Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected and applied to a DEAE-Sephadex A-25 column. The glucosinolates were converted into their desulfo analogs by overnight treated with 100 μL of 0.1% aryl sulfatase, and the desulphoglucosinolates were eluted with 1 mL water. Desulphoglucosinolates was analyzed by HPLC. HPLC analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a VWD detector. Samples were separated at 30°C on a Waters Spherisorb C18 column (250 × 4.6 mm) using acetonitrile and water at a flow rate of 1.0 mL min–1. Absorbance was detected at 226 nm. Result of glucosinolate content was expressed as mmol kg–1 of dry weight (Sun et al., 2018 (link)).
Agilent 1260 hplc instrument
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 HPLC instrument is a high-performance liquid chromatography system designed for analytical and preparative applications. It offers reliable and efficient separation and detection of a wide range of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Spherisorb c18 column, by Waters Corporation (4 mentions)
Deae sephadex a25 column, by Merck Group (3 mentions)
Ortho nitrophenyl β d galactopyranoside, by Merck Group (3 mentions)
Aryl sulphatase, by Merck Group (2 mentions)
Analyst 1, by AB Sciex (2 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Icap 6000, by Thermo Fisher Scientific (1 mentions)
Vwd detector, by Agilent Technologies (1 mentions)
Api5000 triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Api 5000 triple quadrupole lc ms ms mass spectrometer, by AB Sciex (1 mentions)
Agilent sb c18 column, by Agilent Technologies (1 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (1 mentions)
Uvmini 1240, by Shimadzu (1 mentions)
11 protocols using agilent 1260 hplc instrument
1
Quantifying Glucosinolates in Freeze-Dried Samples
2
Evaluating Bone and Inflammatory Markers
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To evaluate the serum biochemical markers of bone turnover and proinflammatory
cytokines, the associated ELISAs were performed according to the manufacturer’s instructions.21 (link) Specifically, ELISA for procollagen 1 N-terminal peptide (P1NP), receptor
activator of nuclear factor kappa-B ligand (RANKL), and cross-linked
carboxy-terminal telopeptide of type 1 collagen (CTX-1; IDS, Frankfurt, Germany)
were detected for bone turnover; tumor necrosis factor alpha (TNF-α) and
interleukin 17 (IL-17) were also measured using commercial ELISA kits
(eBioscience, San Diego, CA, USA) to explore levels of inflammatory markers in
the serum and cell supernatant. Moreover, the serum levels of calcium (Ca) and
phosphorus (P) were measured using a plasma emission spectrometer (iCAP 6000;
Thermo Fisher Scientific, Waltham, MA, USA). In addition, to assess whether LGG
administration disturbed the TDF levels in blood, the serum TDF concentrations
in the five groups were detected using an Agilent 1260 HPLC instrument (Agilent
Technologies, Santa Clara, CA, USA) as previously described.28 (link) Samples were measured at least in duplicate.
cytokines, the associated ELISAs were performed according to the manufacturer’s instructions.21 (link) Specifically, ELISA for procollagen 1 N-terminal peptide (P1NP), receptor
activator of nuclear factor kappa-B ligand (RANKL), and cross-linked
carboxy-terminal telopeptide of type 1 collagen (CTX-1; IDS, Frankfurt, Germany)
were detected for bone turnover; tumor necrosis factor alpha (TNF-α) and
interleukin 17 (IL-17) were also measured using commercial ELISA kits
(eBioscience, San Diego, CA, USA) to explore levels of inflammatory markers in
the serum and cell supernatant. Moreover, the serum levels of calcium (Ca) and
phosphorus (P) were measured using a plasma emission spectrometer (iCAP 6000;
Thermo Fisher Scientific, Waltham, MA, USA). In addition, to assess whether LGG
administration disturbed the TDF levels in blood, the serum TDF concentrations
in the five groups were detected using an Agilent 1260 HPLC instrument (Agilent
Technologies, Santa Clara, CA, USA) as previously described.28 (link) Samples were measured at least in duplicate.
3
Quantitative Analysis of Glucosinolates
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Glucosinolates were extracted and analyzed as previously described (17 (link)). Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected after centrifugation, and the residues were washed once with water, centrifuged and then combined with the previous extract. The aqueous extract was applied to a DEAE-Sephadex A-25 column (Sigma Chemical Co., Saint Louis, USA). The glucosinolates were converted into their desulpho analogs by overnight treatment with 100 μL of 0.1% aryl sulphatase (Sigma Chemical Co., Saint Louis, USA), and the desulphoglucosinolates were eluted with 1 mL water. High performance liquid chromatography (HPLC) analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a variable wavelength detector (VWD) detector (Agilent Technologies, Inc., Palo Alto, USA). Samples were separated at 30°C on a Waters Spherisorb C18 column (250 mm × 4.6 mm i.d.; 5 μm particle size) using acetonitrile and water at a flow rate of 1.0 mL min−1. Absorbance was detected at 226 nm. Glucosinolates were quantified by using ortho-Nitrophenyl β-D-galactopyranoside (Sigma Chemical Co., Saint Louis, USA) as the internal standard and considering the response factor of each glucosinolate.
4
HPLC Analysis of Organic Compounds
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Chromatographic separations were carried out by an Agilent 1260 HPLC instrument (Agilent Technologies, USA), including a quaternary pump, an autosampler, a column temperature controller, and a diode array detector, connected to Agilent ChemStation software. The two chromatographic columns employed were an ACE EXCEL C18 column (250 × 4.6 mm, 5 μm) and a JADE-PAK C8 column (50 × 4.6 mm, 3.5 μm). The mobile phase was comprised of 0.1% aqueous phosphoric acid (A) and acetonitrile (B). The elution condition was set as follows: 5%–5% B for 0–5 min, 5%–14% B for 5–15 min, 14%–18% B for 15–21 min, 18%–21% B for 21–30 min, 21%–24% B for 30–35 min, 24%–30% B for 35–40 min, 30%–36% B for 40–50 min, 36%–95% B for 50–60 min, 95%–95% B for 60–65 min, and 95%–5% B for 65–75 min. A low rate of 0.6 mL/min and a temperature of 25°C were maintained. A wavelength used to detect signal was selected as 230 nm and the injection volume was set as 10 μL.
5
Quantifying Glucosinolates in Plant Samples
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Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected and applied to a DEAE-Sephadex A-25 column (Sigma Chemical Co., Saint Louis, USA). The glucosinolates were converted into their desulpho analogues by treated with aryl sulphatase, and the desulphoglucosinolates were eluted. HPLC analysis was carried out using an Agilent 1260 HPLC instrument equipped with a variable wavelength detector (VWD) detector (Agilent Technologies, Inc., Palo Alto, USA). Samples were separated at 30 °C on a Waters Spherisorb C18 column (250 mm × 4.6 mm i.d.; 5 µm particle size) using acetonitrile and water at a flow rate of 1.0 mL min−1. Absorbance was detected at 226 nm. Glucosinolates were quantified by using ortho-Nitrophenyl β-d -galactopyranoside (Sigma Chemical Co., Saint Louis, USA) as the internal standard and considering the response factor of each glucosinolate. Result of glucosinolate content was expressed as mmol kg−1 of dry weight (Sun et al., 2018 (link)).
6
Quantification of Glucosinolates in Samples
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Glucosinolates were extracted and analyzed as previously described (1 (link)). Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected after centrifugation, and the residues were washed once with water, centrifuged, and then combined with the previous extract. The aqueous extract was applied to a DEAE-Sephadex A-25 column (Sigma Chemical Co., Saint Louis, USA). The glucosinolates were converted into their desulpho analogs by overnight treatment with 100 μL of 0.1% aryl sulphatase (Sigma Chemical Co., Saint Louis, USA), and the desulphoglucosinolates were eluted with 1 mL water. HPLC analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a VWD detector (Agilent Technologies, Inc., Palo Alto, USA). Samples were separated at 30°C on a Waters Spherisorb C18 column (250 × 4.6 mm i.d.; 5 μm particle size) using acetonitrile and water at a flow rate of 1.0 mL min−1. Absorbance was detected at 226 nm. Glucosinolates were quantified by using ortho-Nitrophenyl β-D-galactopyranoside (Sigma Chemical Co., Saint Louis, USA) as the internal standard and considering the response factor of each glucosinolate. Result of glucosinolate content was expressed as μmol g−1 of dry weight.
7
MS/MS precursor ion scanning for m/z 73
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MS/MS precursor ion scanning for m/z 73.0 [C 3 H 5 O 2 ] was performed using an Agilent 1260 HPLC instrument (Agilent Technologies) coupled to an API5000 Triple Quadrupole mass spectrometer (AB Sciex, Darmstadt) equipped with an electrospray ionization (ESI) unit operated in negative mode. A collision energy of -34 was applied. Chromatographic separations were achieved using an Agilent ZORBAX Eclipse XDB-C18 column (50 × 4.6 mm, 1.8 μm particle diameter) (Agilent Technologies) with a flow rate of 1.1 ml min -1 and gradient elution starting at 5% acetonitrile in 0.05% aqueous formic acid (v/v) followed by a linear in-crease to 95% acetonitrile with 0.05% formic acid (v/v) within 10 minutes. Data were analysed with the Analyst 1.6 software (AB Sciex).
8
Size Exclusion Chromatography of Polymers
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. SEC was carried out on an Agilent 1260 HPLC instrument (Agilent Technologies, Waldbronn, Germany) comprising the following: autosampler, on-line degasser, quaternary pump unit and a thermostatic column compartment set to 40 °C. The detectors used were an Agilent VWD ultraviolet (UV) detector at a UV wavelength of 290 nm and an RI detector at 40 °C. Three GRAM columns (PSS Polymer Standards Service, GmbH, Mainz, Germany) with polyester copolymer as a stationary phase and 10 μm particle size and porosities of either 100 Å and 3 000 Å were used in tandem. A guard column with the same packing material was used. The eluent system used was N,Ndimethylformamide (HPLC grade with 0.05% w/v LiBr salt) at a flow rate of 0.8 mL•min -1 .
Calibration was carried out using low dispersity poly(methyl methacrylate) (Polymer Standards Service (PSS), Mainz, Germany) with peak maximum molecular weights (Mp) ranging between 800 g•mol -1 and 2 200 000 g•mol -1 . Therefore, the SEC data obtained are reported as PMMA equivalents. PSS WinGPC Unichrom 8.2 software was used to acquire and process the data.
Calibration was carried out using low dispersity poly(methyl methacrylate) (Polymer Standards Service (PSS), Mainz, Germany) with peak maximum molecular weights (Mp) ranging between 800 g•mol -1 and 2 200 000 g•mol -1 . Therefore, the SEC data obtained are reported as PMMA equivalents. PSS WinGPC Unichrom 8.2 software was used to acquire and process the data.
9
HPLC-MS/MS for m/z 73.1 Screening
10
Synthesis and Characterization of Novel Organic Compounds
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All reagents and solvents were purchased from commercial sources without further purification. The reaction progress was monitored by TLC on Silica Gel 60 F254 plates, and the target compounds were purified by column chromatography with silica gel (200–300 mesh size). Melting points were measured in open capillary tubes using YRT-3 melting point apparatus and were uncorrected. 1H NMR and 13 C NMR spectra were recorded on JNM-ECZR 400 MHz spectrometer with tetramethyl silane (TMS) as an internal standard. High-resolution mass spectra (HRMS) were obtained from a (UHR-TOF) maXis 4 G instrument. Analytical HPLC was run on the Agilent 1260 HPLC instrument, equipped with Agilent SB-C18 column (Agilent Technologies, Palo Alto, CA) and UV detection at 250 nm. Eluent system was: 70% MeOH in H2O; flow rate = 0.2 ml/min.
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