OTA was analyzed by C18 Spherisorb ODS2 column, 150 × 4.6 mm, 5 µm, 120 Å (Waters, Saint-Quentin, France) using a Dionex Ultimate 3000 UHPLC (Thermo Scientific, France) apparatus. An isochratic flow was delivered at 1 mL/min containing 49% acidified water (0.2% acetic acid, v/v) and 51% acetonitrile. The injection volume was 100 µL. OTA was detected using an FLD detector with 332/466 nm excitation/emission wavelengths and spectrum was further confirmed by a diode array detector (DAD) coupled to the apparatus. OTA quantification was calculated according to a standard calibration curve with concentrations ranging between 20 and 500 µg/L.
C18 spherisorb ods2 column
Manufactured by Waters Corporation
Sourced in France, Ireland, United States
The C18 Spherisorb ODS2 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with octadecylsilane (C18) bonded ligands, providing reversed-phase separation capabilities. The spherical particles in the column packing offer efficient mass transfer and good peak shape for optimal chromatographic performance.
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Lab products found in correlation
Dionex ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
Picotag column, by Waters Corporation (1 mentions)
Varian star workstation, by Agilent Technologies (1 mentions)
L ascorbic acid standard, by Merck Group (1 mentions)
Hplc system, by Gilson (1 mentions)
Esquire 6000, by Agilent Technologies (1 mentions)
Agilent 1100 hplc, by Agilent Technologies (1 mentions)
Ltq xl, by Thermo Fisher Scientific (1 mentions)
2695 separations module, by Waters Corporation (1 mentions)
2475 multi λ fluorescence detector, by Waters Corporation (1 mentions)
9 protocols using c18 spherisorb ods2 column
1
Quantification of Ochratoxin A
2
Femur Bone Collagen Crosslink Analysis
3
Quantitative Lipid Profiling by LC-MS
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Lipids were separated on a C18 Spherisorb ODS2 column (150 × 4.6 mm; 5 µm particle; Waters Ltd) using a gradient of 50–90% B over 30 min, followed by 5 min at 90% B (A, water/acetonitrile/acetic acid, 75:25:0.1; B, methanol/acetonitrile/acetic acid, 60:40:0.1) with a flow rate of 1 ml/min. Eicosanoid species were monitored with specific parent to daughter ion transitions in negative ion mode ([M−H]−) for HETEs (m/z 319.2) at 115 (5-HETE), 179.1 (12-HETE), 219 (15-HETE), 155 (8-HETE), and 167 (11-HETE). 15-HETE-d8 was monitored at m/z 327 to 226. Products were identified and quantified using primary standards, and internal standard runs in parallel under the same method conditions.
4
HPLC-based Quantification of β-Alanine
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Determination of β‐ala was carried out by HPLC coupled to a Multi γ–fluorescence detector (Waters 1525/2475) using a reverse‐phase C‐18 Spherisorb ODS2 column of 5 μm particle size and 150 × 4.6 mm (Waters; García‐García, Peña‐Sanabria, Sánchez‐Thomas, & Moreno‐Sánchez, 2018 ). Enzymatic reactions were stopped with perchloric acid (3% v/v) at the indicated times and immediately frozen in liquid nitrogen and kept at −70°C. The acidic supernatants were neutralized with 3 M KOH/0.1 M Tris and centrifuged to remove KClO4. Supernatant was recovered and used for β‐ala determination by derivatization with 37 mM ortho‐phthalaldehyde (OPA). β‐ala and α‐alanine standards (Sigma‐Aldrich, Saint Louis, MO, USA) were used for identifying of chromatographic peaks.
5
Total Ascorbic Acid Extraction Protocol
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Total ascorbic acid extraction was carried out according to Santos, Cunha, and Casal (2017) (link), using an aqueous solution with 8% acetic acid and 3% metaphosphoric acid, together with tris-(2-carboxy-ethyl)phosphine-hydrochloride (2.5 mM) (Carl Roth, Germany) for dehydroascorbic acid reduction, under light protection. Chromatographic analyses were performed using a HPLC system (Gilson, France), with a photodiode array detector (Varian Prostar, USA), controlled by a data processor software (Varian Star Workstation, USA). Chromatographic separation was achieved using a reversed-phase C18 Spherisorb ODS-2 column (Waters, 3 µm, 150 × 4.6 mm, Ireland) with a gradient of acetate buffer (30 mM) and aqueous methanol (30/70 v/v), at 0.6 mL/ min. Quantification was based on external standard method using L- ascorbic acid standard (Sigma-Aldrich, USA), subjected to the entire extraction procedure, with a quantification limit of 5 μg/mL of extract.
6
GSNO Formation from NO2-Ln and GSH
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To study the formation of GSNO from NO2-Ln and GSH, we incubated several concentrations of NO2-Ln (0.1 and 1 mM) with 1 mM GSH in 50 mM phosphate buffer, pH 7.4, containing 0.1 mM DTPA (diethylenetetraminepentaacetic acid) for 1 h at RTa with a gentle agitation. Reactions were conducted in darkness. Formation of GSNO was analyzed by LC-ES/MS (Bruker Esquire 6000, HPLC Agilent 1100) in negative ion mode. The different analytes were separated in a Waters Spherisorb ODS2 C18 column (3 mm × 125 mm, 5 μm). The mobile-phase composition was water (A) and acetonitrile (B) both with 1% of formic acid at a flow rate of 0.6 ml min−1. The gradient profile was as follows: 2–5% B (0–5 min); 40–95% B (6–22 min); and 95–2% B (22–25 min). MS/MS/MS (M3) analysis from GSNO was conducted in 0.40 V (335) and 0.60 V (305). The desolvation temperature was set at 400°C. In all cases, the data were collected, analyzed, and processed using Data Analysis Mass Spectrometry Software (Bruker, Daltonics).
7
Quantification of Phenolic Compounds in OLEU-EE
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Phenolic quantification of ethanolic OLEU-EE was performed using a method previously described by Bouaziz and Sayadi et al. (13 (link)). A YL9100 Plus HPLC (Young IN Chromass, Korea), YL9160 PDA Detector (Young IN Chromass, Korea), YL9150 Plus LC Autosampler (Young IN Chromass, Korea), YL9131 Column Compartment (Young IN Chromass, Korea), and a Spherisorb ODS2 C−18 column (250 × 4.6 mm id, 5 μm) (Waters, USA) were used. Elution was performed at a flow rate of 0.6 mL/min using a mixture of 0.1% phosphoric acid (mobile phase A) and acetonitrile/water 70:30 v/v (mobile phase B) as the mobile phase; flow of 0.6 ml/min. The solvent gradient is as follows: from 90% solvent A in 0–10 min to 75% in 25 min, to 20% in 35 min, to 0% in 40 min, and to 90% solvent A in 50 min. Phenolic quantification was performed at 280 nm using Chromatography Data System (YL-Clarity).
8
Analysis of Thymbra capitata Infusion
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The phenolic profile of the Thymbra capitata infusion was achieved by high-performance liquid chromatography (Finnigan Surveyor, THERMO, Waltham, MA, USA) coupled to a photodiode array (PDA) (Finnigan Surveyor, THERMO) and a linear ion trap mass spectrometer (LIT-MS) (LTQ XL, Thermo Scientific, Waltham, MA, USA). The sample was eluted with the mobile phases of 2% (v/v) aqueous formic acid (solvent A) and acetonitrile (solvent B) through a Waters Spherisorb ODS2 C18 column (150 × 2.1 mm and 3 μm particle size) (Waters Corp., Milford, MA, USA). The gradient was 5–50% (v/v) solvent B for 60 min at a flow rate of 200 μL/min, at 20 °C, and the absorption profile of the compounds was recorded at 280 and 320 nm. MS spectra were acquired by negative electrospray ionization (ESI) mass spectrometry. Helium was used as the collision gas with a collision energy of 35%. Nitrogen was used as the nebulizing gas with a sheath gas flow of 35 (arbitrary units) and as an auxiliary gas with a flow of 20 (arbitrary units). The temperature and voltage of the capillary were 275 °C and −35.00 V, respectively. The source voltage was 5.00 kV. For the analysis, a concentration of 500 μg/mL of T. capitata infusion was injected.
9
Quantitative Analysis of Resveratrol
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Samples were collected at the end of the incubation period, and RES was extracted immediately.
2.4 Solid-phase extraction of resveratrol and high performance liquid chromatography analysis (HPLC)
RES from culture medium samples was extracted by solid-phase extraction using a Bond Elut C18 cartridge and ethanol as eluting solvent. Samples were dried under a nitrogen stream and stored at 4 ºC until analysis.
RES was quantified by HPLC, using a Waters 2695 Separations Module coupled to a Waters 2475 Multi λ fluorescence detector (Waters, Milford, MA, USA). HPLC conditions were a modification of those described by Serra et al. (2009) . Mobile phase was: A) acetic acid 0.2% and B) acetonitrile, according to the following gradient: 0-10 min 95% A, 10-20 min 75% A, 20-25 min 95% A, with a flow-rate of 1 mL/min. A Waters Spherisorb ODS2 C18 column (3 μm, 4.6 x 250 mm) was used and column temperature was maintained at 30 ºC during analysis. Detection was performed at 280 nm, and retention time was 14.66 min. Quantification was achieved with a software integrator (Empower 2, Milford, MA, USA).
RES detection (LOD) and quantification (LOQ) limits were experimentally determined from the calibration curve of a set of seven standards (from 1 to 320 µM RES), which was linear in the range of 10-320 µM RES (r 2 = 0.989). The LOD and LOQ determined was 10 and 25 µM, respectively.
2.4 Solid-phase extraction of resveratrol and high performance liquid chromatography analysis (HPLC)
RES from culture medium samples was extracted by solid-phase extraction using a Bond Elut C18 cartridge and ethanol as eluting solvent. Samples were dried under a nitrogen stream and stored at 4 ºC until analysis.
RES was quantified by HPLC, using a Waters 2695 Separations Module coupled to a Waters 2475 Multi λ fluorescence detector (Waters, Milford, MA, USA). HPLC conditions were a modification of those described by Serra et al. (2009) . Mobile phase was: A) acetic acid 0.2% and B) acetonitrile, according to the following gradient: 0-10 min 95% A, 10-20 min 75% A, 20-25 min 95% A, with a flow-rate of 1 mL/min. A Waters Spherisorb ODS2 C18 column (3 μm, 4.6 x 250 mm) was used and column temperature was maintained at 30 ºC during analysis. Detection was performed at 280 nm, and retention time was 14.66 min. Quantification was achieved with a software integrator (Empower 2, Milford, MA, USA).
RES detection (LOD) and quantification (LOQ) limits were experimentally determined from the calibration curve of a set of seven standards (from 1 to 320 µM RES), which was linear in the range of 10-320 µM RES (r 2 = 0.989). The LOD and LOQ determined was 10 and 25 µM, respectively.
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