Agilent 1260 infinity lc system

Manufactured by Agilent Technologies

Sourced in United States, Germany

The Agilent 1260 Infinity LC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical applications. It provides reliable and efficient separation, detection, and quantification of a wide range of chemical compounds. The system features modular components that can be customized to meet specific analytical requirements.

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50 protocols using agilent 1260 infinity lc system

High-Resolution Mass Spectrometry Analysis

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Samples were analyzed using an Agilent 6530 Accurate-Mass Q-TOF coupled with a 1260 Agilent Infinity LC system equipped with a Sunfire^® C₁₈ column (150 × 2.1 mm ; i. d. 3.5 μm, Waters, Milford, MA, USA) with a flow rate of 0.25 mL/min. Full scan mass spectra were acquired in the positive-ion mode in a mass range of m/z 100 to 1200 Da, with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate at 10 L/min. Capillary, fragmentor, and skimmer voltages were set at 3500 V, 175 V, and 65 V respectively. Four scan events were used: positive MS, mass range encompassing m/z 100–1200, and three data-dependent MS/MS scans of the five most intense ions from the first scan event. Three collision energies (viz. 30, 50, and 70 eV) were used for MS/MS data generation. Purine (C₅H₄N₄, m/z 121.050873 and HP-0921 (hexakis (1 H, 1 H, 3 H-tetrafluoropropoxy)-phosphazene C₁₈H₁₈F₂₄N₃O₆P₃, m/z 922.009798) were used as internal lock masses. Full scans were acquired at a resolution of 10,000 (m/z 922) and 4000 (m/z 121). A permanent MS/MS exclusion list criterion was established to prevent oversampling of the internal calibrant.

Kouamé T., Okpekon A.T., Bony N.F., N’Tamon A.D., Gallard J.F., Rharrabti S., Leblanc K., Mouray E., Grellier P., Champy P., Beniddir M.A, & Le Pogam P. (2020). Corynanthean-Epicatechin Flavoalkaloids from Corynanthe pachyceras. Molecules, 25(11), 2654.

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Dereplication of Crude Plant Extracts

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The HPLC-ESI-QTOF-MS/MS analysis, or dereplication, is a new method that allows identifying very quickly and efficiently the known compounds in a complex mixture, such as crude plant extracts. With this method, it becomes easy to tell the difference between known and unknown compounds in a complex mixture, based on database search [Jongmin et al., 2017] [13] . This analytical method is based on the coupling system ESI-QTOF-MS/MS (electrospray ionizationquadrupole-time of flight-mass spectroscopy), that provides sufficient information to determine the exact structure of a known compound [McFarland et al., 2004] [14] . For the analysis of the crude methanol extract, using the system an Agilent 6530 Accurate-Mass QTOF coupled with a 1260 Agilent Infinity LC system, the flow rate was fixed to 0.25 mL/min, and mass spectra were acquired in positive mode in the mass range of m/z 100 to 1200 Da.

Blaise K.K., Claude K.A.L., Jacques K.D., Raphaël O.K., Kouamé D.B., & Barthélemy A.K. (2021). Identification of limonoids and steroids from the leaves of Turraea heterophylla (Smith) by HPLC‐ESI‐QTOF‐MS/MS methods. Journal of Pharmacognosy and Phytochemistry, 10(6), 50-55.

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Agilent 1,260 Infinity LC-MS/MS Analysis

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Samples were analyzed on an Agilent 1,260 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) comprising a 6,460 triple quadrupole equipped with an electrospray ionization source, vacuum degasser, binary pump and thermostatic column compartment. The LC system, mass spectrometer and data analysis are controlled using Agilent Mass Hunter software version B.05.01 (Agilent Technologies).

Liu J., Shao H., Fang S., Cheng Y., Ling P, & Chen J. (2019). Evaluation of pharmacokinetics and pharmacodynamics of sinomeninehyaluronic acid conjugate after intraarticular administration for osteoarthritis treatment. Drug Design, Development and Therapy, 13, 657-665.

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Quantification of DNA Adducts by LC-MS/MS

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Caecal tissue was harvested as described before and genomic DNA was extracted using QIAamp Fast DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. 25μg of isolated DNA were spiked with stable isotopically labeled adduct standards and hydrolyzed to 2’-deoxynucleosides (dN) according to our optimized protocol46 (link). After analyte enrichment by C₁₈ solid phase extraction, the predominant DNA adducts after 1-MIM-OH exposure, namely N²-(1-MIM)-dG and N⁶-(1-MIM)-dA, were analyzed by isotope-dilution liquid chromatography tandem-mass spectrometry (LC-MS/MS) as described previously47 (link) using an Agilent 1260 Infinity LC system coupled to an Agilent 6490 triple quadrupole-mass spectrometer (Agilent Technologies) interfaced with an electrospray ion source operating in the positive ion mode (ESI+). Further, in the DNA hydrolyzate prior to sample purification, we analyzed the amounts of dC and 5mdC (5-methyl-2’-deoxycytidine) by isotope-dilution LC-MS/MS as recently published48 (link). We calculated the amount of DNA that was actually used for the adduct analysis knowing that dC (in fact the sum of dC and 5mdC) accounts for 21% of the total dN in the mouse genome.

Gronke K., Hernández P.P., Zimmermann J., Klose C.S., Kofoed-Branzk M., Guendel F., Witkowski M., Tizian C., Amann L., Schumacher F., Glatt H., Triantafyllopoulou A, & Diefenbach A. (2019). Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature, 566(7743), 249-253.

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Quantification of DNA Adducts by LC-MS/MS

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Hydrogen-Deuterium Exchange Mass Spectrometry of IgG1-Fc Glycoforms

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Hydrogen exchange was performed using an H/DX PAL robot (LEAP Technologies, Carrboro, North Carolina) and MS measurements were conducted using a QTOF mass spectrometer (Agilent 6530, Santa Clara, California) with Agilent 1260 Infinity LC System as described previously.^{35 (link)} For HX, deuterium labeling of 3 μL each of the IgG1-Fc glycoproteins, held at 20 μM concentration was performed by adding 27 μL of deuterated labeling buffer at 25°C in triplicate. The above exchange reactions were quenched at 1°C by 1:1 dilution of the labelled proteins by a quench buffer (containing 4 M guanidine hydrochloride, 1M TCEP and 0.2 M sodium phosphate at pH 2.5) after incubation for 5 labeling times: 15 s, 10² s, 10³ s, 10⁴ s and 86400 s. Subsequently, the quenched samples were digested on an immobilized pepsin column that was prepared in-house.³⁶ Carry-over from the pepsin column was removed between samples using a two-wash cocktail method as previously described.^{37 (link)} A standard LC-MS procedure for HX-MS^{35 (link)} was used and all the measurements were made relative to HM-Fc glycoform, which was run in parallel on the same day with each of the three other glycoforms as a control for day-to-day variability.

More A.S., Toth RT I.V., Okbazghi S.Z., Middaugh C.R., Joshi S.B., Tolbert T.J., Volkin D.B, & Weis D.D. (2018). Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry (HX-MS). Journal of pharmaceutical sciences, 107(9), 2315-2324.

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Quantification of Fatty Acids by HPLC

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An isocratic reversed phase validated HPLC method [30 (link)] was used for the detection and quantification of FA using an Agilent 1260 infinity LC system (Agilent Technologies, Santa Clara, CA, USA) equipped with quaternary pump, an auto-sampler unit, and a UV detector. The stationary phase was Kromasil C18 (150 mm × 4.6 mm) column packed with a 5-μm-size adsorbent. A mixture of acetonitrile/methanol/0.05 M phosphoric acid (50:10:40 v/v/v) with a pH adjusted to 2.9 was used as the mobile phase at a flow rate of 2 mL/min. The detector was set at 235 nm, and the injection volume was 100 μL. The regression equation for the calibration curve was y = 26617x − 14.87 and the assay was linear (r² = 0.998) in the concentration range of 125 μg/mL–5 ng/mL.

Ahmed I.S., Elnahas O.S., Assar N.H., Gad A.M, & El Hosary R. (2020). Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation. Pharmaceutics, 12(3), 199.

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Ras Protein Quantification Protocol

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20 μg of cell lysate or 40 μg of mouse lysate containing spike-ins of the relevant heavy Ras standard were fractionated using SDS PAGE. The region of the gel containing endogenous and His-Ras standards was excised and dissected into ~1 mm gel cubes. These were reduced using 10 mM DTT in 100 mM Ammonium bicarbonate (Ambic) for 1 hour at 56 º C, alkylated in 55 mM iodoacetamide for 30 mins at room temperature, quenched with 10 mM DTT for 5 minutes at room temperature, then digested with 5 ng / μl Trypsin Gold (Promega) in 9% Acetonitrile and 40 mM Ambic, overnight at 37 º C. Trypsin was quenched using Formic acid and extracted peptides dried using a SpeedVac. Peptides were subjected to C18 desalting using an Agilent 1260 Infinity LC system equipped with an MRP-C18 Hi-recovery column (Agilent, USA) before SpeedVac drying.

Hood F.E., Sahraoui Y.M.E., Jenkins R.E., & Prior I.A. (2021). Ras protein abundance correlates with Ras isoform mutation patterns in cancer.

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Gentiopicroside Quantification by HPLC

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Gentiopicroside concentrations were determined via High Performance Liquid Chromatography (HPLC). All sample solutions and stock solutions of gentiopicroside were prepared as we have described before [23 (link)]. Chromatographic separations were conducted with a C18 column (250 × 4.6 mm, 5 μm particle size; Agilent Technologies Inc., USA) on an Agilent 1260 Infinity LC system, using a solvent system comprising 70% ddH₂O (A) and 30% methanol (B). The flow rate was adjusted to 0.8 mL min^-1 and the detection wavelength was 245 nm. All separations were performed at 25°C.

Cao X., Guo X., Yang X., Wang H., Hua W., He Y., Kang J, & Wang Z. (2016). Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated Gentiana macrophylla Seedlings. PLoS ONE, 11(11), e0166493.

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Quantification of Glutathione Disulfide Reduction

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Ferric cyt c (400 µM) or ABTS (400 µM) was mixed with GSSSG or GS³⁴SSG (400 µM), NADPH (400 µM) and GR (1 U ml⁻¹) in a total volume of 100 µl in PBS. After 5 minutes, the reaction was stopped by the addition of 100 µl of MBB in MeOH (10 mM). The samples were incubated for 5 minutes in the dark, and then 200 µl of CHCl₃ was added to remove GR by precipitation. After centrifugation at 300 RCF, the upper phase was removed for further analysis. The sample (10 µl) was injected into an Agilent 1260 Infinity LC system attached to an Agilent 6120 Single Quadrupole MS with ESI source and evaporative light scattering detector (ELSD). Separation was performed on a Kinetex 2.6 μm C18 100 Å LC column (50 × 2.1 mm) at 40 °C using a flow rate of 0.6 ml min⁻¹. Solvent ‘A’ was 0.01% HCOOH in water; solvent ‘B’ was 0.01% HCOOH in MeCN. The method was: 100% A for 2 minutes, then from 100% to 10% A in 10 minutes and then 1% A for another 10–12 minutes. Data were processed with MestReNova (14.2.1) software.

Barayeu U., Schilling D., Eid M., Xavier da Silva T.N., Schlicker L., Mitreska N., Zapp C., Gräter F., Miller A.K., Kappl R., Schulze A., Friedmann Angeli J.P, & Dick T.P. (2022). Hydropersulfides inhibit lipid peroxidation and ferroptosis by scavenging radicals. Nature Chemical Biology, 19(1), 28-37.

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