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10a hplc system

Manufactured by Shimadzu
Sourced in Japan

The Shimadzu 10A HPLC system is a high-performance liquid chromatography instrument designed for analytical separations. It features a quaternary solvent delivery system, an autosampler, a column oven, and a variety of detectors. The 10A HPLC system is capable of performing a range of analytical techniques, including reversed-phase, normal-phase, and ion-exchange chromatography.

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16 protocols using 10a hplc system

1

Quantifying α-Tocopherol in Microgreens

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The α-tocopherol content was evaluated using high-performance liquid chromatography (HPLC) on a Pinnacle II Silica column (Restek, USA; 5-μm particle size; 150 mm × 4.6 mm), as described previously (Fernandez-Orozco et al., 2003 (link)). Tocopherols were extracted from fresh plant tissues using pure hexane (1:10) by centrifugation at 349 × g for 5 min. The supernatant was filtered through a 0.45-μm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, USA). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 ml min−1. The α-tocopherol was identified according to the analytical standard. The α-tocopherol content is expressed per gram dry weight of microgreens.
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2

Quantifying Carotenoids in Microgreens

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The contents of lutein-zeaxanthin and β-carotene were evaluated using HPLC on a YMC carotenoid column (YMC, Japan; 3-µm particle size; 150 mm × 4.0 mm), as described previously (Edelenbos et al., 2001 (link)). Plant tissue (1 g) was ground in liquid N2. Then, 10 ml of 80% acetone was added to the sample and mixed. The sample was centrifuged at 5,000 × g for 15 min. The supernatant was filtered through a 0.22-µm nylon membrane syringe filter (VWR International, USA). The HPLC 10A system (Shimadzu, Japan), equipped with a diode array (SPD-M 10A VP) detector, was used for the analysis. Peaks were detected at 440 nm. The mobile phase consisted of solvent A (80% methanol and 20% water) and solvent B (100% ethyl acetate). Carotenoids were identified according to the standards. Carotenoid contents are expressed per gram dry weight of microgreens by the calculation of the ratio of fresh weight to dry weight.
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3

Carotenoid Quantification by HPLC

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Contents of lutein and β-carotene were evaluated using HPLC with a diode array detector (at 440 nm), on a YMC Carotenoid column (3 μm particle size, 150 x 4.0 mm; YMC, Japan). Carotenoids were extracted using 80% acetone (1 g of sample grounded with liquid nitrogen 10 ml-1 of solvent), centrifuged (5 min, 349 x g), and filtrated through a 0.45-μm nylon membrane syringe filter (VWR International, USA). The HPLC 10A system (Shimadzu, Japan) equipped with a diode array (SPD-M 10A VP) detector was used for analysis. Peaks were detected at 440 nm. The mobile phase consisted of A (80% methanol, 20% water) and B (100% ethylacetate). Gradient: 0 min; 20% B, 2.5 min; 22.5% B, 20–22.5 min; 50% B, 24–26 min; 80% B, 31–34 min; 100% B, 42–47 min; and 20% B, flow rate 1 ml min-1. The sensitivity of all chromatographic methods was established using a method validation procedure outlined by Edelenbos et al. [24 (link)].
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4

Quantifying Alpha Tocopherol Content

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Alpha tocopherol (α-T) content was evaluated according to Fernandez-Orozco et al. [23 (link)] using high-performance liquid chromatography (HPLC) on a Pinacle II silica column, 5 μm particle size, 150 x 4.6 mm (Restek, USA). Tocopherol homologues were extracted using pure hexane (1g of sample / 10 ml of solvent), centrifuged (5 min, 349 x g) and filtrated through 0.45 μm PTFE membrane using s syringe filter (VWR International, USA). The HPLC 10A system, equipped with RF-10A fluorescence detector (Shimadzu, Japan) was used for analysis. Peak was detected using an excitation wavelength of 295 nm and emission wavelength at 330 nm. The mobile phase was 0.5% isopropanol in hexane, flow rate 1 ml min-1.
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5

Quantifying Chlorophylls and Carotenoids in Lettuce

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Contents of chlorophylls (a, b) and carotenoids (neoxanthin, violaxanthin, lutein and zeaxanthin, αand β-carotenes) were evaluated according to the methods of Edelenbos (2001) using high-performance liquid chromatography (HPLC) on a Chromegabond C30 column (3 µm particle size, 15 × 2.1 mm) (ES Industries, West Berlin, NJ, USA) [20] (link). Carotenoids were extracted using 80% acetone (500 mg of sample grounded with 10 mL liquid N 2 ), centrifuged (10 min, 4000 rpm min -1 ), and filtrated through a 13 mm and 0.22 µm nylon syringe filter (BGB Analytik AG, Böckten, Switzerland). The HPLC 10A system (Shimadzu, Kyoto, Japan) equipped with a diode array (SPD-M 10A VP) detector was used for analysis. Peaks were detected at 440 nm. The mobile phase consisted of A (80% methanol, 20% water) and B (100% ethyl acetate). Gradient: 0 min; 20% B, 2.5 min; 22.5% B, 20-22.5 min; 50% B, 24-26 min; 80% B, 31-34 min; 100% B, 42-47 min; and 20% B, flow rate 1 mL min -1 . The contents of chlorophylls and carotenoids in each group of lettuce were evaluated after 18, 36, 60, and 84 h after inoculation. The results are expressed as mg g -1 in the dry weight of plants.
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6

Lipid Quantification by HPLC-MS/MS

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Lipids were extracted from 50 μl of liver homogenate with an internal standard mixture. The extracted free fatty acids were derivatized by amino methyl phenyl pyridium (AMPP) into FA-AMPP derivatives in order to obtain high sensitivity in MS. Measurement of lipids was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with Xcalibur (Thermo).
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7

Cholesterol Quantification in Lysosomes

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The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.
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8

Quantification of Diacylglycerol Lipids by LC-MS

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Diacylglycerol (DAG) and subspecies were quantitatively and qualitatively detected by LC-MS through Washington University Mass Spec Facility. In brief, lipids were extracted from 5×106 cells using a modified Bligh-Dyer method in the presence of an internal standard DG15:0-15:0 (0.5 µg per sample). Measurement of DAGs were performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with X calibur (Thermo). Quality control (QC) samples were prepared by pooling the aliquots of study samples and were used to monitor instrument stability. The QC was injected seven times at the beginning to stabilize the instrument, and was injected again every five study samples. Only the lipid species with CV < 15% in QC sample were reported. Relative quantification of lipids was provided, and data were reported as the peak area ratios of the analytes to the internal standard. Relative quantification data generated in same batches are appropriate to compare the change of an analyte in AKR1B10 expression or AKR1B10 silencing samples to the corresponding control.
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9

Proximate Composition and Cholesterol Analysis

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The dry matter content was determined [34 ] by drying samples in an oven at 105 °C until a constant weight was obtained (method No. 950.46B) in [34 ]. The crude protein content was determined by the Kjeldahl method using the Kjeltec system 1002 apparatus (Foss-Tecator AB, Höganäs, Sweden), and a conversion factor of 6.25 was used to convert total nitrogen to crude protein (method No. 981.10) in [34 ]. Crude fat was determined by the Soxhlet extraction method (method No. 960.39) in [34 ]. Ash was determined by incineration in a muffle furnace at 550 °C for 24 h (method No. 920.153) in [34 ]. The content of protein, fat and ash was expressed as the weight percentage of dry matter from muscle tissues. The hydroxyproline content was determined by the NMKL-AOAC method [35 (link)].
The cholesterol content in meat was determined according to the extraction method described by Polak et al. [36 (link)], followed by HPLC separation and analysis on a Shimadzu 10 A HPLC system (Shimadzu Corp., Kyoto, Japan). The data collection and evaluation were performed by using the LC Solution (Shimadzu Corp., Kyoto, Japan) operating system. The analytical column was LiChrospher 100 RP-18e, 150 × 4.6 mm, 5 μm (Alltech Associates Inc., Chicago, IL, USA) with a guard column (LiChrospher 100 RP-18, 7.5 × 4.6 mm). The cholesterol content was expressed as mg/100 g of fresh meat.
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10

Quantification of Lipid Profiles in Mouse Hearts

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The abundance of PA, DAG, triglyceride, and CL in mouse hearts was determined as described with modification (22 (link)). The LC-MS analysis was performed either with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole (TQ) mass spectrometer operated in selected reaction monitoring mode or with a Thermo Fisher Scientific Vantage TSQ mass spectrometer with Thermo Accela UPLC operated by Xcalibur software using selected ion monitoring mode. PA-(14:0)2, DAG-(15:0)2, triglyceride-(17:0)3, and CL-(14:0-14:0)2 were used as internal standards. Quantification of lipids was based on the ratio of the peak area of the analyte to the internal standard. For example, the ratio of CL-(18:2/18:2)2 and CL-(14:0-14:0)2 is used for measurement of CL-(18:2/18:2)2.
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