Thermo ultimate 3000

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Thermo Ultimate 3000 is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a modular design, advanced control software, and reliable performance to support efficient and precise sample separation and analysis.

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25 protocols using thermo ultimate 3000

HPLC Analysis of Methanolic L. capensis Leaves

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HPLC analysis of the methanolic extract from L. capensis leaves was performed using a Thermo UltiMate3000 (Thermo Fisher Scientific, USA) gradient chromatograph equipped with quaternary pumps controlled by Chromeleon interface, an autosampler and multidiode array detector (DAD). Solvents were filtered using a Millipore system and analysis was performed on an Accucore XL C18 column (150 × 4.6 mm, 4 μm). All the samples were filtered through 0.22 μm filter before being analysed. The mobile phase was pure acetonitrile (A) and bidistilled water containing 0.1% acetic acid (B), and the gradient setting was 10–23% (A) in 5 min.; 23% (A) isocratic for 10 min. and then 23–35% (A) in 12 min.; 35–70% (A) for 5 min. The injection volume was 20 μl with scanning absorbance wavelengths from 240 to 520 nm, typical for phenols including flavonols, flavones, hydroxycinnamic acids and anthocyanins. The flow rate increased from 0.2 ml to 1 ml/min. HPLC grade solvents and bidistilled water were used in the chromatographic studies. All chromatographic experiments were performed at 25°C. Standard curves for authentic samples of the polyphenols were obtained from purchased reagents (Sigma Chemical Co., St. Louis, USA) of analytical or high‐performance liquid chromatography (HPLC) grade. Each solution was injected in triplicate and the calibration curves were constructed with the averages.

Postu P.A., Noumedem J.A., Cioanca O., Hancianu M., Mihasan M., Ciorpac M., Gorgan D.L., Petre B.A, & Hritcu L. (2017). Lactuca capensis reverses memory deficits in Aβ1‐42‐induced an animal model of Alzheimer's disease. Journal of Cellular and Molecular Medicine, 22(1), 111-122.

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HPLC Analysis of Liver Adenine Nucleotides

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Analyses were carried out by using high-performance liquid chromatography (HPLC) as described in a previous study [24 (link)]. Approximately 80 mg of frozen liver samples were homogenized with 1.5 mol/L perchloric acid and centrifuged to obtain supernatants. The collected supernatants were neutralized with 2 mol/L potassium carbonate and centrifuged again to obtain supernatants for analysis by using a Thermo Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with a C18 chromatographic column (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase (50 mmol/L K₂HPO₄-KH₂PO₄ buffer solution and methanol (HPLC grade); 99:1, v/v; pH = 7.0) was filtered (0.45 µm filter) and degassed before use. All standards and samples were filtered (0.22 µm filter) and injected (20 µL) to the HPLC system. The wavelength for UV detection was 254 nm, the flow rate was 1.0 mL/min, and the column temperature was 40℃. Total adenine nucleotide (TAN) and adenylate energy charges (AEC) were calculated by using the previously reported equations [25 (link)]: TAN = ATP + ADP + AMP and AEC = (ATP + 0.5 ADP)/(ATP + ADP + AMP).

Su W., Xu W., Zhang H., Ying Z., Zhou L., Zhang L, & Wang T. (2017). Effects of dietary leucine supplementation on the hepatic mitochondrial biogenesis and energy metabolism in normal birth weight and intrauterine growth-retarded weanling piglets. Nutrition Research and Practice, 11(2), 121-129.

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HPLC Analysis of Hydroxyalkyl Amides

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The HAAs were analyzed using HPLC (Thermo Ultimate 3000, Thermo Scientific, Waltham, MA, USA) equipped with an autosampler (WPS-3000), a pump (LPG-3400SD), and a diode array detector (DAD-3000). A reversed-phase analytical column (Acclaim^TM 120 C₁₈, 3 μm, 4.6 × 150 mm) from Tosoh Bioscience GmbH (Stuttgart, Germany) was used for separation, and the column temperature was maintained at 35 °C. A gradient program was applied using a mobile phase of methanol: acetonitrile: deionized water: acetic acid (8:14:76:2, v/v/v/v) as a solvent A (pH = 5.0 adjusted with ammonium hydroxide (25%)); and acetonitrile as a solvent B. HAAs were identified by their retention times and quantified using an external calibration curve.

Oz F., Oz E., Aoudeh E., Abd El-Aty A.M., Zeng M, & Varzakas T. (2021). Is Ultra-High Temperature Processed Milk Safe in Terms of Heterocyclic Aromatic Amines?. Foods, 10(6), 1247.

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HA-DCAF16 Affinity Purification and Proteomic Analysis

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HEK293T cells were transfected with HA-DCAF16 plasmid by PEI transfection reagent for 24 h and treated with DMSO or 10 µM KB02-PEG0-SLF for 2 h. Cells were collected and lysed in 1% NP-40 lysis buffer with complete protease inhibitor cocktail. HA immunoprecipitation (40 µL slurry anti-HA agarose, 4 °C, 2 h) was performed with 10 mg of total protein lysates to purify HA-DCAF16. After washing the HA resin three times with IP washing buffer and once with PBS, HA-DCAF16 protein was eluted by heating at 65 ºC for 10 min with 8 M urea in PBS, then reduced with 12.5 mM DTT at 65 ºC for 15 min and alkylated with 25 mM iodoacetamide at 37 ºC for 30 min. The protein solution was diluted with PBS to 2 M urea and digested with 2 µg trypsin at 37 ºC for 6 h. Tryptic peptides were acidified with 5% formic acid and loaded onto a silica capillary column (250 µm) packed with 3 cm of C18 resin (Aqua 5 µm, Phenomenex). Peptides were analyzed on LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) coupled with a Thermo UltiMate 3000 UHPLC system. Peptides were separated on a capillary column packed with 10 cm of C18 resin (Aqua 5 µm, Phenomenex) and a 5 µm tip. MS parameters were set as previously described⁵¹. The raw data was acquired in Xcalibur operation software.

Zhang X., Crowley V.M., Wucherpfennig T.G., Dix M.M, & Cravatt B.F. (2019). Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nature chemical biology, 15(7), 737-746.

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Determination of Heterocyclic Aromatic Amines

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The HAA content was determined according to the method described by Messner and Murkovic [17 ], with minor modifications. Briefly, the barbecued samples and NaOH (1 M) were homogenized and mixed with Extrelut NT packing material (Merck, Darmstadt, Germany). During the solid-phase extraction, ethyl acetate was used for extraction. After washing with HCl and MeOH, the analytes including HAAs were eluted with MeOH-concentrated ammonia. The eluted mixtures were evaporated and the residues were dissolved in MeOH including internal standard. HPLC (Thermo Ultimate 3000, Thermo Scientific, Santa Clara, CA) with diode array detector (DAD-3000) was used for determination and Acclaim^TM 120 C18, 3 μm (4.6 × 150 mm, Tosoh Bioscience GmbH, Stuttgart, Germany) column was used for separation of HAAs. A mobile phase consisting of methanol/acetonitrile/water/acetic acid (8/14/76/2, v/v/v/v) at pH 5.0 (adjusted with ammonium hydroxide 25%) and acetonitrile were used as solvent A and solvent B, respectively.

, & Oz E. (2020). Effects of smoke flavoring using different wood chips and barbecuing on the formation of polycyclic aromatic hydrocarbons and heterocyclic aromatic amines in salmon fillets. PLoS ONE, 15(1), e0227508.

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Comprehensive Metabolic Profiling Protocol

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The blood was collected once a week before the end of the experiment and at 4 h intervals. All the plasma samples were run in duplicate in a single assay. The plasma levels of glucose (Glu), total cholesterol (TC), low-density triglycerides (TG), non-esterified fatty acids (NEFA), high-density lipoproteins, low-density lipoprotein, and apolipoproteins (A1, B, E) were individually measured at the Clinical Biochemistry Service of Nan Jing general hospital (Nanjing, China), using Beckman AU5811 analyzer (Beckman-Coulter, 250S.Kraemer Boulevard Brea, USA). The plasma melatonin level was determined using high performance liquid chromatography (HPLC, Thermo Ultimate 3000; Thermo Fisher Scientific, Waltham, MA, USA) as described previously by Yin et al [16 (link)]. Leptin and insulin levels were measured in plasma using commercial bovine ELISA kit (Shanghai Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions. For plasma insulin analysis, the results with intra-assay coefficient of variation (CV) of 3.0% to 6.0% were taken into consideration. For leptin analysis, results were acceptable with a CV of less than 5%.

Yu Y., Qiu J., Cao J., Guo Y., Bai H., Wei S, & Yan P. (2021). Effects of prolonged photoperiod on growth performance, serum lipids and meat quality of Jinjiang cattle in winter. Animal Bioscience, 34(9), 1569-1578.

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HPLC Analysis of Pharmaceutical Compounds

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Chromatographic analysis was performed on a Thermo UltiMate 3000 HPLC system (Thermo Scientific, United States). The separations were achieved on an Agilent SB-C18 column (250 mm × 4.6 mm, 5 μm) with the column temperature at 40°C. The mobile phases consisted of acetonitrile (A) and an aqueous solution containing 0.05% phosphoric acid (B) using a gradient elution as follows: 0–10 min 96–89% B, 10–25 min 89–87% B, 25–50 min 87–85% B, 50–70 min 85–80% B, 70–100 min 80–67% B, 100–115 min 67–40% B, and 115–140 min 40% B with a flow rate of 1.0 ml/min. The injection volume was 10 μl and the detection wavelength was at 268 nm.

Gao D., Zhao H., Yin Z., Han C., Wang Y., Luo G, & Gao X. (2021). Rheum tanguticum Alleviates Cognitive Impairment in APP/PS1 Mice by Regulating Drug-Responsive Bacteria and Their Corresponding Microbial Metabolites. Frontiers in Pharmacology, 12, 766120.

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Analytical Instrument Protocol Compendium

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The vortex mixer (MX-S) was purchased from Scilogex Co. (USA). The high-speed freezing centrifuge (5810 R) was obtained from Eppendorf Co. (Hamburg, Germany). The Eyela rotary evaporator (N-1100) was obtained from RIKAKIKAI Co. (Tokyo, Japan). Thermo UltiMate^™ 3000 was purchased from Thermo Fisher Scientific Co. (New York, USA). The TSQ Vantage was obtained from Thermo Fisher Scientific Co. (New York, USA). The freeze dryer (Biosafer-10 B) was purchased from Biosafer Co. (Nanjing, CN).

Han X, & Liu D. (2019). Detection and analysis of 17 steroid hormones by ultra-high-performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-MS) in different sex and maturity stages of Antarctic krill (Euphausia superba Dana). PLoS ONE, 14(3), e0213398.

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Targeted LC-HRMS Analysis of Wastewater

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The wastewater samples were analyzed using an LC system (Thermo UltiMate 3000, Thermo Fisher Scientific, USA) coupled to a quadrupole orbitrap MS (Q Exactive Focus, Thermo Fisher Scientific, USA). A C18 column (ACQUITY UPLC BEH C18, 2.1 × 150 mm, 1.7 μm, Waters, USA) was used for LC separation under 2 mM ammonium acetate water (phase A) and methanol (phase B) with a column temperature 40°C and an injection volume of 10 μL. The flow rate is 0.3 ml/min and the gradient was set as follows: 0 to 1 min 10% B, 1 to 36 min 10% to 100% B, 36 to 50 min 100% B, 50 to 50.1 min, 100% B to 10% B, and 50.1 to 55 min 10% B.
HRMS was operated with full scan (under mass resolution of 70,000) followed by data-dependent MS/MS scan (under mass resolution of 17,500) in electrospray ionization negative mode. Sheath gas, aux gas, and sweep gas were set on 48, 11, and 2, separately. Capillary temperature, aux gas–heated temperature was set on 320° and 413°C. Spray voltage was set on −2.5 kV. The S-lens radio frequency level was set on 50. Other settings were as follows: full scan range of m/z, 80 to 1000; full-scan automatic gain control (AGC) target, 1 × 10⁶; MS/MS scan range, 50 to 1000; MS/MS scan AGC target, 5 × 10⁴. Precursor ions were selected by quadrupole with an isolation window of 1.0 Da and fragmentation normalized collision energy of −35 ± 15 eV.

Wang X., Yu N., Jiao Z., Li L., Yu H, & Wei S. (2024). Machine learning–enhanced molecular network reveals global exposure to hundreds of unknown PFAS. Science Advances, 10(21), eadn1039.

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Cardiac Metabolite Profiling by LC-MS

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We analyzed samples from hearts by liquid chromatography‒mass spectrometry (LC‒MS) with a Thermo Ultimate 3000 (Thermo Fisher Scientific, USA) and TripleTOF 5600+ (AB SCIEX, USA). Detailed information on the sample preparation and analysis is available in the Supplemental Materials.

Yu H., Gan D., Luo Z., Yang Q., An D., Zhang H., Hu Y., Ma Z., Zeng Q., Xu D, & Ren H. (2024). α-Ketoglutarate improves cardiac insufficiency through NAD+-SIRT1 signaling-mediated mitophagy and ferroptosis in pressure overload-induced mice. Molecular Medicine, 30, 15.

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