BODIPY-labelled Δ-disaccharides were resolved using a standard binary HPLC system (Cecil, Cambridge, UK) equipped with either an ACE UltraCore 5 SuperC18 column (250 mm × 4.6 mm, 5 μm, Hichrom), SUPELCOSIL LC18 (30 cm × 4 mm, 5 μm, Sigma-Aldrich), or an Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm Agilent technologies) and an in-line fluorescence detector (λex 473 nm, λem 510 nm, Picometrics, Toulouse, France) under the following conditions: isocratic 100% A at a flow rate of 0.5 mL min−1 for 20 min, then linear gradient elution of 0–100% B at a flow rate of 0.5 mL min−1 for 100 min, where solvent A contained 0.1 M ammonium acetate, 30% (v/v) HPLC grade acetonitrile, 10 mM tetraoctylammonium bromide (TOAB) and solvent B contained 30 mM TOAB dissolved in HPLC grade acetonitrile (VWR). The column was subsequently cleaned for 10 min using solvent B (isocratic) before re-equilibration with solvent A for 10 min between separations. Correction factors for equal amounts of bona fide BODIPY-labelled Δ-disaccharides were established through integration of the corresponding peaks from chromatograms obtained as previously described,17 (link) and subsequently applied to sample chromatograms.
Supelcosil lc 18
Manufactured by Merck Group
Sourced in United States
Supelcosil LC-18 is a reverse-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with chemically bonded C18 alkyl ligands, which provide excellent retention and selectivity for non-polar and moderately polar analytes.
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Lab products found in correlation
Lc 250, by PerkinElmer (2 mentions)
0.22 m cellulose acetate membranes, by Corning (2 mentions)
Supelguard 20 4.6 mm id precolumn, by Merck Group (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Hplc grade acetonitrile, by Avantor (1 mentions)
Eclipse xdb c18 column, by Agilent Technologies (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Liquid chromatograph, by Shimadzu (1 mentions)
6120 mass spectrometer, by Agilent Technologies (1 mentions)
Model 1200, by Agilent Technologies (1 mentions)
Fluorescence detector, by Shimadzu (1 mentions)
10 protocols using supelcosil lc 18
1
BODIPY-Labelled Δ-Disaccharide HPLC Separation
2
Vitamin and Phytosterol Analysis of Samples
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Vitamin and phytosterol analysis of the samples was done by revising the method of Aydın et al. (2011) . Samples were weighed in 1g and homogenized with n-hexane/isopropyl at 3/2 (v / v) ratio and after the hydrolysis with 5% KOH at 85 °C (15 min), the tubes were removed from the oven and cooled at room temperature, 5 ml of distilled water was added and mixed well. After this process, the mixture in the tubes was separated into phases and the upper hexane phase was taken into clean centrifuge tubes and its solvent was evaporated. Then, the Acetonitrile / methanol mixture (50% + 50%, v / v) was prepared, and 1 ml was dissolved in this mixture, taken into autosames and prepared for analysis.
Acetonitrile / methanol (60% + 40%, v / v) mixture was used as mobile phase. Mobile phase flow rate was determined as 1 ml / min. DAD-UV detector was used for analysis. Supelcosil ™ LC 18 (15 x 4.6 cm, 5 μm; Sigma, USA) column was used as the colon. Detection wavelength 326 nm for vitamin A, 202 nm for vitamins E, D, K and phytosterols.
Acetonitrile / methanol (60% + 40%, v / v) mixture was used as mobile phase. Mobile phase flow rate was determined as 1 ml / min. DAD-UV detector was used for analysis. Supelcosil ™ LC 18 (15 x 4.6 cm, 5 μm; Sigma, USA) column was used as the colon. Detection wavelength 326 nm for vitamin A, 202 nm for vitamins E, D, K and phytosterols.
3
Analysis of Lipid-soluble Vitamins and Phytosterols
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Lipide-soluble vitamins and phytosterols have been extracted from the lipid fraction by the method of Sánchez-Machado et al. (2002) (link) with minor modifications. The extracted lipids of seed material have been dissolved in acetonitrile/methanol (75/25 v/v) and have been injected 50 μL to HPLC (Shimadzu, Kyota Japan). The used column is a Supelcosil TM LC18 (250 × 4.6 mm, 5 μm, Sigma, USA) and the mobile phase is acetonitrile/methanol (75/25, v/v). The elution has been performed at a flow-rate of 1 mL/min and the temperature of analytical column is kept constant at 40 °C. the detection has been performed at 320 nm for retinol (vitamin A) and retinol acetate, and 215 nm for δ-tocopherol, vitamin D, α-tocopherol, α-tocopherol acetate, 265 nm for vitamin K1 and 202 nm for phytosterols (López-Cervantes et al., 2006) (link). Class Vp 6.1 software assisted at workup of the data. The results of analysis have been expressed as μg/g for samples.
4
Quantification of Lipid-Soluble Vitamins and Phytosterols
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Lipid-soluble vitamins and phytosterols were obtained from the lipid fraction based on the method of Sánchez-Machado et al. (2002) (link). The samples were suspended in acetonitrile/methanol (75/25 v/v), and 50 mL was added to the HPLC (Shimadzu, Japan). A Supelcosil TM LC18 (250 x 4.6 mm, 5 mm, Sigma, USA) was used as column and acetonitrile/methanol (75/25, v/v) for the mobile phase. The temperature of the column was kept at 40°C. The wavelength was 320 nm for retinol (vitamin A) and retinol acetate; 215 nm for d-tocopherol, vitamin D, a-tocopherol, and a-tocopherol acetate; 202 nm for phytosterols; and 265 nm for vitamin K1 (López-Cervantes et al., 2006) . The results of the analyses were measured as μg/g.
5
HPLC Quantification of Nucleotides
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After the enzymatic assay, 20 μL aliquots were analyzed using the HPLC method (Supelcosil LC-18, 25 cm 64.6 mm, Supelco) in a Shimadzu liquid chromatograph (Shimadzu, Japan). Through a linear gradient (first, using a 100% buffer solution named A containing 150 nmol/L PBS and 150mM KCl at pH 6 and, second, using a 100% buffer solution named B containing 15% C2H3N in buffer solution A), molecules were separated. To quantify nucleotides and their subproducts, a 254 nm absorbance wavelength was applied. All peaks were identified by their retention time and quantity and compared against their respective standards. Purine concentration is expressed as nmol of the released product (mean ± S.D.). All experiments were run in triplicates to discount the effect of non-enzymatic nucleotide hydrolysis. When quantifying nucleotides, aliquots containing the incubation medium with nucleotides and without cells were used to standardize the experiment.
6
Purification and Analysis of θ-Amanitin
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We analyzed the amanitin-related peptides by using the Agilent Model 1200 high-performance liquid chromatography (HPLC) system coupled to an ultraviolet detector (monitoring at 280, 295, and 305 nm) and an Agilent 6120 mass spectrometer. The HPLC elution solution A was 0.02 M ammonium acetate:acetonitrile (90:10, vol/vol), adjusted to pH 5 with glacial acetic acid, and solution B was 0.02 M aqueous ammonium acetate:acetonitrile (76:24, vol/vol), pH 5 (38 (link)).
θ-Amanitin was purified from the P450-29 mutant in two steps. The first separation was performed on a semipreparative C18 column (25 cm × 10 mm, 5 mm; Supelcosil LC-18, Supelco). The flow rate was 2 mL/min with a stepwise gradient profile of 100% A for 3 min, 43% A for 7 min, and 0% A for 9 min. Fractions containing θ-amanitin were pooled, lyophilized, and then redissolved in H2O. The second separation was performed on a 250 × 4.6 mm C18 column (Higgins Analytical,http://www.higanalyt.com ). The flow rate was 1 mL/min with a gradient of 100% solution A to 100% solution B in 15 min. The fractions containing θ-amanitin were collected, dried under vacuum, and redissolved in H2O.
θ-Amanitin was purified from the P450-29 mutant in two steps. The first separation was performed on a semipreparative C18 column (25 cm × 10 mm, 5 mm; Supelcosil LC-18, Supelco). The flow rate was 2 mL/min with a stepwise gradient profile of 100% A for 3 min, 43% A for 7 min, and 0% A for 9 min. Fractions containing θ-amanitin were pooled, lyophilized, and then redissolved in H2O. The second separation was performed on a 250 × 4.6 mm C18 column (Higgins Analytical,
7
Quantification of 8-oxodG in DNA
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DNA was extracted from fresh liver samples using the chaotropic-NaI method, as we previously reported [45 (link)]. After DNA quantification using Nanodrop 2000c (Thermo Scientific Inc), samples were digested with nuclease P1 and E. coli acid phosphatase. 8-oxodG detection was performed as we reported previously [45 (link)]. Briefly, 100 μg of digested DNA was loaded into a HPLC coupled with an electrochemical detector (EC, Waters Inc) and the system was connected to a Supelcosil LC-18 (Supelco, Bellefonte, PA, USA) reverse-phase column (250X4.6 mm, i.d. particle size 5 μm). The eluent was 50mM potassium phosphate buffer, pH 5.5, with 8% methanol at 1 ml/min flow rate. The molar ratio of 8-oxodG in each DNA sample was determined based on EC detection at 290 mV for 8-oxodG and absorbance at 254 nm for dG.
8
Quantifying Glutamate Levels in Traumatic Brain Injury
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Glutamate concentration in TBI patients and controls were analyzed in 25 μL of the CSF cell‐free supernatant samples through high‐performance liquid chromatography (HPLC). Briefly, samples were filtered and derivatized with o‐phthalaldehyde and mercaptoethanol. CSF samples were separated by reverse phase column (Supelcosil LC‐18, 250 mm × 4.6 mm, Supelco) in a Shimadzu Instruments liquid chromatograph. The mobile phase flowed at a rate of 1.4 mL/min at 24°C. Buffer composition is A: 0.04 mol/L sodium dihydrogen phosphate monohydrate buffer, pH 5.5, containing 20% of methanol; and B: 0.01 mol/L sodium dihydrogen phosphate monohydrate buffer, pH 5.5, containing 80% of methanol. The gradient profile was modified according to the content of buffer B in the mobile phase: 0% at 0.00 min, 25% at 13.75 min, 100% at 15.00–20.00 min, and 0% at 20.01–25.00 min. Absorbance was read in a Shimadzu fluorescence detector, with excitation and emission being 360 nm and 455 nm, respectively. The concentration was expressed in μmol/L.22
9
Determination of Glutathione Levels
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Heart samples were homogenized with a glass-Teflon homogenizer in a solution containing 1 M HClO4-2 mM EDTA (1:1), and centrifuged at 16,000g for 20 min at 4 • C. Supernatants were filtered through 0.22 m cellulose acetate membranes (Corning Inc., NY, US), and frozen at -80 • C until use. HPLC analysis was performed in a Perkin Elmer LC 250 liquid chromatography (Perkin Elmer, Waltham, MA, US), equipped with a Perkin Elmer LC ISS 200 advanced sample processor, and a Coulochem II (ESA, Bedford, MA, US) electrochemical detector. A Supelcosil LC-18 (250 × 4.6 mm ID, 5 m particle size) column protected by a Supelguard (20 × 4.6 mm ID) precolumn (Supelco, Bellfonte, PA, US) was used for sample separation. GSH and GSSG were eluted at a flow rate of 1.2 mL/min with 20 mM sodium phosphate (pH 2.7), and electrochemically detected at an applied oxidation potential of +0.800 V. Results were expressed as M (Rodriguez- Ariza et al., 1994) (link).
10
Quantifying Glutathione Redox Status
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Lung samples were homogenized with a glass-Teflon homogenizer in solution containing 1 M HClO 4 -2 mM EDTA (1:1), and centrifuged at 16000g for 20 min at 4 • C. Supernatants were filtered through 0.22 m cellulose acetate membranes (Corning Inc., NY, USA) and frozen at -80 • C until use. HPLC analysis was performed in a Perkin Elmer LC 250 liquid chromatograph, equipped with a Perkin Elmer LC ISS 200 advanced sample processor, and a Coulochem II (ESA, Bedford, MA, USA) electrochemical detector. A Supelcosil LC-18 (250 × 4.6 mm ID, 5 m) column protected by a Supelguard (20 × 4.6 mm ID) precolumn (Supelco, Bellfonte, PA, USA) was used for sample separation. GSH and GSSG were eluted at a flow rate of 1.2 mL/min with 20 mM sodium phosphate (pH 2.7), and electrochemically detected at an applied oxidation potential of +0.800 V. Results were expressed as M (Rodriguez-Ariza et al., 1994).
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