Agilent 1200 hplc system

Manufactured by Agilent Technologies

Sourced in United States, Germany, Canada, United Kingdom, Japan

The Agilent 1200 HPLC system is a high-performance liquid chromatography instrument designed for analytical applications. It features a modular design that allows for customization to meet specific analytical requirements. The system includes a solvent delivery system, autosampler, column compartment, and a variety of detectors, such as UV-Vis and diode array detectors.

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387 protocols using agilent 1200 hplc system

Analytical Methods for Metabolite Quantification

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Cell growth was monitored by measuring the absorbance at 600 nm (OD₆₀₀) using an UV–VIS spectrophotometer. Glucose consumption was quantified by a biosensor SBA-90 (Biology Institute of Shandong Academy of Sciences, China). Residual concentration of xylose was measured using Waters 1515 HPLC system, equipped with a Bio-Rad HPX-87H column and a refractive index detector (Waters 2414, Milford, USA), and the column was eluted at 65 °C with 5 mM sulfuric acid at 0.6 mL/min.
The broth samples were centrifuged, and supernatants were filtered through 0.22 μm syringe filter, and injected to the HPLC system. 4-HMA and l-tyrosine were measured using Agilent 1200 HPLC system equipped with a C18 column (250 × 4.6 mm, Agilent) and a PDA detector (Agilent) at 196 nm with a mobile phase (10 % methanol-90 % H₂O, addition of 0.1 % formic acid) at 1.0 mL/min. The structure of 4-HMA was identified using LC–MS (Agilent 1200 HPLC system and 6310 Ion Trap mass spectrum system, Agilent) under negative ion mode. All of the HPLC analysis were quantified using a six point standard curve and the R² coefficient for the standard curve was higher than 0.99.

Li F.F., Zhao Y., Li B.Z., Qiao J.J, & Zhao G.R. (2016). Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose–xylose mixture. Microbial Cell Factories, 15, 90.

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Radiochemical Synthesis and PET/CT Imaging

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HPLC analysis
was performed with
an Agilent 1200 HPLC system equipped with a 1200 series diode array
detector. Radio-HPLC analysis was performed with an Agilent 1200 HPLC
system equipped with a series diode array detector and a Raytest GABI
Star radioactivity detector. ¹⁸F-Fluoride was purchased
from either the PET Center at St. Thomas’ Hospital or Alliance
Medical UK. All reagents were purchased from Sigma-Aldrich and were
used without further purification. The radiochemical yield was calculated
as a percentage of purified tracer to starting activity. The radiochemical
conversion was calculated based on the HPLC peak AUCs. Preclinical
PET/CT images were acquired using a NanoScan PET/CT (Mediso, Budapest,
Hungary) scanner. Data are represented as mean ± SD, unless stated
otherwise. Chromatographs and graphs were plotted on GraphPad Prism
8.

Mota F., Pell V.R., Singh N., Baark F., Waters E., Sadasivam P., Southworth R, & Yan R. (2021). A Reactivity-Based 18F-Labeled Probe for PET Imaging of Oxidative Stress in Chemotherapy-Induced Cardiotoxicity. Molecular Pharmaceutics, 19(1), 18-25.

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Comprehensive Analytical Techniques for Biosimilar Characterization

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SEC was performed on an Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany). RPLC was performed on an Agilent 1100 HPLC system for method evaluation (section 3.1) and on an Agilent 1200 HPLC system for biosimilar comparability exercises (section 3.2). Detection was carried out with a diode-array detector. SEC multiangle light scattering (MALS) was performed on a 1260 Bio-Inert HPLC coupled with a Bio-Dual Angle LS/DLS (Agilent). CGE-UV separations were carried out on a G7100 CE system (Agilent) coupled to a diode-array detector. For RPLC quadrupole time-of-flight (QTOF) experiments, a UHPLC system coupled to a DTIMS-QTOF mass spectrometer 6,560 (Agilent) was used. A Dual Agilent Jet Stream ESI was used as electrospray ionization source.

Demelenne A., Ben Yahia A., Lempereur D., Crommen J., Servais A.C., Fradi I, & Fillet M. (2021). Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein. Frontiers in Chemistry, 9, 782099.

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Metabolomic Profiling by High-Resolution LCMS

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Analyses were performed in a HPLC 1200 Agilent system coupled to an Agilent 6520 qTOF mass spectrometer operated in mode full scan from 50 to 1200 m/z, with a RP C8 column Agilent Poroshell (150 mm 2.1 mm, 2.7 μm) as previously described^{34 (link)}. Mobile phases were composed by 5 mmol/L NH₄HCO₂ in ultra-pure water (phase A) and 5 mmol/L NH₄HCO₂ in MeOH (85%) and IPA (15%) in phase B, in positive ionization mode, and formic acid 0.1% in ultra-pure water (phase A) and formic acid 0.1% in MeOH (85%) and IPA (15%), for the negative ionization mode, pumped at 0.5 mL/min. The capillary voltage (V) was 3500 kV in positive ionization mode and 4500 kV in negative ionization mode. Reference masses were infused in all analyses to perform the mass correction, which were 121.0509 m/z and 922.0098 m/z for the positive ionization mode and 112.9856 m/z and 1033.9881 m/z for the negative ionization mode. Data files were collected in centroid mode at a scan rate of 1.02 scans/s.

Gramage E., Sáiz J., Fernández-Calle R., Martín Y.B., Uribarri M., Ferrer-Alcón M., Barbas C, & Herradón G. (2022). Metabolomics and biochemical alterations caused by pleiotrophin in the 6-hydroxydopamine mouse model of Parkinson’s disease. Scientific Reports, 12, 3577.

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Plasma Metabolite Extraction and LC-MS Analysis

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50 μl of plasma samples were aliquoted and added 350 μl of a mixture of MTBE/Ethanol (50:50 v/v), followed by vortex for approximately 30s and centrifugation at 15 min 4000 g, at 15 °C [18 (link)]. The supernatant was collected and transferred to the sterile vials to LC-MS analysis. Analysis was performed by HPLC 1200 Agilent system accoupled to mass spectrometry, with a RP C8 column Agilent Poroshell (150 mm × 2.1 mm, 2.7 μm) as previously described [19 (link)]. Mobile Phases were composed by 5 mmol L⁻¹ NH4HCO2 in ultra-pure water (phase A) and 5 mmol L⁻¹ NH4HCO2 in MEOH (85%) and IPA (15%) in phase B in positive mode; formic acid 0.1% in ultra-pure water (phase A) and formic acid 0.1% in MEOH (85%) and IPA (15%) for the negative mode, pumped 0.5 mL/min. Quadrupole-time of flight (qTOF) mass spectrometry (6520, Agilent Technologies) was performed in both polarities ESI (positive and negative) in mode full scan from 50 to 1200 m/z. The capillary voltage (V) was 3500 kV in positive mode and 4500 kV in negative mode. Reference masses were applied in all analyses, which were 121.0509 m/z and 922.0098 m/z for the positive mode and 112.9856 m/z and 1033.9881 m/z for the negative mode to performe the mass correction. Data files were collected in centroide mode, scan rate of 1.02 scans/s.

Mendes-Frias A., Santos-Lima B., Furtado D.Z., Ruperez F.J., Assunção N.A., Matias M.J., Gomes V., Gaifem J., Barbas C., Castro A.G., Capela C, & Silvestre R. (2020). Dysregulation of glycerophospholipid metabolism during Behçet’s disease contributes to a pro-inflammatory phenotype of circulating monocytes. Journal of Translational Autoimmunity, 3, 100056.

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Chemical Fingerprint Analysis of SBP

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One g of SBP_water or SBP_EtOH was sonicated in 10 ml of EtOH. The extract was filtered; the supernatant was collected and analyzed for chemical fingerprint analysis. The analysis was performed on an Agilent HPLC 1200 system (Agilent, Waldbronn, Germany), equipped with a degasser, a binary pump, an auto sampler, a thermostatic column compartment, and a DAD. The samples were separated on a PLATISIL C₁₈ column (4.6 mm × 250 mm, 5 μm i.d.) after filtered with a guard column. The mobile phase was composed of acetonitrile (A) and 0.03% phosphoric acid solution (B) according to pre-set gradient program: 0 to 25 min, linear gradient 15% to 40% (A), 85% to 60% (B); 25% to 55 min, linear gradient 40% to 75% (A), 60% to 25% (B); 55 to 65 min, linear gradient 75% to 100% (A), 25% to 0% (B); 65% to 75 min, 100% (A). The injection volume was 10 μl; the flow rate was 0.8 ml/min; and the column temperature was 25°C. The detector was set to 280 nm.

Xu M.L., Zheng Z.Y., Xia Y.J., Liu E.Y., Chan S.K., Hu W.H., Duan R., Dong T.T., Zhan C.S., Shang X.H, & Tsim K.W. (2019). Shexiang Baoxin Pill, a Formulated Chinese Herbal Mixture, Induces Neuronal Differentiation of PC12 Cells: A Signaling Triggered by Activation of Protein Kinase A. Frontiers in Pharmacology, 10, 1130.

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HPLC Analysis of Organic Acids

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For this determination an Agilent HPLC 1200 system (Agilent, Palo Alto, CA, USA), comprising a DAD detector, quaternary pump, and a thermostatic auto sampler, was used. A Luna 2 Phenomenex (size: Φ 4.6 × 150 mm) column served as a stationary phase at 25 °C. The mobile phase consisted of: (A) 10mM NaH₂PO₄·H₂O (pH 2.50 ± 0.02) and (B) acetonitrile. A volume of 10 µL was injected into the system and the elution was performed using an isocratic method with 90% (A) and a variable flow: 0.6 mL/min until 1.5 min, 0.4 mL/min at 2 min, 0.25 mL/min from 3 to 15 min, 0.3 mL/min at 16 min, 0.6 mL/min at 17 min, 1 mL/min from 18 to 23 min, 0.6 mL/min until 25 min, for a better separation. The absorbance was monitored at 220 nm. The separation of organic acids was achieved in 25 min and the identification was obtained by comparing their retention times with those of standards using a Chemstation (Agilent, Palo Alto, CA, USA) software. All used reagents were HPLC grade reagents, purchased from EMD Millipore, Merck Group.

Uțoiu E., Matei F., Toma A., Diguță C.F., Ștefan L.M., Mănoiu S., Vrăjmașu V.V., Moraru I., Oancea A., Israel-Roming F., Cornea C.P., Constantinescu-Aruxandei D., Moraru A, & Oancea F. (2018). Bee Collected Pollen with Enhanced Health Benefits, Produced by Fermentation with a Kombucha Consortium. Nutrients, 10(10), 1365.

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Stability Analysis of GdCho-Len Nanoparticles

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GdCho-Len nanoparticles were placed at 4, 15, 25 and 37°C for 7 days. Stability of GdCho-Len was analyzed by high performance size exclusion chromatography performed using a TSKgel G3000SWxl column (Tosoh Bioscience) and an Agilent HPLC 1200 system (Agilent Technologies Gmbh).

Zhou T., Hang D., Li Y., Zhang J., Wu H., Wang H., Tian E, & Yan J. (2019). Role of Gd2O3-doped carbon-11-choline-lenvatinib nanoparticles contrast agent PET/CT in the diagnosis of patients with lung cancer. Oncology Letters, 19(2), 1117-1124.

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HPLC Analysis of SBP Extracts

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One gram of SBP_water or SBP_EtOH was dissolved in 10 ml EtOH. The extract was filtered, and the supernatant was collected for chemical analysis (Gao et al., 2008 (link); Xu et al., 2019 (link)). The analysis was performed with an Agilent HPLC 1200 system (Agilent, Waldbronn, Germany), equipped with a binary pump, a degasser, an auto sampler, a thermostatic column compartment, and a diode array detector (DAD). The samples were analyzed by using a PLATISIL C₁₈ column (4.6 mm × 250 mm, 5 μm i.d.). Acetonitrile (A) and 0.03% phosphoric acid solution (B) were used as the mobile phase according to pre-set gradient program: 0–25 min, linear gradient 15–40% (A), 85–60% (B); 25–55 min, linear gradient 40–75% (A), 60–25% (B); 55–65 min, linear gradient 75–100% (A), 25–0% (B); 65–75 min, 100% (A). The column temperature was 25°C; the injection volume was at 10 μl; the flow rate was set at 0.8 ml/min; and the wavelength was 280 nm.

Hu W.H., Mak S.H., Zheng Z.Y., Xia Y.J., Xu M.L., Duan R., Dong T.T., Li S.P., Zhan C.S., Shang X.H, & Tsim K.W. (2020). Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice. Frontiers in Pharmacology, 11, 1045.

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Hybrid LC-MS Proteomic Workflow

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LC-MS was performed on a hybrid LTQ-Orbitrap Velos mass spectrometer (Thermo Fischer Scientific, San Jose, CA, USA). An Agilent HPLC 1200 system (Agilent Technologies, Santa Clara, CA, USA) was used for online reversed-phase nano-LC at a flow of 0.4 µl/min. Solvent A was 97% water, 3% ACN, 0.1% formic acid; and solvent B was 5% water, 95% ACN, 0.1% formic acid. The curved gradient went from 2% B up to 40% B in 45 min, followed by a steep increase to 100% B in 5 min. Samples (3 µl from each IPG fraction) were trapped on Zorbax 300SB-C18, 5 µm, 5×0.3 mm (Agilent Technologies, Santa Clara, CA, USA) and separated on a NTCC-360/100-5-153 C18 column (Nikkyo Technos Co., Tokyo, Japan) installed on to the nano electrospray ionisation (NSI) source of the Orbitrap Velos instrument. Acquisition proceeded in ∼3.5 s scan cycles, starting by a single full scan MS at 30000 resolution (profile mode), followed by two stages of data-dependent tandem MS (centroid mode): the top 5 ions from the full scan MS were selected firstly for collision induced dissociation (CID, at 35% energy) with MS/MS detection in the ion trap, and finally for high energy collision dissociation (HCD, at 50% energy) with MS/MS detection in the orbitrap. Precursors were isolated with a 2 m/z width and dynamic exclusion was used with 60 s duration.

Kjellin H., Johansson H., Höög A., Lehtiö J., Jakobsson P.J, & Kjellman M. (2014). Differentially Expressed Proteins in Malignant and Benign Adrenocortical Tumors. PLoS ONE, 9(2), e87951.

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