For chromatographic separation of plasmalogen species, an Accela pump system (Thermo Fisher Scientific Inc., Waltham, MA, USA) (coupled to a triple quadrupole MS) or a Prominence pump system (Shimadzu Corp., Kyoto, Japan) (coupled to an Orbitrap MS) was utilized. A Hypersil GOLD C8 column (1.9 µm, 50 mm × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA, USA) was equipped and kept at 40 °C. The mobile phase consisted of methanol/water 5:1 v/v (with 10 mM ammonium acetate) (A) and methanol (B) at a flow rate of 200 µL/min, with the elution gradient shown in Figure S1 . The tray temperature and the injection volume were set at 4 °C and 10 µL, respectively.
Hypersil gold c8 column
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
The Hypersil Gold C8 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. The column features a silica-based stationary phase with C8 bonded ligands, which provides a moderate level of hydrophobicity for effective separation of both polar and non-polar compounds. The column dimensions and particle size can vary depending on the specific application requirements.
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4 protocols using hypersil gold c8 column
1
Chromatographic Separation of Plasmalogens
2
Quantification of Baicalin in Scutellaria Radix
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Dried roots of S. baicalensis Georgi was purchased from a drug store in Nanjing. The plant material was authenticated by in-store pharmacist and confirmed by HPLC method following the protocol given in Chinese Pharmacopeia. An extract from the radixes of S. baicalensis was prepared and chemical contents were determined following a reported procedure22 (link) on a Survit STI-501Plus HPLC system (Hangzhou, China) using a Hypersil Gold C8 column (150 × 4.6 mm, particle diameter of 5 μm, Thermo Fisher). The mobile phase was 18% methanol, 25% acetonitrile, and ultra-pure water with 0.2% acetic acid. The flow rate was set at 0.8 ml per min and the UV detector was set at 275 nm.
3
Quantification of Iloprost Deposition
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Iloprost filter deposition (mfilter) was quantified by reversed-phase ultrahigh-pressure LC on an Ultimate 3000 HPLC (Thermo Scientific, Germany) with an analytical flow of 0.35 mL/min using a Hypersil Gold C8 column (3 μm, 2.1 × 100 mm, Thermo Scientific, Germany). The column was maintained at 60 °C. The mobile phase composition was 0.1% formic acid (FA, Sigma Aldrich, Darmstadt, Germany) in water (LC-MS Grade, Merck, Darmstadt, Germany) for solution A and 100% acetonitrile (LC-MS Grade, Merck, Germany) in 0.1% FA. The gradient was 1:25, 2:35, 2.5:45, 4.5:50, 6:80, 6.5:80 and 7:25 (time, %B). The LC system was coupled to an Iontrap amaZon speed (Bruker Daltonics, Bremen, Germany) via electrospray ionization source. The analytes were analyzed using multi reaction monitoring (MRM) with transmission of 378.3 > 325.3 for iloprost and 400.3 > 365.2 for the internal standard.
4
UPLC-HRMS Analysis of Metabolites
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UPLC-HRMS data were acquired on an UltiMate 3000 system coupled to a Q-Exactive hybrid Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a HESI-II probe for electrospray ionization. Separation was achieved on a Thermo Hypersil Gold C8 column (100 × 2.1 mm, 1.9 μm) at 40 °C. Through a flow-splitter, approximately 1/15 of LC eluent was sent to the mass spectrometer. A makeup solution consisting of 5 mM ammonium formate in IPA/de-ionized/methanol 1/2/7 (v/v) was delivered constantly at 100 µL/min to MS. The solvent system was composed of acetonitrile and IPA. Initial gradient was 100% acetonitrile, which increased linearly to 5% IPA in 1 min, and then linearly to 70% IPA in 8 min, and was held for 2 min, at a flow-rate of 750 µL/min.
MS data were acquired in positive ion mode in data dependent mode, alternating between full MS and MSMS scans, where the three most abundant precursor ions were subjected to MSMS using 25 eV collision energy. The source parameters were set as follows: sheath gas: 15, auxiliary gas flow: 4, sweep gas: 0, spray voltage: 2.1 kV, capillary temperature: 375 °C, heater temperature: 300 °C.
MS data were acquired in positive ion mode in data dependent mode, alternating between full MS and MSMS scans, where the three most abundant precursor ions were subjected to MSMS using 25 eV collision energy. The source parameters were set as follows: sheath gas: 15, auxiliary gas flow: 4, sweep gas: 0, spray voltage: 2.1 kV, capillary temperature: 375 °C, heater temperature: 300 °C.
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