Shim pack gist c18 column

The prepared samples and standards were analysed on an UFLC system, equipped with a Shim-pack GIST C18 column (2 μm; 100 × 2.1 mm l.D) (Shimadzu, Kyoto, Japan), thermostatted at 40 °C. Chromatographic separation was achieved using a gradient elution system consisting of eluent A (MilliQ water with 0.1% formic acid) and eluent B (methanol with 0.1% formic acid) (Romil Chemistry, UK) at a constant flow rate of 0.2 mL min⁻¹. Each metabolite class (amino acids, phytohormones, flavonoids and phenolics) had a specific elution gradient (Supplementary Table 6). The total chromatographic run time was 10, 40, 31 and 30 min; and injection volume 3, 1, 2, and 3 µL for amino acids, phytohormones, flavonoids and phenolic acids, respectively. The MRM-MS detection parameters developed and optimised (Supplementary Table 5) were then applied, and the MS conditions were as follows: nitrogen gas was used as a drying gas and a nebulising gas at flow rates of 10 L min⁻¹ and 3 L min⁻¹ respectively. The heating gas flow was set at 10 L min⁻¹, interface temperature at 300 °C, interface voltage at 4 kV, DL temperature at 250 °C, and heat block temperature at 400 °C.

For the HPLC analysis, a Shim-pack GIST C18 column (100 mm × 2.1 mm, 1.9 μm, Shimadzu, Kyoto, Japan) was adopted. The mobile-phase that contained methanol (A) and ultrapure water (B) was ultrasonically degassed prior to use. The samples were chromatographed under the following gradient conditions: 0–4 min, linear from 50% to 90% A; 4–8 min, 90% A; 8–11 min, linear from 90% to 100% A; 11–20 min 100% A; and 20–24 min, linear from 100% to 50% A; and 24–25 min, 50% A for equilibrating the column. The flow rate was 0.30 mL/min, the sample-tray temperature was maintained at 4 °C, and the sample volume was 1.0 μL.
MS was carried out on an LC-MS/MS8045 (Shimadzu, Kyoto, Japan) equipped with an ESI source manipulated in both positive-ion and negative-ion mode. The scan range for MS was varied from m/z 200 to m/z 650. Analysis was performed in PIS mode. The source parameters were shown below: DL temperature, 250 °C; heat block temperature, 400 °C; drying gas flow, 10.0 L/min; spray gas pressure, 35 psi; and capillary voltage, 4.0 kV.

The drug loading of MSNs-COS and MSNs-COS-MAC was determined by HPLC (Nexera-i LC-2040C 3D, Shimadzu, Japan). The chromatographic conditions used in the analysis were as follows: a Shim-pack GIST C18 column (50 mm × 2.1 mm, 2 μm, Shimadzu), a mobile phase of a methanol/pH 2.0 phosphoric acid solution (70:30), a flow rate of 0.3 mL/min, a detection wavelength of 225 nm, a column temperature of 25 °C and an injection volume of 5 μL. COS in the drug-loaded samples was extracted with methanol under an ultrasonic condition and then filtered using a 0.22 μm membrane filter before running the HPLC analysis. Drug loading (%) = (Weight of costunolide in samples /Weight of samples) × 100.

The samples were analyzed by HPLC (Nexera-i LC-2040C 3D, Shimadzu, Japan). Analysis was carried out on a Shim-pack GIST C18 column (50 × 2.1 mm, 2 μm, Shimadzu). The mobile phase consisted of an 80:20 (% v/v) mixture of methanol and pH 2.0 phosphoric acid solutions, the flow rate was 0.3 mL/min and the detection wavelength was 258 nm. The column temperature was 25 °C. The injection volume was 5 μL. The retention times are about 3.07 min for IMB16-4. The samples were filtered using a 0.22 μm membrane filter before running the HPLC analysis. Drug loading (%) = (weight of IMB16-4 in samples/weight of samples) × 100.

Optical rotations and UV spectra were obtained on a JASCO P-2000 polarimeter and a JASCO V-650 spectrophotometer (JASCO Corporation, Tokyo, Japan), respectively. IR spectra were acquired by KBr disc method on a Shimadzu FTIR-8400S spectrometer (Shimadzu Co. Ltd., Tokyo, Japan). ¹H NMR (500 MHz), ¹³C NMR (125 MHz), and HMBC spectra were run on Bruker AVIII-500 spectrometer with TMS (tetramethylsilane) as the internal standard (Bruker Biospin Corporation, Fallanden, Switzerland) in CD₃OD. HRESIMS were performed on an Agilent 6520 HPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, United States). Semi-preparative HPLC was conducted on Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, United States) with a YMC-Pack ODS-A column (250 mm × 10 mm, 5 μm; YMC co., Ltd., Kyoto, Japan). The LC–MS experiments were accomplished by Shimadzu LCMS-2020 (Shimadzu Co. Ltd., Tokyo, Japan) equipped with a Shim-pack GIST C18 column (100 mm × 2.1 mm, 5 μm, Shimadzu Co. Ltd., Tokyo, Japan).