Cosmosil 5c18 ms 2 column

Determination of HMF and chlorogenic acid was performed using RP-HPLC (reversed phase-high performance liquid chromatographic method) method with UV detection, on Cosmosil 5C18-MS-II column (Nacalai Tesque, Inc., Kyoto, Japan), 150 mm long with an internal diameter of 4.6 mm. The analysis was performed with gradient elution with acetonitrile as phase A and 1% aqueous formic acid as phase B, at room temperature, with a flow rate of 1.0 mL/min, injection volume of 20 µL, and UV detection wavelength of 280 nm. Gradient conditions were: 0−9 min 5%−20% of A, 9−10 min holding 20% of A, 10−15 min 20%−5% of A, with an equilibration time of 10 min. The stock solutions of HMF and chlorogenic acid standard were prepared in a solvent and calibration was obtained at eight concentrations (concentration range 10.0, 20.0, 30.0, 50.0, 75.0, 100.0, 150.0, and 200.0 mg/L). Linearity of the HMF calibration curve was confirmed by R² = 0.9996 with limit of detection (LOD) of 1.79 mg/mL, limit of quantification (LOQ) of 5.9 mg/mL, and HMF retention time of 10.7 min. Linearity of the chlorogenic acid calibration curve was confirmed by R² = 0.9998 with limit of detection (LOD) of 0.016 mg/L, limit of quantification (LOQ) of 0.054 mg/L, and retention time of 14.3 min.

The ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) spectra were measured in CDCl₃ or DMSO-d₆ using a JNM-ECZ600R spectrometer (JEOL, Tokyo, Japan) at room temperature, and the chemical shifts given as a δ (ppm) scale with tetramethylsilane (TMS) as the internal standard. The FAB-MS was measured using a JEOL JMS-HX110 mass spectrometer and acquired in a glycerol matrix. HPLC was conducted using a Waters machine equipped with a 1525 binary pump and a 2489 UV/Vis detector (Waters, Massachusetts, USA). Separation was carried out using a Cosmosil 5C₁₈ MS-II column (20.0 mm × 250 mm, ODS, 5 µm; Nacalai Tesque, Kyoto, Japan). Apoptosis analyses were carried out using a Muse Cell Analyzer (Merck KGaA, Darmstadt, Germany).

Oligopeptides were separated at 40 °C using a linear gradient of 0.1% formic acid (FA) in MS grade water (solvent A) and 0.1% FA in acetonitrile (ACN) (solvent B) for 20 min at a flow rate of 0.2 mL/min, and an Agilent 1200 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Cosmosil 5C₁₈-MS-II column (Φ 2.0 mm × 150 mm, Nacalai Tesque Inc., Kyoto, Japan). The MS conditions comprised: drying gas, N₂; flow rate, 8.0 L/min; drying gas temperature, 200 °C; drying gas pressure, 1.6 bar; HV capillary voltage, −4500 V; capillary exit, 70.0 V; skimmer 1, 50.0 V; hexapole 1, 23.0 V; hexapole RF, 100.0 Vpp; skimmer 2, 23.0 V; lens 1 transfer, 52.0 μs, and mass range m/z 100–1000. A calibration solution of 10 mM sodium formate in 50% ACN was injected at the beginning of each run. Data were analyzed using Bruker Data Analysis version 3.2 software (Bruker, Billerica, MA, USA). Calibration curves were prepared using 0.1–10 μM standards.

The STR-His16 peptide was purchased from Biologica Co. (Shanghai, China). His16 peptide was synthesized by solid-phase peptide synthesis, and STR-His16 peptide was prepared by treatment of the His16 peptide on resins with stearic acid (C₁₇H₃₅COOH) and diisopropylcarbodiimide in the presence of N-hydroxybenzotriazole. STR-His16 peptide with a stearyl moiety at the N-terminus and an amide group at the C-terminus was purified by high-performance liquid chromatography (HPLC) using a Waters 2489 UV/visible detector and 1524 binary pump (Waters, Milford, MA, USA) and a reversed phase COSMOSIL 5C₁₈-MS-II column (10 mm × 250 mm; Nacalai Tesque, Kyoto, Japan) as previously described [4 (link)]. The column was run for 40 min at 3 mL/min with a linear gradient from 0% to 80% (v/v) acetonitrile in water containing 0.1% (v/v) trifluoroacetic acid. The purity of STR-His16 peptide was calculated from peak areas of HPLC chart, and final purity of the peptide was >95% (Figure S1A). The molecular masses were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (AutoFlex II; Bruker Daltonics, Billerica, MA, USA) (Figure S1B).

In the preparation of PC 16:0/18:2;OOH and CE 18:2;OOH isomers, linoleic acid hydroperoxide (FA 18:2;OOH) isomers (FA 18:2(10E,12Z);9OOH, FA 18:2(10E,12E);9OOH, FA 18:2(8E,12Z);10OOH, FA 18:2(9Z,13E);12OOH, FA 18:2(9Z,11E);13OOH, and FA 18:2(9E,11E);13OOH) were firstly prepared following our previous report [5 (link),10 (link)]. The hydroperoxyl group of the resulting FA 18:2;OOH isomers was then protected with MxP for subsequent esterification [12 (link)]. Protected FA 18:2;OOH isomers were esterified with LPC 16:0/0:0 or cholesterol [12 (link),13 (link)]. Protected PC 16:0/18:2;OOH and CE 18:2;OOH isomers were finally deprotected and purified as in a previous study [12 (link),13 (link)].
The concentration of the standard of PC 16:0/18:2;OOH isomers was determined by quantifying inorganic phosphorus according to Bartlett method [14 (link)]. To determine the concentration of CE 18:2;OOH isomers, HPLC with chemiluminescence detection was used [1 (link)]. The separation was achieved using a COSMOSIL 5C₁₈-MS-II column (5.0 µm, 4.6 × 250 mm, nacalai tesque, INC., Kyoto, Japan) with methanol/2-propanol (3:2, v/v) as the mobile phase (1 mL/min) at 40 °C. The concentration of CE 18:2;OOH isomers was calculated by using the standard of triacylglycerol hydroperoxide, whose concentration was known [15 (link)].

An overnight culture of Variovorax sp. H002 in 2xR2A liquid media was transferred to small baffled acrylic flasks containing 15 mL of freshly prepared modified M9 media fortified with 0, 0.5, 1, 5, 10, or 50 µM (final concentration) of FeCl₃·6H₂O. Upon cultivation at 30 °C/140 rpm for seven days, the cultures were centrifuged, and the supernatants were applied to pre-washed Sep-Pak C₁₈ cartridges, which were then washed with water and eluted with methanol. The collected methanolic fractions were dried and resuspended in 0.5 mL of methanol. The particulate-free sample solutions (up to 100 µL) were injected into a Cosmosil 5C₁₈-MS-II column (10 mm ID × 250 mm, Nacalai Tesque) attached to an HPLC with an autosampler, at a flow rate of 0.8 mL/min at 40 °C. Each analysis was performed using an aqueous acetonitrile mobile phase.