Zorbax rrhd eclipse plus c18 column

Manufactured by Agilent Technologies

Sourced in United States, Germany

The ZORBAX RRHD Eclipse Plus C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of compounds in various applications. The column features a C18 stationary phase and is suitable for use in rapid resolution high-definition (RRHD) HPLC systems.

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45 protocols using zorbax rrhd eclipse plus c18 column

Quantitative Analysis of Pseudomonas Metabolites

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This experiment was also performed by LC/Q-ToF (Agilent) using ZORBAX RRHD Eclipse Plus C18 column (Agilent) in MS mode, with 1 μL injection of experimental samples and standards for pyochelin, phenazine-1-carboxylic acid, and rhamnolipids. The mobile phase was water (A) and acetonitrile (B), both with 0.1% (v/v) formic acid. The elution procedure was 5% B between 0 and 1 min; 5−95% B between 1 and 11 min; 95% B between 11 and 13 min; 95−5% B between 13 and 14 min; and 95% B between 14 and 16 min with a flow rate of 0.4 mL/min.
The standards were dissolved in methanol to make stock solutions at a concentration of 1 g/L for pyochelin, 0.1 g/L for phenazine-1-carboxylic acid, and 10 g/L for rhamnolipids. The stock solutions were diluted to appropriate concentrations for sample preparation.

Donadt T.B., Wu Y., Lang J., Li S, & Yang R. (2021). Design of Polymeric Thin Films to Program Microbial Biofilm Growth, Virulence and Metabolism. Biomacromolecules, 22(12), 4933-4944.

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Quantitative Analysis of Infant Fecal Monosaccharides

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The monosaccharides analyzed in this method are those that are unconjugated and not constituents of di-, oligo-, or polysaccharides. The separation of monosaccharides used a binary solvent system, solvent A contained 5% acetonitrile (ACN) and 25 mM ammonium acetate (NH₄Ac) pH adjusted with ammonium hydroxide (NH₄OH) to 8.2. Solvent B consisted of 95% ACN. A flow rate of 0.500 mL/min was used with the following gradient: 0–7 min, 12–15% B; 7–7.10 min, 15–99% B; 7.10–8.50 min, 99% B; 8.50–8.60 min, 99–12% B. Quantitation of monosaccharides in infant feces was conducted using a calibration curve constructed with a range from 1 µg to 100 mg monosaccharide per mg solution. The column used for separation was an Agilent ZORBAX RRHD ECLIPSE PLUS C18 column (150 × 2.1 mm) with 1.8 µm particle size. The UHPLC-QqQ system was an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6495B QqQ mass spectrometer and 1 µL of each sample was injected for a run time of 10 min. Further details on instrument parameters and monosaccharide standards have been described by Xu et al. (26 ).

Ranque C.L., Stroble C., Amicucci M.J., Tu D., Diana A., Rahmannia S., Suryanto A.H., Gibson R.S., Sheng Y., Tena J., Houghton L.A, & Lebrilla C.B. (2020). Examination of Carbohydrate Products in Feces Reveals Potential Biomarkers Distinguishing Exclusive and Nonexclusive Breastfeeding Practices in Infants. The Journal of Nutrition, 150(5), 1051-1057.

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Broccoli Phytosterol Extraction and HPLC Quantification

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Ground broccoli head samples were placed into an Eppendorf tube (50 mL) containing 30 mL of ethanol. After vortexing and shaking for 3 min, the samples were ultrasonicated for 60 min using an ultrasonic cleaner (Xianou Instrument Co., Nanjing, China) and then centrifuged at 8000× g for 10 min when the samples cooled down. The supernatant passed through a 0.22 μm PVDF membrane.
The phytosterol was analyzed by an Agilent 1260 Series HPLC system (Agilent Technologies, Wilmington, DE, USA). The analytes were separated by an Agilent ZORBAX RRHD Eclipse Plus C18 column (50 × 3 mm, 1.8 μm) held at 30 °C. The mobile phase ratio of methanol/water (90:10% v/v) maintains a flow rate of 0.5 mL/min with a temperature of 350 °C. The injection volume was 10 μL. The identification and quantification parameters of mass spectrometry are shown in Table S2. The mass spectrum detector was operated in positive electrospray ionization (ESI).

Shi L., Li Y., Lin M., Liang Y, & Zhang Z. (2024). Profiling the Bioactive Compounds in Broccoli Heads with Varying Organ Sizes and Growing Seasons. Plants, 13(10), 1329.

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Metabolite Profiling by HPLC-QTOF MS

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Metabolites were identified and quantified by HPLC/Q-TOF (Agilent 1260 Infinity II/Agilent G6545B) in MS mode using positive ionization. For analysis, 1 μL of each culture sample was injected and separated in the ZORBAX RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm) (Agilent) with water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) as the mobile phase. The gradient program was set as 0–1 min, 95% A; 1–11 min, 95%–5% A; 11–13 min, 5% A; 13–14 min, 5%–95% A; and 14–16 min, 95% A at a flow rate of 0.4 mL/min. The m/z value of the [M+H]⁺ adduct was then used to extract the ion chromatogram (with a mass error below 20 ppm) for compound identification and quantification (see Supplementary Fig. S1 for extracted ion chromatograms of DDK, DMY, and Y standard). For compounds with available chemical standards, including cinnamic acid, hydrocinnamic acid, p-coumaric acid, p-hydrocoumaric acid, DDK, DMY, and Y, concentrations were quantified by comparing the integrated peak areas to standard curves (Supplementary Fig. S2 and Fig. S3). For identification of compounds of which no standards are available, target MS/MS mode with three different levels of collision energy (10, 20 and 40 eV) was used in addition to high-resolution MS analysis.

Wu Y., Chen M.N, & Li S. (2022). De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae. Metabolic Engineering Communications, 14, e00195.

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Purification and Analysis of Disaccharide-Depsipentapeptide from L. plantarum

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Disaccharide-depsipentapeptide (GlcNAc-MurNAc-L-Ala-D-Gln-mDAP-L-Ala-L-Lac) was purified from PG of dacA1dacA2 double mutant of L. plantarum^NC8. PG was digested with mutanolysin (Sigma-Aldrich), and the resulting soluble muropeptides were reduced by NaBH₄ as described previously (Bernard et al., 2011 (link)). The reduced muropeptides were separated by reverse-phase ultra-high-pressure liquid chromatography (RP-UHPLC) with a 1290 chromatography system (Agilent Technologies) and a Zorbax RRHD Eclipse Plus C18 column (100 by 2.1 mm; particle size, 1.8 µm; Agilent Technologies) at 50°C using ammonium phosphate buffer and methanol linear gradient. The eluted muropeptides were detected by UV absorbance at 202 nm. The peak corresponding to the disaccharide-depsipentapeptide was collected. Purified muropeptide was then incubated with 10 µg of purified DltE_extra or DacA1 in 50 mM Tris-HCl, 100 mM NaCl buffer overnight at 37°C. The reaction mixtures were analyzed by RP-UHPLC as described above and by LC-MS with an UHPLC instrument (Vanquish Flex, Thermo Scientific) connected to a Q Exactive Focus mass spectrometer (Thermo Scientific). Mass spectra were collected over the range m/z = 380–1400. Data were processed using Xcalibur QualBrowser v2.0 (Thermo Scientific).

Nikolopoulos N., Matos R.C., Ravaud S., Courtin P., Akherraz H., Palussiere S., Gueguen-Chaignon V., Salomon-Mallet M., Guillot A., Guerardel Y., Chapot-Chartier M.P., Grangeasse C, & Leulier F. (2023). Structure–function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth. eLife, 12, e84669.

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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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Quantitative Analysis of PFOA and PFBA

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The PFBA samples were analyzed at wavelength of 262 nm using ultraviolet−visible spectrophotometry (Unico, model 2008A). PFOA was analyzed directly via LC-MS/MS by two methods. First, The PFOA samples without SDS employed a Thermo Ultimate 3000 HPLC (Thermo Fisher Scientific, Breme, Germany) and AB-Sciex Qtrap 4500 mass spectrometer (AB Sciex Pte. Ltd., Singapore). A Waters column (C18, 2.1 × 50 mm, 1.7 μm) was maintained at 35 °C. The mobile phase of water and 100% acetonitrile applied in the ratio of 40:60 was set at a flow rate of 0.25 mL/min. Second, PFOA samples with SDS employed an Agilent HPLC 1260 and mass spectrometer 6150. An Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm) was maintained at 40 °C. The mobile phase of 0.01M ammonium acetate and 100% acetonitrile applied in the ratio of 60:40 was set at a flow rate of 0.3 mL/min. All the samples were passed through a 0.45 μm filter to remove suspended particulate impurities. Mass-labeled internal standard ¹³C₄-PFOA was added to monitor recovery. The R² of the calibration curves were >0.999 for each measurement. The quantifiable detection limit of the two systems is ~ 0.1 μg/L. These methods have been used successfully in our prior studies.^{26 (link),33 (link),34 (link)}

Ji Y., Yan N., Brusseau M.L., Guo B., Zheng X., Dai M, & Liu H. (2021). The Impact of Hydrocarbon Surfactant on the Retention and Transport of PFOA in Saturated and Unsaturated Porous Media. Environmental science & technology, 55(15), 10480-10490.

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Oxylipin Analysis by UPLC-MS/MS

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Oxylipins were analyzed on an Agilent 1290 Infinity UPLC system coupled to an Agilent 6460 triple-quadrupole tandem mass spectrometer (UPLC-MS/MS) with electrospray ionization (ESI) (Agilent, Palo Alto, CA, USA). Analyte separation was achieved using an Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 × 150 mm, 1.8 μm Agilent Corporation). Mobile phase A consisted of MilliQ water containing 0.1% acetic acid, and mobile phase B consisted acetonitrile/methanol (v/v, 80/15) containing 0.1% acetic acid. Samples were maintained at 4 °C during the analysis. The column temperature was 45 °C. The injection volume was 10 μL and the run time was 20 min. The mobile phase gradient and flow rate were set as follows: 1) 0–2 min, 65% A, 0.25 mL/min; 2) 2–12 min, 65–15% A, 0.25 mL/min; 3) 12–15 min, 15% A, 0.25 mL/min; 4) 15.1–17 min, 0% A, 0.4 mL/min; 5) 17.1–19 min, 65% A, 0.4 mL/min; and 6) 19–20 min, 65% A, 0.3 mL/min. The MS ion source parameters were set as follows: gas temperature 300 °C, gas flow 10 L/min, nebulizer gas 35 psi, sheath gas heater temperature 350 °C, and capillary voltage 4000 V. Oxylipins were captured using optimized dynamic multiple reaction monitoring parameters shown in Supplementary Table 1.

Liang N., Emami S., Patten K.T., Valenzuela A.E., Wallis C.D., Wexler A.S., Bein K.J., Lein P.J, & Taha A.Y. (2022). Chronic exposure to traffic-related air pollution reduces lipid mediators of linoleic acid and soluble epoxide hydrolase in serum of female rats. Environmental toxicology and pharmacology, 93, 103875.

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Ultra-sensitive Glycopeptide Analysis

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An ultra performance liquid chromatography system Nexera UPLC LC-30A (Shimadzu Corporation, Kyoto, Japan) equipped with a ZORBAX RRHD Eclipse Plus C18 column (1.8 μm, 2.1 mm × 100 mm, Agilent Technologies, Santa Clara, CA) was coupled online to a 6500 plus Qtrap Mass Spectrometer (AB Sciex, CA, USA). Mobile phase A was 0.1% FA and 3% acetonitrile in nanopure water (v/v/v), and mobile phase B was 0.1% FA in 90% acetonitrile. Peptides and glycopeptides were separated by a binary gradient at 40°C with 0.5 mL/min flow rate consisting of (1) 0 min at 2.0% B; 0.5-1.0 min at 5.0% B; 6.0 min at 30.0% B; (2) 6.1-8.0 min at 100.0% B), (3) 8.1-10.0 min at 2.0% B. The MS was operated in the positive mode. Curtain gas: 30.0 psi, collision gas: high, ion spray voltage: 5500.0 V, temperature: 300.0°C, ion source gas 1: 55.0 psi, ion source gas 2: 60.0 psi, declustering potential: 20.0 V, entrance potential: 10.0 V, and collision cell exit potential: 14 V. The scheduled MRM mode was used. Q1 and Q3 were set to unit resolution. The cycle time was fixed to 500 ms, the MRM detection window was set to 30.0 s, and the target scan time was 0.5 s. CE for each MRM transition was optimized by a 5 V step followed by a 2 V step fine-tuning. The analysis was controlled using the AB Sciex Analyst software (version 1.6.3).

Han J., Zhou Z., Zhang R., You Y., Guo Z., Huang J., Wang F., Sun Y., Liu H., Cheng X., Su Y., Shi H., Hu Q., Teng J., Yang C., Ren S, & Ye J. (2022). Fucosylation of anti-dsDNA IgG1 correlates with disease activity of treatment-naïve systemic lupus erythematosus patients. EBioMedicine, 77, 103883.

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UHPLC-MS/MS Analysis of Substrates and Metabolites

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Chromatographic separations were carried out on an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) using an Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 × 50 mm inner diameter,1.8 μm particle size) (Agilent Technologies, Santa Clara, CA, USA). The elution profile was composed of an initial isocratic step with water (0.1% formic acid): acetonitrile (ACN) (95:5) for 1.5 min and then increasing ACN to 30% over 4 min to separate the substrates and metabolites at 30 °C. The column was washed with 90% of ACN for 2.5 min followed by the equilibration of the column for 5 min with 5% ACN. The flow rate was 0.4 mL/min and the injection volume was 5 μL. An Agilent 6460 triple quadrupole-mass spectrometer with an electrospray ion source (Agilent Technologies, Santa Clara, CA, USA) was used for flow injection analysis with optimized fragmentor and source parameters. The optimized source parameters for MS analysis were drying gas temperature, 350 °C; gas flow, 10 L/min; nebulizer gas flow pressure, 35 psi; and capillary voltage, 4500 V.

Lu Y., Xie T., Zhang Y., Zhou F., Ruan J., Zhu W., Zhu H., Feng Z, & Zhou X. (2017). Triptolide Induces hepatotoxicity via inhibition of CYP450s in Rat liver microsomes. BMC Complementary and Alternative Medicine, 17, 15.

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