and quantified through the coupled Agilent 6495 Triple Quadrupole equipped with an electrospray ion (ESI) source. The coefficient of variation for PG from 14-blinded repeat samples was 15.4%.

Song M.A., Reisinger S.A., Freudenheim J.L., Brasky T.M., Mathé E.A., McElroy J.P., Nickerson Q.A., Weng D.Y., Wewers M.D, & Shields P.G. (2019). Effects of Electronic Cigarette Constituents on the Human Lung: A Pilot Clinical Trial. Cancer prevention research (Philadelphia, Pa.), 13(2), 145-152.

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Targeted Metabolite Extraction and UPLC-MS Analysis

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Samples were acidified to a pH of 1 with HCl (Sigma) with GCA (Steraloids) as the internal standard and extracted twice with ethyl acetate (Sigma). The organic phase was collected and dried down using a turbovap (Biotage). Dried extracts were resuspended in 75% HPLC-grade methanol in dH₂O and analyzed by UPLC-MS (Agilent Technologies 1290 Infinity II UPLC system coupled online to an Agilent Technologies 6120 Quadrupole LC/MS spectrometry in negative electrospray mode) using a published method^{69 (link),75} with modifications outlined as follows. Extracted molecule solutions were injected onto a Phenomenex 1.7 μm, C18 100 Å, 100 × 21 mm LC column with a flow rate of 0.350 mL/min using 0.05% formic acid in water as mobile phase A and acetone as mobile phase B. The following gradient was applied: 0–1 min: 25–60% B, 1–5 min: 60–70% B, 5–6 min: 70–100% B, 6–7 min: 100% B isocratic, 7–8 min: 100–25% B, 8–10 min: 25% isocratic. Extracted ion chromatograms and areas under the curve were generated using Agilent ChemStation C.01.07 SR3. Exported data were analyzed in Microsoft Excel and GraphPad Prism V9.

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UPLC-QTOF-MS Analysis of Samples

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An Agilent 1290 Infinity II UPLC system connected to a G6545B Q-TOF MS system with a dual ESI source (Agilent Technologies, USA) was used to assess the samples. Using 0.1% formic acid-deionized water (A) and acetonitrile (B), all samples went through separation on an Agilent ZORBAX RRHD Eclipse Plus C18 column (50 × 2.1 mm, 1.8 μm) linked to an Agilent A-Line Quick Connect LC Fitting. Temperature: 20 °C; injection volume: 1 μL; data rate: 10 Hz; flow rate: 0.3 mL/min; split ratio: 1:1; wavelength: 210, 254, and 315 nm were the parameters of the UPLC. 0–0.3 min, 10% B; 0.3–10 min, 10%–100% B; 10–12 min, 100% B; 12–12.1 min, 100%–10% B; 12.1–15 min, 10% B, was the optimal gradient elution program.

Rahmat E., Yu J.S., Lee B.S., Lee J., Ban Y., Yim N.H., Park J.H., Kang C.H., Kim K.H, & Kang Y. (2024). Secondary metabolites and transcriptomic analysis of novel pulcherrimin producer Metschnikowia persimmonesis KIOM G15050: A potent and safe food biocontrol agent. Heliyon, 10(7), e28464.

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Quantifying Fecal Short-Chain Fatty Acids

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The levels of SCFA (acetic acid, propionic acid, and butyric acid) were measured using a 3-nitrophenylhydrazine derivatization strategy. About 100 mg of feces were extracted with 20 × 70% methanol (m/v) and then processed with TissueLyzer after adding zinc beads. The fecal samples were then centrifuged at 14,000 rpm at 4°C for 15 min. About 200 μL of upper supernatant was transferred to new tubes for LC-MS analysis. An Agilent 1290 Infinity II UPLC system coupled with a triple quadrupole 6470 mass spectrometer was used for targeted metabolomic profiling. A Waters BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with a precolumn was used. The MS data were collected and processed using Agilent software. The chromatographic condition was used as described previously by Dei Cas et al 22 , a and mass spectrometry detection was performed as described in our previous study 23 (link).

Huan Q., Peng J., Chang Y., Zhang Q., Xing T., Jiang D., Chen W., Shen X., Bian Z, & Xiao H. (2023). Activation of P2Y1R impedes intestinal mucosa repair during colitis. International Journal of Biological Sciences, 19(14), 4360-4375.

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Rapid Glycoside Separation and Quantification

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Derivatized glycosides were separated on an Agilent Poroshell HPH-C18 column (2.1 × 50 mm, 1.9 µm) and guard using an Agilent 1290 Infinity II UPLC system. A constant flow rate of 1.050 mL/min was employed on a 2 min isocratic elution at 12% solvent B followed by a 1.6 min flush at 99% solvent B and 0.79 min equilibration for a total run time of 4.6 min for the separation of compounds. Solvent A consisted of 25 mM ammonium acetate in 5% acetonitrile with pH adjusted to 8.2 using concentrated ammonia solution. Solvent B consisted of 95% acetonitrile in water. The separated glycosides were then detected on an Agilent 6495B triple-quadrupole mass spectrometer (QqQ-MS) operated in positive ion mode using dynamic multiple reaction monitoring (dMRM).

Castillo J.J., Couture G., Bacalzo NP J.r., Chen Y., Chin E.L., Blecksmith S.E., Bouzid Y.Y., Vainberg Y., Masarweh C., Zhou Q., Smilowitz J.T., German J.B., Mills D.A., Lemay D.G, & Lebrilla C.B. (2022). The Development of the Davis Food Glycopedia—A Glycan Encyclopedia of Food. Nutrients, 14(8), 1639.

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Bacterial Metabolite Extraction and UPLC-MS Analysis

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Bacterial cultures or cell lysates were acidified to pH=1 using HCl (Sigma) and extracted twice with ethyl acetate (Sigma). The organic phase was collected and dried using a SpeedVac (Thermo Scientific) for 96-well plate cultures or a TurboVap (Biotage) for bacterial tube cultures or microcentrifuge tube lysates, respectively. Dried extracts were solubilized in 75% HPLC-grade methanol (EMD Millipore) in dH₂O and analyzed by UPLC-MS (Agilent Technologies 1290 Infinity II UPLC system coupled online to an Agilent Technologies 6120 Quadrupole LC/MS spectrometry in negative electrospray mode) using a published method^{39 ,40 (link)} with modifications outlined as follows. Extracted BA solutions were injected onto a Phenomenex 1.7 μm, C18 100 Å, 100 × 21 mm LC column with a flow rate of 0.350 mL/min using 0.05% formic acid in water as mobile phase A and acetone as mobile phase B. The following gradient was applied: 0–1 min: 25–60% B, 1–5 min: 60–70% B, 5–6 min: 70–100% B, 6–7 min: 100% B isocratic, 7–8 min: 100–25% B, 8–10 min: 25% isocratic.

Paik D., Yao L., Zhang Y., Bae S., D’Agostino G.D., Zhang M., Kim E., Franzosa E.A., Avila-Pacheco J., Bisanz J.E., Rakowski C.K., Vlamakis H., Xavier R.J., Turnbaugh P.J., Longman R.S., Krout M.R., Clish C.B., Rastinejad F., Huttenhower C., Huh J.R, & Devlin A.S. (2022). Human gut bacteria produce TH17-modulating bile acid metabolites. Nature, 603(7903), 907-912.

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Molecular networking of LC-MS data

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Aliquots (1 μL) of dried fraction (100 μg/mL in MeOH) were analysed on an Agilent 6545 Q-TOF LC/MS equipped with an Agilent 1290 Infinity II UPLC system, utilising an Agilent SB-C8 1.8 μm, 2.1 × 50 mm column, eluting with 90% H₂O/MeCN to MeCN at a 0.417 mL/min over 2.5 min with an isocratic 0.1% formic acid modifier. UPLC-QTOF-(+)MS/MS data acquired for all samples at collision energy of 10, 20, and 40 eV were converted from Agilent MassHunter data files (.d) to mzXML file format using MSConvert software and transferred to the GNPS server (gnps.ucsd.edu). Molecular networking was performed using the GNPS data analysis workflow [22 (link)] using the spectral clustering algorithm with a cosine score of 0.6 and a minimum of 5 matched peaks. The resulting spectral network was imported into Cytoscape software (version 3.7.1) [23 (link)] and visualized using a ball-stick layout where nodes represent parent masses and the cosine score was reflected by edge thickness. Furthermore, group abundances were set as pie charts, which reflected the intensity of MS signals.

Elbanna A.H., Khalil Z.G, & Capon R.J. (2021). Oxandrastins: Antibacterial Meroterpenes from an Australian Mud Dauber Wasp Nest-Associated Fungus, Penicillium sp. CMB-MD14. Molecules, 26(23), 7144.

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