The traditional preparation of RHTR was performed by decoction with 25 g of stems in 500 mL of boiling distilled water for 30 min (aqueous extract; AE). Subsequently, the AE was filtered and centrifuged at 2500 rpm for 15 min at 4 °C in conical tubes to recover the supernatants and concentrated under negative pressure with a rotary vacuum evaporator at 40 °C and 5 rpm (Rotavapor® R-300, Büchi). Next, the AE was freeze-dried (FreeZone Triad, Labconco®) and stored at − 20 °C in an amber vial. The fractionation of AE was performed by solid phase extraction with the aid of a vacuum Manifold (Visiprep™, Sigma®) and ENVI™-C18 cartridges (Supelclean™, Sigma®) to concentrate compounds of a flavonoid nature. The cartridge was activated with 20 mL of MeOH (J.T. Baker®) and 1% acetic acid in water. Subsequently, 15 mL of ethyl acetate (flavonoid fraction, FF) HPLC-grade from J.T. Baker® was eluted through the cartridge and collected for concentration by rotary evaporation. Finally, the fraction was weighed and resuspended in 2 mL of 50% MeOH (MS grade, J.T. Baker®) and filtered with a 0.20 μm PTFE syringe filter (Corning®) for storage as a stock solution in an amber vial at − 20 °C for later use. Extracts and fractions were prepared according to the method disclosed in the Mexican patent: MX/E/2018/078316.
Visiprep
Manufactured by Merck Group
Sourced in United States, Germany
The Visiprep is a laboratory equipment designed for sample preparation. It is used for vacuum filtration and extraction of samples. The device facilitates the efficient separation of solid and liquid components within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Freezone triad, by Labconco (2 mentions)
Envi c18 cartridges, by Merck Group (2 mentions)
Ptfe syringe filter, by Corning (2 mentions)
Rotavapor r 300, by Büchi (2 mentions)
Supelclean lc 18, by Merck Group (1 mentions)
Waters 515 hplc pump, by Waters Corporation (1 mentions)
Waters 2487 uv vis detector, by Waters Corporation (1 mentions)
Phenolic standards, by Merck Group (1 mentions)
Zinc sulfate heptahydrate, by Merck Group (1 mentions)
Potassium hexacyanoferrate 2 trihydrate, by Merck Group (1 mentions)
Milli q ro4 water purification system, by Merck Group (1 mentions)
Millex, by Merck Group (1 mentions)
Minivap, by Merck Group (1 mentions)
10 protocols using visiprep
1
Extraction and Fractionation of RHTR Flavonoids
2
Solid-Phase Extraction of Environmental Samples
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Samples were spiked with the mix of internal standards at 100 ng/L, 24 h prior to extraction and stored at 4 °C. Experimental conditions for conditioning, loading, and elution on each cartridge followed recommendations of suppliers, or the method developed by Kern et al. for the Multilayer cartridge [27 (link)], and are described in Table 5 . All extractions were carried out on Visiprep (Sigma-Aldrich, Augsburg, Germany) and Autotrace (AT280, Caliper) SPE systems. All cartridges were loaded with 1 L of sample at pH 6–7, except for Strata X-A (pH 2–3 recommended) and Oasis HLB, for which both pHs were tested. Before elution, the cartridges were dried for 30 min. After elution, the extracts were stored in the dark at 4 °C prior to their analysis. SPE extracts were evaporated under a stream of nitrogen, then reconstituted in 1 mL of Milli-Q water and MeOH (80/20, v/v), and filtered through 0.2 µm PTFE filters before a 10 µL injection on the analytical system.
3
Aqueous Extract Fractionation of RHTR
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An aqueous extract (AE) of RHTR was elaborated by decoction (25 g of stems and 500 mL of boiling distilled water, 30 min), centrifugation (2500 rpm, 15 min, 4 °C), concentration (negative pressure, 40 °C, 5 rpm) (Rotavapor® R-300, Büchi, Flawil, C.H.), and lyophilization (freezing: –40 °C, 2 h; sublimation: –15 °C, 3 h; desorption: 40 °C, 1 h; temperature ramp: 1 °C/min) (FreeZone Triad, Labconco®, Kansas City, MO, USA). Later, AE was fractionated with ENVI™-C18 cartridges (Supelclean™, Sigma®, St. Louis, MO, USA) in a vacuum manifold (Visiprep™, Sigma®, St. Louis, MO, USA). The solvents used were 1% acetic acid (aqueous fraction-01; AF01), ethyl acetate (aqueous fraction-02; AF02), and ethylic ether (aqueous fraction-03; AF03) (15 mL each, HPLC grade, J.T. Baker®, Edo. de México, M.X.). Finally, fractions were evaporated, weighed, resuspended in 2 mL 50% MeOH (MS grade, J.T. Baker®, Edo. de México, M.X.), and filtered with a 0.22 µm PTFE syringe filter (Corning®, Corning, NY, USA). Extract and fractions were prepared according to the method disclosed in the Mexican patent MX/E/2018/078316.
4
Extraction and Quantification of Environmental Pollutants
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All chemicals used were of HPLC grade and purchased from Sigma-Aldrich (Johannesburg, South Africa). These include acetone, methanol, nonylphenol (Technical grade), dichlorophenol (99%), estrone (99%), 17β- estradiol (98%), bisphenol A (97%), octylphenol (99%), triclosan (99%), atrazine (99%), (99%) and 1,2,4-and triazole (98%). De-ionized water was produced with Millipore (Millipore SA, France). Freezing was on a rotary evaporator (Büchi Rotavapor R-210 with Büchi Bath B-491, Büchi Labotechnik, Switzerland). Drying of the frozen water samples was with Vir Tis BenchTop K freeze dryer, equipped with Elnor vacuum pump (SP Scientific, Pennsylvania USA). Solid-phase extraction (SPE) tubes (Supelclean LC-18) and vacuum manifold (Visiprep) were purchased from Sigma-Aldrich (Johannesburg, South Africa).
5
Phenolic Compound Extraction and Analysis Protocol
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An aliquot (5-15 mL) of the various supernatants was passed through a conditioned Varian (Varian Associates, Harbor City, CA) Bond Elut PPL (3 mL size with 200 mg packing) solid-phase extraction tube at 5 mL/min attached to a Visiprep (Supelco, Bellefonte, PA). The tubes were then placed under vacuum (-60 kPa) until the resin was thoroughly dried after which the phenolic compounds were eluted with 1 mL of ethyl vials. The PPL tubes were conditioned by first passing 2 mL of ethyl acetate followed by 2 mL of water (pH < 2.0). Purified phenolic extracts (1 μL: 10:1 split) were analyzed for composition by comparison with phenolic standards (Aldrich Chemical Co, Milwaukee, WI) and chromatography with standards on a Waters 600 High Performance Liquid Chromatograph LCD System equipped with Waters 515 HPLC pump, Waters 2487 UV/VIS detector, C18 column with dimensions 5 μm, 4.6 × 250 mm with Hamilton microliter syringe, and injection volume of 20 μL. The following conditions were employed per separation: wavelength, 280 nm; flow rate, 1.0 mL/min; gradient elution total run time of 31 minutes, having Solvent A as acetonitrile, solvent B as 0.1% phosphoric acid in de-ionized water, which was started with 85% A and held at this for 13 minutes. This was followed by 75% eluent B for 10 minutes and then the concentration of B was increased to 85% for another 8 minutes.
6
Antibiotics Extraction from Plasma with SPE
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The extraction of
antibiotics from human plasma samples was carried out with SPE using
Discovery DSC-18 cartridges. Before extraction, cartridges were preconditioned
twice with 2 mL of methanol accompanied by 2 mL of 0.5% v/v FA in
ultrapure water aspirating with a Supelco Visiprep equipment. All
of the CS and QC samples were diluted with 600 μL of 0.5% v/v
FA in ultrapure water, vortexed on a cyclomixer (Spinix Tarsons, India)
for 5 min, and loaded on preconditioned cartridges. To suppress the
matrix effect, the cartridges were then washed with 1 mL of 5% v/v
methanol in ultrapure water, and the solution accumulated in the tubes
was discarded. Analytes were eluted in fresh tubes with 2 mL of pure
methanol and then dried using a nitrogen dryer (TurboVap) at 50 °C
for 30 min. Dried samples were reconstituted with 100 μL of
methanol, vortex-mixed, and injected (10 μL) into LC-MS/MS for
analysis.
antibiotics from human plasma samples was carried out with SPE using
Discovery DSC-18 cartridges. Before extraction, cartridges were preconditioned
twice with 2 mL of methanol accompanied by 2 mL of 0.5% v/v FA in
ultrapure water aspirating with a Supelco Visiprep equipment. All
of the CS and QC samples were diluted with 600 μL of 0.5% v/v
FA in ultrapure water, vortexed on a cyclomixer (Spinix Tarsons, India)
for 5 min, and loaded on preconditioned cartridges. To suppress the
matrix effect, the cartridges were then washed with 1 mL of 5% v/v
methanol in ultrapure water, and the solution accumulated in the tubes
was discarded. Analytes were eluted in fresh tubes with 2 mL of pure
methanol and then dried using a nitrogen dryer (TurboVap) at 50 °C
for 30 min. Dried samples were reconstituted with 100 μL of
methanol, vortex-mixed, and injected (10 μL) into LC-MS/MS for
analysis.
7
HPLC Quantification of Ascorbic Acid
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All organic solvents, methanol and acetonitrile, were from J.T. Baker (Deventer, Holland) HPLC-grade. Water was purified by a Milli-Q-RO4 water purification system (Millipore, Bedford, MA, USA) with a resistivity of 10 MΩ·cm. The standard AA was purchased from Fluka (Deisenhofen, Germany). Potassium hexacyanoferrate (II) trihydrate and zinc sulfate heptahydrate were obtained from Merck (Darmstad, Germany). Oasis HLB 200 mg/6 mL SPE cartridges were obtained from Waters (Milford, MA, USA). Bond Elut-Accucat (200 mg/3 mL) SPE cartridges were purchased from Varian (Chicago, IL, USA) and used with a SPE vacuum manifold (Visiprep, Supelco, Bellefonte, PA, USA). Amber glass auto-sampler vials with septum screw caps were obtained from Agilent Technologies (Wilmington, DE, USA). The analytical column (Atlantis dC18, 250 × 4.6 mm/5) with pre-column and syringe filters (0.45 μm PVDF) were from Waters (Milford, MA, USA).
A standard AA stock solution (1.0 mg/mL) was prepared by dissolving 1.0 mg of AA in 1.0 mL of Milli-Q water. To weigh AA, an electronic analytical balance (Radwag, PCE group, 0.01 mg Radom, Poland) was used. The AA stock solution was diluted in amber glass volumetric flasks to prepare calibration standards at 50, 100, 200, 500, 800 and 1000 ng/mL respectively and stored at 4°C until use.
A standard AA stock solution (1.0 mg/mL) was prepared by dissolving 1.0 mg of AA in 1.0 mL of Milli-Q water. To weigh AA, an electronic analytical balance (Radwag, PCE group, 0.01 mg Radom, Poland) was used. The AA stock solution was diluted in amber glass volumetric flasks to prepare calibration standards at 50, 100, 200, 500, 800 and 1000 ng/mL respectively and stored at 4°C until use.
8
Solid-Phase Extraction of BPA from Real Samples
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The PPMIP particles (200 mg) were packed into a SPE cartridge (3 mL) using an upper frit and a lower frit to prevent sorbent loss. The obtained cartridges were connected to a SPE manifold (Supelco Visiprep, Bellefonte, PA, USA) equipped with a vacuum pump (Jinteng, Tianjin, China). Prior to sample loading, the SPE cartridges were equilibrated with ACN (3 mL) and water (3 mL). Then, the prepared real samples were percolated at a flow rate of 1.0 mL·min−1. After drying under vacuum for 30 min, the SPE cartridges were rinsed with 2 mL of can, and the retained BPA were eluted with 4 mL of methanol/TFA (98:2, v/v). Finally, the eluates were evaporated to nearly dryness under nitrogen gas, and the residues were diluted to 1.0 mL with methanol/water (65:35, v/v) for further HPLC analyses.
9
Phytochemical Extraction Using MSPD
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Phytochemicals, including betalains, were extracted via the matrix solid-phase dispersion (MSPD) method published by Araujo-León et al. (2023) [32 (link)]. A uniform powder was obtained by grinding 100 mg of lyophilized tissue at room temperature in a mortar with 400 mg of C18 stationary phase (BondElut-C18). The solid mixture was placed into cartridges for solid-phase extraction. With the aid of a vacuum manifold (Visiprep, SUPELCO, Bellefonte, PA, USA), 9 mL of 0.1% acetic acid-water was eluted, followed by a mixture of 0.1% acetic acid-water:methanol (1:1, v/v) mixture at −15 mmHg. Both eluates were collected and evaporated to dryness. The resulting extract was resuspended in 0.1% acetic acid-water:methanol (1:1, v/v) mixture and transferred to an amber vial for analysis by HPLC-UV-DAD.
10
Wastewater Analyte Extraction and Purification
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Aliquots of 100 mL of wastewater were vacuum-filtered through 0.7 μm glass microfiber filters GF/A (47 mm diameter, Whatman, Kent, UK) and 0.45 μm cellulose filters (47 mm diameter, Merck Millipore).Samples were spiked with 20 ng of every IS after filtration. SPE was performed using a standard vacuum manifold Visiprep™ (12-port model, Supelco, Steinheim, Germany) maintaining a pressure of approx. 5 inches Hg.
Samples were loaded onto mixed-mode reversed-phase strong anion-exchange cartridges (Oasis MAX-60 mg, Waters, Milford, MA, USA) previously rinsed with 3 mL of MeOH followed by 3 mL of ultrapure water. Sorbents were dried under nitrogen during 30 min and analytes eluted with 5 mL of 2% formic acid in MeOH. Eluates were evaporated to dryness under nitrogen using a Turbo-Vap II (Zymark, Hopkinton MA, USA) and a Mini-Vap (Supelco, Steinheim, Germany) concentrator. Extracts were reconstituted in 100 μL of MeOH, filtered through 0.22 μm PVDF syringe-driven filters (Millex, Merck Millipore) and injected into the UPLC ® system.
Samples were loaded onto mixed-mode reversed-phase strong anion-exchange cartridges (Oasis MAX-60 mg, Waters, Milford, MA, USA) previously rinsed with 3 mL of MeOH followed by 3 mL of ultrapure water. Sorbents were dried under nitrogen during 30 min and analytes eluted with 5 mL of 2% formic acid in MeOH. Eluates were evaporated to dryness under nitrogen using a Turbo-Vap II (Zymark, Hopkinton MA, USA) and a Mini-Vap (Supelco, Steinheim, Germany) concentrator. Extracts were reconstituted in 100 μL of MeOH, filtered through 0.22 μm PVDF syringe-driven filters (Millex, Merck Millipore) and injected into the UPLC ® system.
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