The pretreatments of the samples for the analysis of sinigrin in BJD and BJJ were carried out by modifying the method of Kim et al. [25 (link)]. First, 20 g of freeze-dried BJD and BJJ and 400 mL 80% ethanol was added, and the mixture was refluxed for 2 h at 70 °C, concentrated under reduced pressure and lyophilized. The instruments used for the analysis were a Waters 2695 Separation Module HPLC system and a Waters Photodiode Array Detector (Waters Co., Milford, MA, USA). Table 2 shows the conditions used for the analysis. The column used for the analysis was Sunfire (TM) C18 (4.6 mm × 250 mm, 5.0 μm, Waters Co., Milford, MA, USA).
Waters 2695 separation module hplc system
Manufactured by Waters Corporation
Sourced in United States
The Waters 2695 separation module is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative separations. It features a quaternary solvent delivery system, an autosampler, and a column oven. The system is capable of performing a variety of HPLC techniques, including reversed-phase, normal-phase, and ion-exchange chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Empower software, by Waters Corporation (2 mentions)
Sunfire c18, by Waters Corporation (1 mentions)
Waters photodiode array detector, by Waters Corporation (1 mentions)
0.45 μm membrane filter, by Cytiva (1 mentions)
Capcell pak c18 ug120, by Shiseido (1 mentions)
Hypurity c18 column, by Thermo Fisher Scientific (1 mentions)
Quattro micro api triple quadrupole tandem mass spectrometer, by Waters Corporation (1 mentions)
Hesperidin, by Merck Group (1 mentions)
Sunfire c18 column, by Waters Corporation (1 mentions)
Waters 996 photodiode array detector, by Waters Corporation (1 mentions)
Quercetin, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Vanillic acid, by Merck Group (1 mentions)
Rutin, by Merck Group (1 mentions)
Naringin, by Merck Group (1 mentions)
Capcell pak c18 ug120 column, by Shiseido (1 mentions)
6 protocols using waters 2695 separation module hplc system
1
Extraction and HPLC Analysis of Sinigrin in BJD and BJJ
2
Quantitative HPLC Analysis of Antioxidants
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The HPLC apparatus was a Waters 2695 separation module HPLC system (Waters Co., Milford, MA, USA) equipped with a pump, an autosampler, a column oven, and a Waters 996 photodiode array detector. The analytical column was a Shiseido Capcell Pak C18 UG120 (Shiseido, 4.6 mm × 250 mm, 5.0 μm, Tokyo, Japan). The column temperature was maintained at 30 °C. The mobile phase was composed of A (1% acetic acid in water) and B (methanol) with a gradient elution as follows: 0–20 min, linear from 10 to 65% A; 20–40 min, linear from 65 to 100% A; 40–45 min, maintained at 100% A; 45–47 min, linear from 100 to 10% A; and then finally, holding for 3 min. The mobile phase was filtered through a 0.45 μm membrane filter (Whatman, Amersham, UK) and degassed under vacuum. The flow rate was set 1.0 mL/min, and the injection volume was 20 μL. The antioxidants were determined at 284 nm. Data acquisition and remote control of the HPLC system were performed using Empower software (Waters Co., Milford, MA, USA).
3
Quantification of CYP Substrate Metabolites
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The concentrations of the CYP substrates and their metabolites were quantified using a Waters 2695 separation module HPLC system (Waters Corp., Milford, Massachusetts, USA) coupled to a Quattro micro API triple quadrupole tandem mass spectrometer (Waters Corp., Milford, Massachusetts, USA) with an electrospray ionization source. The samples were separated on a HyPURITY C18 column (150 mm×2.1 mm, 5 µm, Thermo, USA) with a C18 security guard column (4.0 mm×3.0 mm, ID 5 µm). The mobile phases consisted of 20 mM ammonium formate and acetonitrile at a ratio of 60∶40. Aliquots of 20 µL were injected at a mobile phase flow rate of 0.3 mL/min. Multiple reaction monitoring was performed in the positive mode. The transitions are listed in table 1 . The mass spectra of the metabolites formed in the incubations were identical to those of the corresponding authentic standards.
4
HPLC Analysis of Phenolic Compounds in AME
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HPLC was conducted in order to qualitatively analyze the AME according to a previously described method with minor modifications. Briefly, the test sample (AME) and the standard sample (phenolic compounds: caffeic acid, catechin, chlorogenic acid, p-coumaric acid, ferulic acid, hesperidin, naringin, quercetin, rutin, vanillic acid; Sigma-Aldrich Co., St. Louis, MO, USA) were dissolved in 100% dimethyl sulfoxide (DMSO) and 5 mg/mL of stock solution was prepared, respectively. Using Waters 2695 Separation Module HPLC System (Waters Co., Milford, MA, USA) and Sunfire™ C18 column (inner diameter, 4.6 mm; length, 250 mm; Waters Co., Milford, MA, USA) filled with octadecylsilyl silica gel (diameter, 5 μm), 10 μL of test and standard samples were, respectively, chromatographed at 40 °C and 1.0 mL/min of flow rate. A (acetonitrile) and B (phosphoric acid, H3PO4) solutions were set as mobile phases under concentration gradient as follows: 0–23 min (A, 8; B, 92), 23–26 min (A, 15%; B, 85%), 26–36 (A, 30%; B, 70%), 36–40 min (A, 45%; B, 55%), 40–43 min (A, 45%; B, 55%), 43–45 min (A, 8%; B, 92%) and 45–53 min (A, 8%; B, 92%). The ingredients of AME were detected via using a Waters 996 Photodiode Array Detector (wavelength, 280 nm; Waters Co., Milford, MA, USA).
5
HPLC-PDA Analysis of Compounds
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HPLC-PDA analysis was performed on a Waters 2695 separation module HPLC system (Waters Co., Milford, MA, USA) coupled with a Waters 996 photodiode array detector (Waters Co.). Chromatography was separated on an Osaka soda Capcell Pak C18 UG120 column (4.6 mm × 250 mm, 5.0 μm, Shiseido, Tokyo, Japan), and the column oven temperature was maintained at 30 °C. For detection, the mobile phases were 0.1 % (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B), and the following gradient was used: 0–5 min, maintained at 90 % A; 5–15 min, linear from 90 to 70 % A; 15–20 min, linear from 70 to 90 %; 20–25 min, maintained at 90 % A. The mobile phase was filtered using a 0.45 μm membrane filter and degassed prior to use. Marker compounds were monitored at 254 nm.
6
Soluble Aggregates Determination by SEC
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The formation of soluble aggregates was determined by size exclusion chromatography (SEC), using a Waters 2695 separation module HPLC system. Heated and control samples were diluted to 0.25% (wt/wt) protein in 20 mM sodium phosphate buffer at pH 7 (mobile phase). Filtration of samples though 0.45 μm low protein binding filters (Sartorius Stedim Biotech GmbH, Germany) ensured removal of larger, non-soluble aggregates. A TSK Gel G2000SW XL run-in series with a G3000SW XL , 7.8 × 300 mm column (TosoHaas Bioscience GmbH, Stuttgart, Germany), under isocratic conditions, at a flow rate of 0.5 mL•min -1 over 1 h, was used to elute 20 μL of sample to a Waters 2487 dual wavelength absorbance detector at wavelengths of 214 and 280 nm. The following M w standards were used for column calibration: BSA (~66.5 kDa), α-LA (~14 kDa), β-LG (~18 kDa), aldolase (158 kDa), ferritin (44 kDa), cytochrome c (12 kDa), and carbonic anhydrase (29 kDa). Data analysis and integration were carried out using Waters Empower software. HPLC-grade Milli-Q water was used in the preparation of all buffers and samples. Buffers were vacuum filtered through 0.45-μm high velocity filters [Millipore (UK) Ltd., Durham, UK] before analysis.
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