Extraction and quantification of retinoids by HPLC-fluorescence detection (FD) was performed as previously described with some modifications (10 (link)). In brief, cells were washed twice with PBS, and retinoids were extracted twice with 1 ml n-hexane:2-propanol (3:2; v/v) for 10 min under constant shaking at room temperature. Organic phases were combined and dried in a speed-vac (Labconco, Kansas City, MO). For retinoid analysis by HPLC-FD, retinoid extracts were dissolved in methanol:toluene (1:1; v/v) and separated on a YMC-Pro C18 column (150 × 4.6 mm, S-3 μl, 12 nm, YMC Europe GmbH, Dinslaken, Germany) using a gradient solvent system (flow, 2 ml/min; gradient, 1–2 min 100% methanol, 2–4.2 min 60%/40% methanol/toluene, and 4.2–6 min 100% methanol). Fluorescence was detected at excitation 325 nm/emission 490 nm. The HPLC consisted of a Waters e2695 separation module, a column oven (at 25°C), and a Waters 2475 fluorescence detector (Waters Corporation, Milford, MA). Area under the peak was standardized against known amounts of reference compounds using Empower 3 chromatography data software (Waters Corporation). For normalization of cellular retinoid content, cells were dissolved in NaOH/SDS, and cell protein was determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
Pro c18 column
Manufactured by YMC
Sourced in Germany, Japan
The Pro C18 column is a laboratory chromatography column designed for the separation and purification of various compounds. It features a C18 stationary phase, which is commonly used for the analysis and purification of a wide range of organic molecules, including pharmaceuticals, natural products, and environmental contaminants. The column provides high-performance liquid chromatography (HPLC) separations with consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
E2695 separation module, by Waters Corporation (4 mentions)
Empower 3 chromatography data software, by Waters Corporation (4 mentions)
2489 uv vis detector, by Waters Corporation (2 mentions)
2475 multi λ fluorescence detector, by Waters Corporation (2 mentions)
Waters 2475 fluorescence detector, by Waters Corporation (1 mentions)
Speedvac, by Labconco (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Waters hplc system, by Waters Corporation (1 mentions)
Labsolution software, by Shimadzu (1 mentions)
2695 separations module, by Waters Corporation (1 mentions)
Lc dad system, by Shimadzu (1 mentions)
Lc 20ad, by YMC (1 mentions)
Uv 2075 plus, by Jasco (1 mentions)
18 protocols using pro c18 column
1
HPLC-Fluorescence Retinoid Quantification
2
Ascorbic Acid Extraction and Analysis
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The extraction and analysis of ascorbic acid (vitamin C) was made as previously described by Wahyuni et al. (2011) (link). An extraction solution of 5% metaphosphoric acid in purified water containing 1 mM diethylenetriaminepentaacetic acid was prepared. Three hundred milligrams of frozen and grinded material was weighed in cold 2-ml Eppendorf tubes and 1.2 ml of ice-cold extraction solution added. The extracts were vortexed, sonicated for 15 min and centrifuged at 2500 g for 20 min. The supernatants were filtered over through 0.2-µm polytetrafluoroethylene filters and pipetted into amber vials for HPLC-PDA analysis using the same Waters HPLC system as described above. Separation was made at 30 °C using a YMC-Pro C 18 column (YMC Europe GmbH; 150 × 3.9 mm) with 50 mM KH 2 PO 4 buffer (pH 4.4) as eluent at a flow of 0.5 ml/min. Quantification was made based on absorbance at 260 nm, using a calibration curve of an authentic l-ascorbic acid standard from Merck. Retention time of chromatographic peak of ascorbic acid in plant extracts was verified by co-elution with the authentic standard (Supplementary material 2). As performed for the previous analysis, quality control samples from pooled leaf material of all accessions were extracted using the same procedure and injected after every eight accession sample extracts.
3
Lipid Extraction and HPLC Analysis
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Dried lipid extracts of the n-hexane extraction were dissolved in 1 ml of methanol/toluene (1:1, v/v) and separated on a YMC-Pro C18 column (150 × 4.6 mm, S-5 μl, 12 nm, YMC Europe GmbH, Dinslake, Germany) using a gradient solvent system (flow, 1 ml/min; gradient, 1–5 min 100% methanol, 5–14 min 60:40% methanol/toluene, and 14–18 min 100% methanol). Fluorescence was detected at excitation 325 nm/emission 490 nm. The HPLC consisted of a Waters e2695 separation module, including a column oven (at 25 °C) and a Waters 2475 fluorescence detector (Waters). Data were analyzed using Empower 3 chromatography data software (Waters).
4
HPLC Lipid Compound Separation and Analysis
5
HPLC Lipid Compound Separation and Analysis
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Lipid extracts, dissolved in methanol:toluene (1:1, v/v), were
separated on a YMC-Pro C18 column (150 × 4.6 mm, S-5 μl, 12
nm, YMC Europe GmbH, Dinslaken, Germany) using a gradient solvent system
(flow, 1 ml/min; gradient, 1–5 min 100% methanol, 5–14 min
60%/40% methanol/toluene, and 14–18 min 100% methanol). Fluorescence
was detected at excitation 325 nm/emission 490 nm. The HPLC consisted of a
Waters e2695 separation module, a column oven (at 25 °C), and a
Waters 2475 fluorescence detector (Waters Corp., Milford, MA). Data were
analyzed using Empower 3 chromatography data software (Waters Corp.).
separated on a YMC-Pro C18 column (150 × 4.6 mm, S-5 μl, 12
nm, YMC Europe GmbH, Dinslaken, Germany) using a gradient solvent system
(flow, 1 ml/min; gradient, 1–5 min 100% methanol, 5–14 min
60%/40% methanol/toluene, and 14–18 min 100% methanol). Fluorescence
was detected at excitation 325 nm/emission 490 nm. The HPLC consisted of a
Waters e2695 separation module, a column oven (at 25 °C), and a
Waters 2475 fluorescence detector (Waters Corp., Milford, MA). Data were
analyzed using Empower 3 chromatography data software (Waters Corp.).
6
Phenolic Compound Profiling of Korean Seaweeds
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SPW was obtained from ground SP extracted in water (85 °C, 8 h), filtered, and dried using a spray dryer. SPW was cultivated in Gyeongsangbuk-do, Korea and provided by FromBio (Gyeonggi-do, Korea). SPE, SPM, SJM, SOM, SPM, and SPW were dissolved in MeOH (20 mg/mL). Phenolic acid and flavonoid standard compounds were also dissolved in MeOH (1 mg/mL). All samples were sonicated for 20 min and filtered through a syringe filter (0.45-μm) prior to analyses. The chromatographic separation of individual components was conducted using an YMC Pro C18 column (250 × 4.6 mm, 5μm). The mobile phase comprised a mixture of 0.5% acetic acid in water (0.5:95.5, v/v) (solvent A) and ACN (solvent B). Gradient elution started with 95% of solvent A and decreased to 75% after 20 min. Solvent A was decreased to 50% at 45 min and further decreased to 10% at 55 min. It was then increased to 65% at 65 min and maintained until 70 min. The injection volume was 10 μL and the flow rate was 1.0 mL/min. The UV detection wavelength was 280 nm.
7
LC-MS/MS Quantification of Pioglitazone
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The LC-MS/MS analysis of PGZ was performed using Shimadzu 8030 system with CTO – 20 AC column oven, LC – 20 AD pump, CBM – 20 controllers, SPD – M 20 PDA detector, electrospray ionization (ESI) interface, and SIL – 20 AC autosampler.
The separation of the PGZ and IS was performed on a YMC Pro C18 column (100 mm × 4.6 mm, 3μ) with a mobile phase consisting of formic acid (0.1% v/v) and acetonitrile (5 : 95, v/v) at a flow rate of 0.7 mL min−1 and injection volume of 10 μL. Mass spectrometric detection was done on a triple quadrupole mass spectrometry using the ESI interface operating in a positive ionization mode. The control of the instrument and acquisition of data was carried using Lab solution software (Shimadzu Ltd., Mumbai, India). The block temperature of 350 °C and desolvation temperature of 250 °C was maintained with a detector voltage and CID gas set at 1.3 kV and 230 kPa, respectively. For nebulization and collision, nitrogen (99.95%) and argon gases (99.99%) were used. Multiple reaction monitoring (MRM) mode was used for the quantification of PGZ. MRM transitions of m/z 357.95 → 135.15 and m/z → 94.05 with the collision energy of −30 eV and −47 eV were chosen for quantification of PGZ and IS, respectively (Fig. 1 ).
The separation of the PGZ and IS was performed on a YMC Pro C18 column (100 mm × 4.6 mm, 3μ) with a mobile phase consisting of formic acid (0.1% v/v) and acetonitrile (5 : 95, v/v) at a flow rate of 0.7 mL min−1 and injection volume of 10 μL. Mass spectrometric detection was done on a triple quadrupole mass spectrometry using the ESI interface operating in a positive ionization mode. The control of the instrument and acquisition of data was carried using Lab solution software (Shimadzu Ltd., Mumbai, India). The block temperature of 350 °C and desolvation temperature of 250 °C was maintained with a detector voltage and CID gas set at 1.3 kV and 230 kPa, respectively. For nebulization and collision, nitrogen (99.95%) and argon gases (99.99%) were used. Multiple reaction monitoring (MRM) mode was used for the quantification of PGZ. MRM transitions of m/z 357.95 → 135.15 and m/z → 94.05 with the collision energy of −30 eV and −47 eV were chosen for quantification of PGZ and IS, respectively (
8
Quantitative Analysis of Neurotransmitters
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For catecholamine and serotonin analysis, mice were euthanized, and the brains were rapidly removed, frozen in liquid nitrogen, and maintained at − 80 °C until assay. The brain was homogenised in 200 μL of 0.2 M perchloric acid containing 3 mM cysteine. The homogenate was centrifuged (12,000 × g, 10 min, 4 °C), and the resulting supernatant was left on ice for 5 min and analysed by HPLC-fluorescence detection (Waters Alliance 2,695 separations module, Milford, MA, USA) as previously described60 (link). Catecholamine and serotonin levels were measured using the YMC-Pack Pro C18 column (250 × 4.6 mm, 5 μm) with acetate buffer (pH 3.5, 12 mM acetic acid, 0.26 mM Na2EDTA)–methanol (86:14, v/v) as the mobile phase. The flow rate was 1.0 mL/min. Catecholamine and serotonin detection was achieved using native fluorescence with excitation at 279 nm and emission at 320 nm.
9
Analytical and Preparative HPLC Purification
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Analytical HPLC was carried out using a Shimadzu LC-DAD system (Shimadzu, Kyoto, Japan; Degaser: DGU-20A3R prominence degassing Unit, Liquid Chromatograph: LC-20 AD prominence liquid chromatograph, SIL-20A HAT prominence autosampler, and SPA-M20A prominence diode-array detector) with a YMC-Pack Pro C18 column (S-5 µm, 12 nm, 150 × 10.0 mm). The preparative HPLC was performed according to Buckett et al. (2020) [43 (link)]. Regarding the biosynthesised compounds, each peak was collected using the analytical method and, after approximately 20 runs, this resulted in enough material for NMR.
10
Carotenoid Purification by SPLC
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Semi-preparative liquid chromatography (SPLC) of carotenoids preparation was performed on a LC-3000 system. A YMC-Pack Pro C18 column (250 mm × 10.0 mm, I.D.; S-5 μm, 12 nm; AS 12S05-2510WT; Ser. No. 104xA60076, United States) was used. The mobile phases were ethyl acetate (A) and mixed solvent of CH3CN - H2O (90:10, v/v; B). The gradient elution program was as follows: 60% A from 0 to 5 min; 70% A from 5.01 to 25 min; 75% A from 25.01 to 30 min; and 70% A from 30.01 to 40 min. The flow rate was 1.0 ml/min. The detection wavelength was set at 450 nm. The fractions were collected on the basis of main marker distribution as follows: 0–8 min (Fr-1), 8–16 min (Fr-2), 16–24 min (Fr-3), 24–32 min (Fr-4), and 32–36 min (Fr-5).
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