Agilent poroshell 120 ec c18 column

Manufactured by Agilent Technologies

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The Agilent Poroshell 120 EC-C18 column is a high-performance liquid chromatography (HPLC) column. It features a silica-based stationary phase with a particle size of 2.7 μm, designed for the separation and analysis of a wide range of analytes.

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11 protocols using agilent poroshell 120 ec c18 column

Photolytic Degradation of Tetracycline

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The FMN photolytic reaction on TC degradation was analyzed using an LC-MS/MS method. The separation of TC and FMN in the solutions is described in Section 2.3. The samples were maintained under blue light irradiation at 20 W/m² for 1 h. TC, FMN, and D-TCF were eluted by an Agilent Poroshell 120 EC-C18 column (2.7 µm, 4.6 mm × 150 mm, Agilent Technologies, Palo Alto, CA, USA), followed by LC-MS/MS analysis. Using an Agilent 1200 Series HPLC System, an electrospray ionization (ESI) source was connected to an Agilent 6410B triple quadrupole MS in this study.
The mobile phase was composed of 0.1% methanoic acid (A) and methanol (B). The process of separation was completed by a mobile phase, the profile of which is depicted below. The linear gradient started with 0–2 min, 95%–80% A; 2–8 min, 80% A; 8–12 min, 80%–40% A; 12–15 min, 40% A; 15–19 min, 40%–5% A; 19–24 min, 5% A; and 24–27 min, 5%–95% A. The final mobile phase was set at 95% solvent A from 27 to 30 min. The reaction solution at a volume of 10 μL was injected at a flow rate of 0.4 mL per minute. TC or D-TCF was investigated by a positive-ion mode for charged fragments. The data were obtained and inspected using the MassHunter Workstation software, version B.06.00.

Huang S.T., Lee S.Y., Wang S.H., Wu C.Y., Yuann J.M., He S., Cheng C.W, & Liang J.Y. (2019). The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5′-Phosphate Photolysis on Microbial Viability. Microorganisms, 7(11), 500.

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Synthesis of Biotinylated Cholesterol

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Commercially available 27-alkyne cholesterol (800 μg, 2 μM) and Azide-PEG₃-biotin (400 μg, 0.9 μM) were mixed in 100 mL dimethyl sulfoxide (DMSO) and water solution (v/v: 1/1). DMSO/water (50 μL, v/v: 1/1) containing CuSO₄ (0.25 μM) and sodium L-ascorbate (0.5 μM) was added in the reaction mixture. The solution was incubated at room temperature under N₂ protection for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The product was purified using a reversed-phase high performance liquid chromatography (HPLC) instrument (Shimadzu, Kyoto, Japan) equipped with an SPD-M40 photo diode array detector (Shimadzu, Kyoto, Japan and an Agilent poroshell 120 EC-C₁₈ column (2.7 mm, 4.6 × 250 mm), using water/methanol/formic acid = 20/80/0.1 (v/v/v) as the mobile phase, at a flow rate of 0.3 mL/min to afford biotinylated cholesterol as a white solid powder. The structure of the resulting biotinylated cholesterol conjugate was characterized by an 800 MHz nuclear magnetic resonance spectroscopy (Bruker Corporation, Rheinstetten, Germany) an LTQ-Orbitrap XL FT-MS spectrometer (Thermo Scientific, Bremen, Germany).

He P., Faris S., Sagabala R.S., Datta P., Xu Z., Callahan B., Wang C., Boivin B., Zhang F, & Linhardt R.J. (2022). Cholesterol Chip for the Study of Cholesterol–Protein Interactions Using SPR. Biosensors, 12(10), 788.

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Engineered Pseudomonas Strain for Ligand Depletion

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The plasmid pJCBAtG^{45 (link)} developed previously was transformed into Pseudomonas putida KT2440 ∆pcaGH, which is unable to metabolise protocatechuate. The pJCBAtG plasmid carries tphC/tpiBA and the full tphAB_II catabolic operon. Resting cell conversion assays were performed as previously described^{45 (link)}. Briefly, P. putida ∆pcaGH cells harbouring pJCBAtG were grown in terrific broth medium supplemented with 25 µg/mL tetracycline for 10 h after induction with m-toluate, harvested by centrifugation, washed with 50 mM Tris-HCl buffer (pH 7.5) and re-suspended to an OD₆₀₀ of 40 in the same buffer. Depletion of ligands was investigated in 1 mL assays containing 1 mM of the corresponding substrate and cells to a final OD₆₀₀ of 30 in 50 mM Tris-HCl buffer. The assays were incubated at 30 °C for 60 min. Supernatant samples were collected at time 0 and after 60 min, filtrated and ligand concentration analysed by high-performance liquid chromatography (HPLC; Agilent 1100 HPLC system, Agilent Technologies, UK). Separation was achieved using an Agilent Poroshell 120 EC-C18 column (4 µm, 100 mm, Agilent Technologies, UK) with a gradient 5–95% acetonitrile containing 0.1% formic acid as the mobile phase. All ligands were detected at 254 nm.

Gautom T., Dheeman D., Levy C., Butterfield T., Alvarez Gonzalez G., Le Roy P., Caiger L., Fisher K., Johannissen L, & Dixon N. (2021). Structural basis of terephthalate recognition by solute binding protein TphC. Nature Communications, 12, 6244.

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HPLC-FLD Quantification of OTA

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The OTA was quantified by HPLC-FLD using an Agilent 1260 Infinity II LC system (Agilent Technologies, Little Fall, DE, USA) and an Agilent Poroshell 120 EC-C18 column (3.0 mm × 150 mm, 2.7 μm) (Agilent Technology, Santa Clara, California, USA). The mobile phase consisted of 0.1% formic acid dissolved in water (A) and acetonitrile (B). The injection volume was 10 µL, and the flow rate was set at 0.5 mL/min. The column was maintained at 30 °C. The elution program was as follows: B: 0–5 min, 50%; 5–10 min, 70%; 10–15 min, 90%; 15–18 min, 50%. The excitation and emission wavelengths for fluorescence detection were 334 nm and 460 nm, respectively.

Guo Y., Wang Z., He Y., Gao H, & Shi H. (2024). Profiling of Volatile Compounds in ‘Muscat Hamburg’ Contaminated with Aspergillus carbonarius before OTA Biosynthesis Based on HS-SPME-GC-MS and DLLME-GC-MS. Molecules, 29(3), 567.

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Insulin Glargine UHPLC Analysis

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Insulin glargine was analyzed using UHPLC techniques as previously described.18 Briefly, each insulin sample was diluted 1:50 in 0.01 N hydrochloric acid. Two microliters of the sample were then injected into an Agilent 1290 Infinity LC System equipped with an Agilent Poroshell 120 EC-C18 column (3.0×50 mm, 1.9 µm; Agilent Technologies, Santa Clara, California, USA). The separation was performed at a column temperature of 40°C and a flow rate of 0.75 mL/min with isocratic elution using a mobile phase consisting of acetonitrile and solution A (26:74 v/v). Solution A was prepared by dissolving 28.4 g of anhydrous sodium sulfate in 1000 mL of water, and 2.7 mL of phosphoric acid was subsequently added. If necessary, the pH was adjusted to 2.3 with ethanolamine. The analysis was run for 7 min, and the eluted insulin was detected at a wavelength of 214 nm. Each sample was assayed in duplicate.

Kongmalai T., Orarachin P., Dechates B., Chanphibun P., Junnu S., Srisawat C, & Sriwijitkamol A. (2022). The Effect of high temperature on the stability of basal insulin in a pen: a randomized controlled, crossover, equivalence trial. BMJ Open Diabetes Research & Care, 10(6), e003105.

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Serum Glycine Levels in Subclinical Atherosclerosis

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Circulating glycine levels were measured in serum samples from patients with and without sCAD by investigators blinded to the study groups as we previously described [8 ,30 (link)]. Briefly, liquid chromatography (LC)-mass spectrometry (MS)/MS was used to measure serum amino acids using isotope-labeled amino acids (MSK-CAA-1, Cambridge Isotopes Laboratory) as internal standard. The LC-MS/MS analysis was performed using a Shimadzu LC-20AD HPLC system (Shimadzu Corporation) coupled in-line to an AB Sciex QTrap 5500 system (Applied Biosystems) equipped with an electrospray ionization source. Chromatographic separation was conducted on an Agilent Poroshell 120 EC-C18 column (50 mm × 2.1 mm I.D., 2.7 μm; Agilent) at a flow rate of 0.4 mL/min. The mobile phase consisted of water (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The mass spectrometer was operated in positive ionization mode with multiple reaction monitoring (MRM). Analyst version 1.6.2 software (Applied Biosystems) was used for data acquisition and analysis. In mice, blood glucose levels were measured using glucometer and test strips (Contour Next). Plasma samples were analyzed for total cholesterol (TC) and triglycerides (TG) using the Wako Cholesterol E kit (999-02601, FUJIFILM medical system) and LabAssay Triglyceride kit (290-63701, FUJIFILM medical system).

Rom O., Liu Y., Finney A.C., Ghrayeb A., Zhao Y., Shukha Y., Wang L., Rajanayake K.K., Das S., Rashdan N.A., Weissman N., Delgadillo L., Wen B., Garcia-Barrio M.T., Aviram M., Kevil C.G., Yurdagul A J.r., Pattillo C.B., Zhang J., Sun D., Hayek T., Gottlieb E., Mor I, & Chen Y.E. (2022). Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis. Redox Biology, 52, 102313.

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Glycosaminoglycan Profile of Dental Epithelium in Fam20B-Deficient Mice

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GAG profile of E11.5 dental epithelium in WT and Fam20B-deficient (KO) mice were characterized regarding the GAG type, amount, sulfation, and disaccharide composition using a recently developed method MRM-LCMS (multiple reaction monitoring liquid chromatography mass spectrometry) [70 (link)]. Briefly, the dental epithelium of lower incisors was isolated from the mandibles of E11.5 KO and WT embryos after dispase digestion (1.8 U/ml in Ca- and Mg-free PBS, Gibco) at 37 °C for 30 min. The epithelium pooled from 6 embryos of each group were lysed in digestion buffer (50 mM ammonium acetate, 2 mM calcium chloride) and digested by cocktail of GAG-lyases (heparin lyase I, II, III, and chondroitin lyase ABC (10 mU each), then placed in 37 °C incubator overnight. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone (AMAC), and analyzed by liquid chromatography mass spectrometry (LC-MS/MS, Thermo Inc.) running at multiple reaction monitoring mode. The separation was carried out with an Agilent 1200 HPLC separation system on an Agilent Poroshell 120 ECC18 column (3.0 × 150 mm, 2.7 μm, Agilent, USA) at 45 °C. The analytical error for GAG profiling was < 3%.

Wu J., Tian Y., Han L., Liu C., Sun T., Li L., Yu Y., Lamichhane B., D’Souza R.N., Millar S.E., Krumlauf R., Ornitz D.M., Feng J.Q., Klein O., Zhao H., Zhang F., Linhardt R.J, & Wang X. (2020). FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling. BMC Biology, 18, 87.

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Glycan and Fatty Acid Analysis via FTMS

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Glycan samples were dissolved in HPLC grade water as 0.2–0.5 μg/μL, and each sample (5 μL) was run in direct infusion mode by the standard ESI source of LTQ-Orbitrap XL FTMS (Thermo Fisher Scientific, San-Jose, CA, USA). LC parameters: Agilent Poroshell 120 ECC18 column (2.7 μm, 3.0 × 50 mm), mobile phase A was 5-mM ammonium acetate prepared with HPLC grade water, and mobile phase B was 5-mM ammonium acetate prepared in 98% HPLC grade acetonitrile with 2% of HPLC grade water. The flow was used 50% A and 50% B at a rate of 250 μL/min. The source parameters for FTMS were in the negative-ion mode, a spray voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens voltage of −50 V, a capillary temperature of 275 °C, a sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6 L/min. All FT mass spectra were acquired at a resolution 60,000 with 200–2000 Da mass range. Fatty acids sample was dissolved in HPLC acetonitrile and used 100% B as flow; all other conditions were the same.

Yan L., Fu L., Xia K., Chen S., Zhang F., Dordick J.S, & Linhardt R.J. (2020). A Revised Structure for the Glycolipid Terminus of Escherichia coli K5 Heparosan Capsular Polysaccharide. Biomolecules, 10(11), 1516.

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RP-HPLC Analysis of Protein Samples

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RP-HPLC was performed with minor modifications as described previously^{27 (link)}. Briefly, RP-HPLC was performed using an Agilent 1100 Series HPLC System (Agilent Technologies, USA) equipped with an Agilent Poroshell 120EC-C18 column, 4 µm 4.6 × 100 mm (Agilent Technologies, USA). About 210 pmol of protein (nonreduced) was injected and eluted (flow rate 1 ml/min) with a linear gradient of 3–95% over 20 min of 0.1% trifluoroacetic acid (TFA), 20% isopropanol, and 70% acetonitrile. The absorbance was measured at 214 nm and chromatographic peaks were integrated by HPLC ChemStation (Agilent Technologies, USA).

Singh S.K., Plieskatt J., Chourasia B.K., Singh V., Bengtsson K.L., Reimer J.M., van Daalen R.C., Teelen K., van de Vegte-Bolmer M., van Gemert G.J., Jore M.M, & Theisen M. (2021). Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine. NPJ Vaccines, 6, 120.

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Quantifying Melatonin Metabolites in Broccoli

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About 1 g of fresh samples of broccoli seedlings were collected and ground. Melatonin and its precursors, tryptamine, serotonin, and N-acetylserotonin, were then extracted using methanol and quantified with LC-MS/MS (API 4000), as described in Yu et al. [27 (link)]. After centrifugation for 5 min at 8000× g and 4 °C, the supernatants were dried with a Termovap Sample Concentrator and dissolved in 200 μL of methanol. Melatonin and its precursors in the extraction solutions were separated using an Agilent Poroshell 120 EC-C18 column (150 mm × 2.1 mm, 2.7 μm, Agilent Technologies). Solvent A consisted of water with 0.1% (v/v) formic acid, while solvent B consisted of methanol. The LC gradient program was set as follows: 0–2 min, 20% of solvent A; 2–5 min, 20% of solvent A; and 5.1–12 min, 80% of solvent A; the flow rate was 0.3 mL min⁻¹. The ion modes and m/z values of the precursor and product ions for each metabolite are listed in Table S1. The method for the calculation of melatonin and its precursors was adopted from Kang et al. [28 (link)].

Li R., Zhou Z., Zhao X, & Li J. (2024). Application of Tryptophan and Methionine in Broccoli Seedlings Enhances Formation of Anticancer Compounds Sulforaphane and Indole-3-Carbinol and Promotes Growth. Foods, 13(5), 696.

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