20 μL of ethanolic extract were injected into the HPLC system (Jasco, Tokyo, Japan; PU-4180 pump, MD-4015 PDA detector, AS-4050 autosampler). The stationary phase was an Agilent (Santa Clara, CA, USA) Zorbax Eclipse Plus C18 reversed-phase column (100 × 3 mm I.D., 3.5 μm). The chromatographic method for the analysis of phenolic acids was adapted from Mattila and Kumpulainen [42 (link)] as detailed in Tubon et al. [34 (link)]. Gradient elution was carried out with a mixture of acidic phosphate buffer (50 mM, pH 2.5) and acetonitrile flowing at 0.7 ml/min. The signals at 254, 280, and 329 nm were used for analyte quantitation. The recovery values of phenolic acids in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6). The chromatographic method for the analysis of flavonoids was adapted from Wojdyło et al. [43 (link)], as previously reported [34 (link)]. Gradient elution was carried out with a mixture of 4.5% formic acid and acetonitrile. Runs were monitored at the following wavelengths: flavan-3-ols and flavanones at 280 nm and flavonol glycosides at 360 nm. PDA spectra were measured over the wavelength range of 200−600 nm in steps of 2 nm. Retention times and spectra were compared with those of pure standards. Calibration curves were constructed for all standards at concentrations ranging from 1.0 to 100.0 μg/ml (r2 ≥ 0.9998). Results are expressed as mg/g extract.
Zorbax eclipse plus c18 reversed phase column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax Eclipse Plus C18 reversed-phase column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a chemically bonded C18 stationary phase on a high-purity, silica-based support material.
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5 protocols using zorbax eclipse plus c18 reversed phase column
1
HPLC Analysis of Phenolic Acids and Flavonoids
2
Quantification of Phenolic Acids via HPLC
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20 µL of each extract were injected into the HPLC system (Jasco, Tokyo, Japan; PU-4180 pump, MD-4015 PDA detector, AS-4050 autosampler). The stationary phase was an Agilent (Santa Clara, CA, USA) Zorbax Eclipse Plus C18 reversed-phase column (100 mm × 3 mm I.D., 3.5 μm).
The chromatographic method for the analysis of phenolic acids was adapted from Mattila et al. [30 (link)]. Gradient elution was carried out with a mixture of acidic phosphate buffer and acetonitrile flowing at 0.7 mL/min. The signals at 254, 280 and 329 nm were used for analyte quantitation. The recovery values of phenolic acids in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6).
The chromatographic method for the analysis of phenolic acids was adapted from Mattila et al. [30 (link)]. Gradient elution was carried out with a mixture of acidic phosphate buffer and acetonitrile flowing at 0.7 mL/min. The signals at 254, 280 and 329 nm were used for analyte quantitation. The recovery values of phenolic acids in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6).
3
Comprehensive Oxylipin Profiling in Liver Tissue
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Oxylipin extraction and measurement was performed as described. 31, 32 In brief, to 50 ± 5 mg liver tissue 10 µL antioxidant mixture, 10 µL deuterated IS and 300 µL ice-cold MeOH were added and tissue samples were homogenized using a ball mill as described above. After centrifugation of tissue homogenates (10 min, 20 000g, 4 °C), supernatants were diluted with 2. injected into the LC-MS/MS system (Agilent 1290 binary pump coupled to AB Sciex 6500 QTRAP MS) and separated on a Zorbax Eclipse Plus C18 reversed phase column (Agilent, Waldbronn, Germany) with a binary gradient using 0.1% HOAc with 5% solvent B as solvent A and ACN/MeOH/HOAc (800/150/1, v/v/v) as solvent B. Measurement was carried out in scheduled multiple reaction monitoring (MRM) mode and quantification was carried out by external calibration (analyte/ IS area ratio). A total of 137 oxylipins was analyzed in the liver samples.
4
HPLC-DAD Analysis of Phenolic Compounds
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HPLC-DAD determination of phenolic acids and flavonoids was performed as previously described [38 (link)]. 20 μL of SSEE was injected into the HPLC system (Jasco Italy; PU-4180 pump, MD-4015 PDA detector, AS-4050 autosampler). The stationary phase was an Agilent (Santa Clara, CA, USA) ZORBAX Eclipse Plus C18 reversed-phase column (100 mm × 3 mm I.D., 3.5 μm). The chromatographic method for the analysis of phenolic acids was adapted from Mattila and Kumpulainen [39 (link)]. Gradient elution was carried out with a mixture of acidic phosphate buffer (50 mM, pH 2.5) and acetonitrile flowing at 0.7 mL/min. Signals at 254, 280, and 329 nm were used for analyte quantitation. The recovery values of phenolic acids in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6). The chromatographic method for the analysis of flavonoids was adapted from Wojdyło et al. [40 (link)]. Gradient elution was carried out with a mixture of 4.5% formic acid and acetonitrile. Runs were monitored at 280 nm for flavan-3-ols and 360 nm for flavonol glycosides. Retention times and spectra were compared with those of pure standards. Calibration curves were constructed for all standards at concentrations ranging from 1.0 to 100.0 ppm (r2 ≤ 0.9998). Results were expressed as mg/g extract or per g of plant material (DW).
5
HPLC Analysis of Phenolic Acids
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Twenty µL of each extract were injected into an HPLC system (Jasco, Tokyo, Japan; PU-4180 pump, MD-4015 PDA detector, AS-4050 autosampler). The stationary phase was an Agilent (Santa Clara, CA, USA) Zorbax Eclipse Plus C18 reversed-phase column (100 mm x 3 mm I.D., particle size 3.5 µm).The chromatographic method for the analysis of phenolic acids was adapted from Mattila et al. (2005) (link). Gradient elution was carried out with a mixture of acidic phosphate buffer and acetonitrile flowing at 0.7 mL min -1 . The signals at 254, 280, and 329 nm were used for analyte quantitation. Identification and quantification were performed based on standard compounds (gallic, p-hydroxybenzoic, syringic, ferulic, p-coumaric, cinnamic, and caffeic acids) . The recovery values in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6).
The sum of all individual phenolic acid concentrations was calculated and used to express the total phenolics acid index (TPAI) for each extract.
The sum of all individual phenolic acid concentrations was calculated and used to express the total phenolics acid index (TPAI) for each extract.
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