The peptide mixture was separated into 10 fractions by high-pH RPLC using ethylene bridged hybrid (BEH) C18 columns (1.7 µm, 1.0 mm × 150 mm) attached to the H-class system (Waters). The second-dimensional liquid chromatography separation of the peptides was conducted on an Ekspert nano LC 415 instrument (AB Sciex, Toronto, Canada). The peptides were eluted using 2% acetonitrile with 0.1% formic acid as buffer A and 98% acetonitrile with 0.1% formic acid as buffer B with a gradient of 100 min. The eluted peptides were subsequently analyzed using a TripleTOF 5600+ mass spectrometer (AB Sciex). Data were obtained in positive ion mode. For the TOF-MS scan, the mass range was set as 350–1500 m/z. The MS/MS scan was carried out with a mass range of 100–1500 m/z.
H class system
Manufactured by Waters Corporation
Sourced in United States
The H-Class system is a high-performance liquid chromatography (HPLC) solution designed for analytical and preparative separations. It offers precise control of mobile phase flow, temperature, and pressure to ensure reliable and reproducible chromatographic results.
Automatically generated - may contain errors
Lab products found in correlation
Kinetex 5 m c18 column, by Phenomenex (2 mentions)
Ekspert nanolc415, by AB Sciex (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Xevo tq s, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Kinetex 5 μm c18 column, by Phenomenex (1 mentions)
0.2 μm membrane, by Merck Group (1 mentions)
Lichrospher si60 column, by Merck Group (1 mentions)
Transwell culture plate, by Corning (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
Rf 5301pc fluorescence spectrophotometer, by Shimadzu (1 mentions)
9 protocols using h class system
1
High-pH Reversed-Phase Liquid Chromatography-Based Peptide Separation
2
UPLC-MS/MS Analysis of Metabolites
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The samples were separated using an ACQUITY UPLC BEH C18 Column (2.1 × 50 mm, 1.7 µm, Waters) on an LC system (ACQUITY H‐Class System, Wnters). The LC mobile phase consisted of (C) water containing 0.1% (v/v) formic acid and (D) acetonitrile. The gradient program was isocratic at 10% D, Initial; linear 1at 0%–85% D, 0–15 min; isocratic at 100% D, 15–16 min; and isocratic at 100% D, 16–20.5 min. The injection volume of each sample was 5 µl, and the flow rate was 0.2 ml min. The isolated samples were detected using a tandem quadrupole MS (Xevo TQ‐S, Waters) in the Multiple Reaction Monitoring (MRM) mode. The MRM conditions for the respective compounds are listed in Table S1 .
3
Quantitative Analysis of RPV and M3RPV
4
Solubility Determination of CAB and MCAB
5
UPLC-PDA Kinetic Analysis Protocol
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Kinetic analyses were performed using a Waters acquity ultra performance liquid chromatography (UPLC) H-Class system with photodiode array (PDA) detector. Instrument control and data processing were performed using Empower software. acquity UPLC BEH C18 column, 2.1×50 mm with a 1.7 µm size particle was used. A mixture of MeOH:H2O with a gradient of 5:95 → 95:5 over 5 min was used as mobile phase. Calibration showing linear fitting was obtained (Supplementary Figure 11 ). See supplementary information for more details, including chromatograms (Supplementary Figures 12 –13 ).
6
Solubility Determination of CAB and MCAB
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The solubilities of CAB and MCAB in water and 1-octanol were determined by adding excess drug to each solution at room temperature then mixing for 24 h. Samples were centrifuged at 20,000 × g for 10 min to pellet insoluble drug. The supernatants containing solubilized drug were vacuum-dried then re-dispersed in methanol for drug concentration measurement using a Waters ACQUITY ultra performance liquid chromatography (UPLC) H-Class System with TUV detector and Empower 3 software (Milford, MA, USA). CAB and MCAB samples were separated on a Phenomenex Kinetex 5 µm C18 column (150 × 4.6 mm) (Torrance, CA) using either 65% 5.0 mM KH2PO4, pH 3.2/35% ACN or 90% ACN/10% water with a flow rate of 1.0 mL/min and detected at 254 and 230 nm, respectively. Drug content was quantitated by comparison of peak area to those of known standards (0.05–50 µg/mL in methanol).
7
UHPLC Analysis of Compound Separation
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The samples were analyzed by ultra-high-performance liquid chromatography (UHPLC), using a Waters Acquity Ultra-performance liquid chromatography (UPLC) H-Class system (Milford, MA, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler and a column heater. Chromatographic separation was carried out at 45 °C on a Waters Cortecs UPLC C18 column (4.6 × 50 mm, 2.7 μm). A mobile phase with two eluent solvents were used: eluent (A) acetonitrile and eluent (B) 5% methanol and 0.1% formic acid in Mili-Q water. The gradient elution was as follows: 0–2% A at 0–2 min, 2–7% A at 2–5 min, 7–13% A at 5–7 min, 13–20% A at 7–9 min, 20–55% A at 9–11.5 min, 55–90% A at 11.5–13.5 min, 90% A at 13.5–14.5 min, 90–3% A at 14.5–14.95 min, 3% A at 14.95–18 min, and 3–0% A at 18–20 min. The flow rate was kept at 0.25 mL/min, and the injection volume was 10 μL. The eluate was monitored at 520 nm on the PDA.
8
Vitamin E Quantification in Dehulled Seeds
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Vitamin E in de-hulled (palea and lemma removed) seeds was analysed following Ko et al. (2003) . Briefly, the ground sample (50 g) was mixed with 300 ml n-hexane for 2 h, then concentrated by evaporating hexane using nitrogen gas. The lipid extract (0.5 g) was mixed with 2 ml 5% pyrogallol solution in ethanol and 20 ml ethanol. After boiling at 70°C, 1 ml of 50% aqueous KOH was added for the 5 min saponification. The sample was extracted by 50 ml diethyl ether, washed with 20 ml distilled water, filtered through anhydrous sodium sulphate and evaporated at 30°C. The residue was diluted with 10 ml n-hexane and filtered through a Millipore 0.2 μm membrane. Individual vitamin E homologues were quantified by ultra-performance liquid chromatography (UPLC, H-Class System, Waters, Massachusetts, USA) at 298 nm excitation and 325 nm emission with a Lichrospher Si-60 column (250 × 4.6 mm i.d.; Merck Co., Gernsheim, Germany) . Descriptive statistics of all traits and correlations were analysed using STAR v2.0.1 (International Rice Research Institute).
9
UPLC and Fluorescence Spectroscopy Protocol
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Equipment. The ultra performance liquid chromatography (UPLC) H-class system (Singapore) was equipped with Empower 3 software (Waters Corporation, Milford, MA, USA), and an H-class Waters column (ACQUITY UPLC BEH C18; 2.1x100 mm; 1.7 µm; Waters Corporation) for UPLC separation. The RF-5301PC fluorescence spectrophotometer was purchased from Shimadzu Scientific Instruments (Columbia, MD, USA). The Bio-Tech Synergy 22100 microplate reader (cat. no. 168-1002XC) was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), and the CF16RX-Ⅱ centrifuge was purchased from Hitachi Koki Co., Ltd (Tokyo, Japan). A Nikon ECLIPSE Ti-U biological microscope (Nikon Corporation, Tokyo, Japan) was used to count the number of cells. Transwell culture plates were purchased from Corning Costar (Corning Incorporated, Corning, NY, USA) and the Pre-Coated PAMPA Plate system was obtained from BD Biosciences (Bedford, MA, USA).
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