High-performance liquid chromatography was used to investigate NDP (HPLC; model Jasco PU-2089 equipped with detector model Jasco UV-2070 plus multi-wavelength UV–vis, Jasco, Tokyo, Japan). According to a prior publication, the separation and analytical method were used [23 (link)]. A C18 column was used (5 µm, 4.6 mm × 250 mm) ACE® column. The detector wavelength was 235 nm. To illustrate NDP, an isocratic flow (1 mL/min) condition was used. A mobile phase containing methanol–acetonitrile–water (25:25:50 v/v) was filtered via a 0.45 μm nylon membrane filter and degassed in a sonicator bath before use. A 20 μL of sample was injected. NDP peak was computed using ChromNav software (Jasco, Tokyo, Japan). These analyses were performed in triplicate.
Chromnav software
Manufactured by Jasco
Sourced in Japan
ChromNav is a software application designed for the management and analysis of chromatographic data. It provides a suite of tools for the visualization, processing, and reporting of chromatographic results.
Automatically generated - may contain errors
Lab products found in correlation
Pu 2089, by Jasco (2 mentions)
Pu 4180 pump, by Jasco (2 mentions)
Uv 4075 uv vis detector, by Jasco (2 mentions)
Pu 4080i infusion pump, by Jasco (2 mentions)
Hplc system, by Jasco (2 mentions)
Co 2060 plus, by Phenomenex (1 mentions)
Kinetex pfp column, by Phenomenex (1 mentions)
Precimat glycerol, by Roche (1 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (1 mentions)
Lux cellulose 1 column, by Phenomenex (1 mentions)
Hplc chromatograph, by Jasco (1 mentions)
Uv 2070 detector, by Jasco (1 mentions)
Lc net 2 adc, by Jasco (1 mentions)
Pu 2089 plus pump, by Jasco (1 mentions)
As 2057 plus autosampler, by Jasco (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
Cobas 6000 analyzer, by Roche (1 mentions)
As 4150, by Jasco (1 mentions)
Md 4015, by Jasco (1 mentions)
Lc 4000 series system, by Jasco (1 mentions)
19 protocols using chromnav software
1
Quantitative Analysis of NDP via HPLC
2
HPLC Analysis of Tocochromanol Extracts
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Clear supernatants (20 μL) of the tocochromanol extract for liquid chromatography were injected into a Jasco HPLC system (controller LC-Net II/ADC, pumps P-U2080 Plus, auto injector AS-2059-SF Plus, column oven co-2060 Plus, mixer LG-2080-02S, degasser DG-2080-53 and fluorescence detector FP-2020 Plus) equipped with a Phenomenex Kinetex PFP column (2.6 μm particle size, 150 × 4.6 mm) maintained at 40 °C, using methanol/water (76/24, v/v) as the mobile phase at a flow rate of 1.2 mL/min for a total run time of 90 min. The fluorescence detector was operated at excitation and emission wavelengths of 296 and 325 nm, respectively. Peaks were recorded and integrated using ChromNAV software (version 1.19, JASCO, Pfungstadt, Germany) and identified and quantified using 11′-αT1 isolated from palm oil and commercial standards of tocopherols and tocotrienols [32 (link)].
3
Plasma Lipid Profiling by FPLC
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Blood samples were collected. Plasma was separated by centrifugation and plasma triglyceride (TG) and free fatty acid (FFA) levels were measured using enzymatic kits (157.109.910.917 and 157.819.910.935, respectively; Diasys Diagnostic Systems, Holzheim, Germany) with Precimat Glycerol or FFA standard FS (10,166,588; Roche, Mannheim, Germany and 157.809.910.065; Diasys Diagnostic Systems, respectively) for the calibration curve. Lipoprotein TG distribution was measured by fast performance liquid chromatography (FPLC) using a system containing a PU-4180 pump with a linear degasser and UV-4075 UV/VIS detectors (Jasco, Tokyo, Japan). Pooled plasma samples (n = 8–12) were injected onto a Superose 6 Increase 10/300 GL column (GE Healthcare, Hoevelaken, The Netherlands) and eluted at a constant flow rate of 0.31 ml/min in PBS (pH 7.4). Triglycerides were measured in line by the addition of TG reagent (157.109.910.917; Diasys, Holzheim, Germany) at a constant flow rate of 0.1 ml/min using an additional PU-4080i infusion pump (Jasco, Tokyo, Japan). Data acquisition and analysis were performed using ChromNav software (version 1.0; Jasco, Tokyo, Japan).
4
Chiroptical Analysis of Flavanones
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Electronic circular dichroism and optical rotation analyses were made using a Shimadzu HPLC chromatograph coupled to a JASCO CD-2095plus CD detector and a PDR advanced laser polarimeter detector, and data were acquired using JASCO ChromNAV software version 2.01.05. Chiroptical measurements were obtained through a chromatographic analysis using a Phenomenex Lux Cellulose-1 column (150 mm x 4.5 mm, 5 µm) with a mobile phase composed of n-hexane/isopropanol 9:1 at a flow rate of 1.0 ml/min. Optical rotation response was measured at 670 nm, while UV and ECD spectra were measured between 220 and 420 nm by stop-flow while each compound was in the CD detector cell. Although this HPLC-CD-OR measurement methodology was not used for the quantitative analyses of the pure isolated flavanones, a validation procedure was performed using pure racemic flavanone and obtaining consistent results with those described in a previous work (Muñoz et al., 2013 (link)).
5
Quantification of Melittin in Apitoxin
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The melittin content of the 5 apitoxin samples was determined according the method developed by Rybak-Chmielewska and Szczêsna [26 ] with some modifications. A melittin standard with 96.5% purity was obtained from Sigma-Aldrich (Germany). Briefly, 5 mg of apitoxin was mixed with 5 mL of ultrapure water and sonicated for 5 min, and the liquid was filtered through a 0.45 μm polytetrafluoroethylene syringe filter and collected in an amber glass vial. A volume of 5 µL of 85% phosphoric acid was added to the vial. The samples were analyzed by HPLC in a Jasco LC-Net II/ADC (Jasco, Spain) coupled with a UV-2070 detector (Jasco). A Machery-Nagel C-18 column with a length of 250 mm, internal diameter of 4 mm, and particle size of 5 µm was used. Gradient chromatography was performed by the linear method with 5–80% of eluent (acetonitrile in 20% phosphoric acid) for 45 min with flow velocity of the moving phase at 1 mL·min−1. Melittin was identified at 220 nm wavelength. The data were collected through the use of Chrom NAV software (Jasco).
6
Extraction and HPLC Analysis of Neurotransmitters
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For NA, DA, and 5-HT extraction, 400 μl of buffer containing 5% ascorbic acid, 200 mM sodium phosphate, 2.5 mM l -cysteine, and 2.5 mM EDTA was added. To precipitate the protein 100 μl of 0.4 M perchloric acid was added, with an incubation at 20 °C for 20 min. Then, after centrifugation at 12,000 rpm for 10 min (4 °C) the collected supernatants were filtered by 0.22 μm then used for the evaluation of NA, DA, and 5-HT by RP-HPLC in a system that consisted of a PU-2089-plus pump (Jasco, Inc.), an AS-2057-plus autosampler (Jasco, Inc.), and an X-LC™3120FP fluorescence detector (Jasco, Inc.). ChromNav software (Jasco, Inc.) was used to control all instruments. Chromatographic runs were performed using a Júpiter C18 column (300 Å, 5 μ, 4.6 × 250 mm, Phenomenex®) at 30 °C. The column was equilibrated with mobile phase A containing 0.1% trifluoracetic acid in water. Then a linear gradient was performed from minute 5 to minute 20 with mobile phase B containing 0.1% trifluoroacetic acid in acetonitrile at a flow rate of 0.8 ml/min. The fluorescence detector was set to a gain of 1000, an attenuation of 32, a response time of 20 s, and 280 nm and 315 nm for excitation and emission, respectively. Using 50 μl as sample injection volume.
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7
Plasma Lipid Profile Quantification
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Plasma triglycerides, free fatty acids, total cholesterol, and free cholesterol were measured using commercially available kits (DiaSys Diagnostic Systems, Holzheim, Germany; Roche Diagnostics, Basel, Switzerland). Plasma lipoproteins were separated by fast protein liquid chromatography using a system containing a PU-4180 pump with a linear degasser and UV-4075 UV/VIS detectors (Jasco, Tokyo, Japan), as described.38 (link) Plasma (25 μL) was diluted with phosphate-buffered saline (PBS) (pH 7.4) in a 1:1 ratio before being loaded onto the column (Superose 6 Increase 10/300 GL; GE Healthcare, Hoevelaken, the Netherlands). Lipoproteins were then separated using PBS (pH 7.4, flow rate of 0.31 mL/min) as eluent. Total cholesterol concentrations were quantified using a colorimetric reagent (11489232; Roche Diagnostics) that was added in line at a rate of 0.1 mL/min using an additional PU-4080i infusion pump (Jasco). Data acquisition and analysis were performed using ChromNav software (version 1.0; Jasco).
Plasma transaminases were analyzed using a Cobas 6000 analyzer with standard reagents (Roche Diagnostics). Endotoxin was measured using Endosafe limulus amebocyte lysate cartridges for the Nexgen-PTS (Charles River, Leiden, the Netherlands) after a dilution of 30× in limulus amebocyte lysate water.
Plasma transaminases were analyzed using a Cobas 6000 analyzer with standard reagents (Roche Diagnostics). Endotoxin was measured using Endosafe limulus amebocyte lysate cartridges for the Nexgen-PTS (Charles River, Leiden, the Netherlands) after a dilution of 30× in limulus amebocyte lysate water.
8
Quantification of Flavonol Compounds in Flour
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F50 and F80 samples were dissolved in methanol to a concentration of 10 mg/mL, and the solutions were injected (20 μL) into a reverse-phase HPLC-PDA system described above. The mobile phase comprised 0.5% HCOOH in H2O (A) and 0.5% HCOOH in acetonitrile (B), and the following gradient was used: initial 10% B for 10 min, 15% B at 30 min, 25% B at 45 min, 35% B at 55 min, 45% B at 60 min, and 55% B at 70 min. The flow rate was set to 1 mL/min, and detection was performed at 330 nm corresponds to the absorbance of the flavonol backbone. Quantification was performed using the area under each peak determined by ChromNAV software (JASCO, Tokyo, Japan). Kaempferol compounds diluted in methanol with concentrations of 0.0017, 0.0035, 0.0175, 0.0349, 0.1747, 0.3494, and 3.4937 μM were used as standards. The content of each compound was expressed as milligram per 100 g dry flour weight.
9
HPLC Analysis of Organic Acids
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Quantification of trimesic acid (H3BTC), H4TazBz, AzA and Nic was performed using HPLC using a reversed phase Jasco LC-4000 series system, equipped with a PDA detector MD-4015 and a multisampler AS-4150 controlled by ChromNav software (Jasco Inc., Easton, MD, USA). A Purple ODS reverse-phase column (5 µm, 4.6 × 150 mm2 Análisis Vínicos) was employed. Note here that AzA was analyzed after its derivatization (der-AzA), as previously described [42 (link)]. The mobile phase consisted of a 50:50 solution (v/v) of buffer (0.04 M, pH = 2.5) and methanol (MeOH) for H3BTC and H4TazBz; 20:80 solution (v/v) acetonitrile:buffer (0.5 mM, pH = 4) for der-AzA; and 5:95 solution (v/v) acetonitrile:buffer pH = 4 for Nic. The injection volume was set at 30 µL with a flow rate of 1 mL min−1 and the column temperature fixed at 25 °C (H3BTC and H4TazBz) and 40 °C (der-AzA and Nic). The standard calibration curves showed a good correlation coefficient ≥0.99. The chromatogram of standard solutions showed a retention time (rt) of 3.51 min (identified as H3BTC, λmax at 225 nm, Figure S1 ), 21.36 min (identified as H4TazBz, λmax at 205 nm, Figure S2 ), 9.12 min (identified as der-AzA, λmax at 255 nm, Figure S3 ), and 5.56 min (identified as Nic, λmax at 213 nm, Figure S4 ).
10
Monitoring Metabolite Concentrations in Microbial Growth
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Medium was sampled at regular time points to measure pH and determine the concentration of certain metabolites (such as glucose, ethanol and acetic acid) throughout growth and lifespan. The samples were filtered (0.2 µm PFTE filter (Sigma-Aldrich, Stockholm, Sweden)) and analysed using a Jasco LC-4000 high-performance liquid chromatography system (JASCO international Co. ltd, Tokyo, Japan). The system is equipped with an AS-4150 autosampler, a PU-4180 pump, a CO-4061 column oven, an UV-4075 detector and a refractive index (RI-4030) detector. The liquid phase consisted of an isocratic concentration of sulfuric acid (5 mM) in water and had a constant flow rate of 0.8 mL/min. The column used was an analytical Rezex ROA Organic acid H+ column (Phenomenex, Aschaffenburg, Germany) and was maintained at 80 °C. Analysis was performed using Chromnav software (JASCO international CO. ltd, Tokyo, Japan) and concentrations were determined using standard calibrations.
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