The modification efficiency of LF on LF-Lipo was determined by SDS-PAGE and quantitatively analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The particle size, polydisperse index (PDI), and zeta potential of Lipo and LF-Lipo were measured by a Zeta Sizer Nanoparticle Analyzer (Malvern Panalytical, Malvern, UK). The morphological observation of the liposomes was performed using transmission electron microscopy (TEM). The drug encapsulation efficiency (EE%) and drug-loading capacity (DL%) of the liposomes were determined by high-performance liquid chromatography (HPLC) (1260 Infinity, Agilent technologies, Santa Clara, CA, USA) equipped with Diamonsil-C18 (5 μm, 250 mm × 4.6 mm, Dikma, Beijing, China). The chromatographic conditions are listed in Supporting Information Tables S1 and S2 .
Diamonsil c18
Manufactured by Dikma Technologies
Sourced in China, United States
Diamonsil C18 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with chemically bonded C18 alkyl chains, providing excellent retention and selectivity for non-polar and moderately polar analytes.
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Lab products found in correlation
Av 400 spectrometer, by Bruker (3 mentions)
Q star elite esi lc ms ms mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Agilent 1100, by Agilent Technologies (2 mentions)
Asi 100 autoinjector, by Dikma Technologies (2 mentions)
Zetasizer nano particle analyzer, by Malvern Panalytical (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
5432 vortex mixer, by Eppendorf (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
L 2130 pump, by Hitachi (1 mentions)
L 2420, by Hitachi (1 mentions)
Rf 5301 spectrofluorometer, by Shimadzu (1 mentions)
Lc 10at pump, by Shimadzu (1 mentions)
Spd 10a uv detector, by Shimadzu (1 mentions)
Av 500 spectrometer, by Bruker (1 mentions)
Agilent lc esi ms system, by Agilent Technologies (1 mentions)
Sciex x500r qtof mass spectrometer, by AB Sciex (1 mentions)
13 protocols using diamonsil c18
1
Liposome Formulation Characterization
2
HPLC Analysis of Plasma Samples
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HPLC analysis was performed using a Dikma Diamonsil C18 (5 μm, 150 × 4.6 mm; Dikma, Beijing, China) on a Shimadzu® HPLC system (LC-20A; Shimadzu Corporation, Tokyo, Japan) with an ultraviolet detector at room temperature. The wavelength of the ultraviolet detector was set at 220 nm. Methanol, acetonitrile and 0.05 mol/L KH2PO4 solution (30:20:50, v/v/v) was used as the mobile phase at a flow rate of 1 mL/min.
A 500 µL volume of the plasma sample was transferred to a 5 mL plastic test tube together with 10 µL of internal standard (I.S.) solution (2 mg/mL chlorpromazine). After vortex shaking for 1 min (5432 vortex mixer, Eppendorf, Hamburger, Germany), 2 mL of diethyl ether was added and the mixture was vortexed for 3 min. After centrifugation at 12,000 rpm for 10 min (Thermo IEC, Micromax, Boston, MA, USA), the upper organic layer was quantitatively transferred to a 10-mL glass tube and evaporated to dryness using an evaporator at 40 °C. The residue was reconstituted in 100 µL of the mobile phase, and then vortex-mixed. After centrifugation at 12,000 rpm for 5 min, a 10 µL aliquot of the solution was injected into the HPLC system for analysis.
A 500 µL volume of the plasma sample was transferred to a 5 mL plastic test tube together with 10 µL of internal standard (I.S.) solution (2 mg/mL chlorpromazine). After vortex shaking for 1 min (5432 vortex mixer, Eppendorf, Hamburger, Germany), 2 mL of diethyl ether was added and the mixture was vortexed for 3 min. After centrifugation at 12,000 rpm for 10 min (Thermo IEC, Micromax, Boston, MA, USA), the upper organic layer was quantitatively transferred to a 10-mL glass tube and evaporated to dryness using an evaporator at 40 °C. The residue was reconstituted in 100 µL of the mobile phase, and then vortex-mixed. After centrifugation at 12,000 rpm for 5 min, a 10 µL aliquot of the solution was injected into the HPLC system for analysis.
3
Phytochemical Extraction and HPLC Analysis
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SB samples were dried at 50°C to a constant weight. The dried samples (leaves, stems, and roots) were comminuted and passed through 60-mesh sieves. Accurately weighed (18.75 mg) sample powders were extracted using 1.5 mL of 60% ethanol by ultrasonication for 40 min and then centrifuged at 13000 rpm for 10 min. The supernatant was then collected. The sample solution was filtered through a 0.22 μm pore-size polytetrafluoroethylene filter.
The extracts were analyzed on a Diamonsil C18 (4.6 × 250 mm, 5.0 µm, DiKMA, Beijing, China) column connected to a SHIMADZU LC-20AD high-performance liquid chromatography (HPLC) system (SHIMADZU, Kyoto, Japan). The mobile phases comprised A (water containing 0.1% formic acid) and B (acetonitrile). The gradient elution program was as follows: 25% B at 0–10 min, 25–45% B at 10–30 min, 45–55% B at 30–55 min. The detection wavelength was set at 254 nm. The sample injection volume was 10 μL and the column temperature was maintained at 25°C.
The extracts were analyzed on a Diamonsil C18 (4.6 × 250 mm, 5.0 µm, DiKMA, Beijing, China) column connected to a SHIMADZU LC-20AD high-performance liquid chromatography (HPLC) system (SHIMADZU, Kyoto, Japan). The mobile phases comprised A (water containing 0.1% formic acid) and B (acetonitrile). The gradient elution program was as follows: 25% B at 0–10 min, 25–45% B at 10–30 min, 45–55% B at 30–55 min. The detection wavelength was set at 254 nm. The sample injection volume was 10 μL and the column temperature was maintained at 25°C.
4
HPLC Analysis of Tea Catechins
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Catechins were determined by HPLC, as described previously (Wang, Yang, Wang, Zeng, Xu, & Yin, 2020 ). The samples of tea infusion were filtered through a 0.45 μm Millipore filter before injection. The detection conditions were as follows: column with 5 μm Diamonsil™ C18 (4.6 mm × 250 mm; Dikma Technologies Inc., Lake Forest, CA); temperature at 35 °C; post-run time for 5 min; injection volume was 10 μL; the flow rate was 1.0 mg/mL; detection wavelength at 280 nm. The mobile phase comprised A: 2% v/v acetic acid; mobile phase B: 100% acetonitrile. The elution gradient was as follows: 0–16 min, 6.5% B; 16–25 min, 15% B; and 25–30 min, 6.5% B. The standard catechins, GA and caffeine (GC: y = 1*e6x, R2 = 0.9994; EGC: y = 2*e6 ×, R2 = 0.9995; C: y = 7*e6 ×, R2 = 0.9991; EC: y = 8*e6x, R2 = 0.9998; GCG: y = 2*e7x, R2 = 0.9997; EGCG: y = 1*e7x, R2 = 0.9998; CG: y = 2*e7x, R2 = 0.9990; ECG: y = 2*e7x, R2 = 0.9999; GA: y = 2*e7x, R2 = 0.9993; caffeine: y = 3*e7x, R2 = 0.9993) were used for identification and quantitative analysis.
5
HPLC Quantification of Compounds
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Quantitative determination of the ESP and complexes was performed using a HPLC system consisting of an L-2420 ultraviolet absorbance detector and an L-2130 pump (Hitachi High-Technologies Corporation, Tokyo, Japan). The HPLC column (200 mm × 4.6 mm × 5 μm) was Diamonsil C18 (Dikma Technologies, Beijing, China). The mobile phase consisted of methanol and 0.5% triethylamine in distilled water (55:45, v/v), and the pH was adjusted to 3.0 with phosphoric acid. The flow rate was 1 mL/min and the detection wavelength was 238 nm. Retention times for ESP, BA, IB, SA, PABA, and BSA were 4.5, 6.5, 6.0, 7.5, 3, and 3.6 min, respectively.
6
Purification and Characterization of Organic Compounds
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All reagents and solvents were used directly as purchased from commercial sources. Flash chromatography was performed using silica gel (200–300 mesh). All reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F254 and UV light visualization. 1H NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to an internal control (TMS). Low-resolution ESI–MS was recorded on an Agilent 1200 HPLC-MSD mass spectrometer and high-resolution ESI–MS on an Applied Biosystems Q-STAR Elite ESI–LC–MS/MS mass spectrometer. HPLC instrument, Dionex Summit HPLC (column: Diamonsil C18, 5.0 μM, 4.6 × 250 mm (Dikma Technologies); detector, PDA-100 photodiode array; injector, ASI-100 autoinjector; pump, p-680A). A flow rate of 0.5 ml/min was used with a mobile phase of ACN in H2O with a 0.1% modifier (TFA, v/v).
7
HPLC-based Quantification of Encapsulated DNR
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The amount of DNR encapsulated in the vesicles was measured using high performance liquid chromatography (HPLC) (Agilent 1100) with a C18 reverse phase column (Diamonsil C18, 150 × 4.6 mm, Dikma Technologies, IL) at a detection wavelength of 481 nm. Briefly, following elution to remove the free drug, the equal purified and untreated samples were spin-dried and dissolved in 1 ml of chloroform. The solution then was filtered using 0.22 μm syringe filter prior to HPLC analysis. The concentration of Coumarin-6 was quantified using fluorescence spectroscopy at 466Ex/504Em (RF-5301 spectrofluorometer, Shimadzu, Japan) likewise. Encapsulation efficiency (% EE) was calculated by comparing the total response value (peak area or fluorescence intensity) of DNR/Coumarin-6 pre- and post gel filtration, diluted to the same final lipid concentration.
8
HPLC Analysis of LPM Compounds
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The mass analysis of LPM was performed by reverse-phase HPLC. The HPLC system consisted of a model LC-10AT pump (Shimadzu, Kyoto, Japan) and a model SPD-10A UV detector (Shimadzu; Mo et al., 2011 (link)). The stationery phase was Diamonsil C18 (200 × 4.6 mm, 5 μm; Dikma, USA). The injection volume was 20 μl; a mixture of 0.1 mol/l ammonium carbonate and acetonitrile (90:10, v/v) was used as a mobile phase with a flow rate of 1.0 ml/min. The UV detection wavelength was 264 nm; the column temperature was 30°C.
9
HPLC Method for Compound Analysis
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An Agilent 1100 liquid chromatography system (Agilent, USA) equipped with a binary gradient pump, diode array detector, column oven, and degasser was used to analyze the samples. The HPLC method was developed using Diamonsil C18 (DiKMA, 250 × 4.6 mm, 5 µm). The mobile phase was composed of 0.1% (volume percent) phosphoric acid water solution (A) and acetonitrile (B). The step gradient was as follows: 10%–15% B at 0–10 min, 15% – 25% B at 10–40 min, 25%–35% B at 40–55 min, 35%−55% B at 55–70 min, 55%–70% B at 70–75 min, 70%–100% B at 75–85 min, 100%–100% B at 85–90 min. The flow rate was set at 1.0 mL/min, and the injection volume was 10 µL. The column temperature was set at 35 °C, and the detection wavelength was chosen to be 327 nm.
10
Characterization of Organic Compounds
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Reagents and solvents were purchased
from commercial sources and used directedly. Flash chromatography
was performed using 300-mesh silica gel. Reactions were monitored
by thin-layer chromatography using silica gel plates with fluorescence
F254 and UV light visualization. Low-resolution electrospray
ionization mass spectrometry (ESI-MS) was performed on an Agilent
1200 high-performance liquid chromatography (HPLC)-mass selective
detector mass spectrometer and high-resolution ESI-MS on an Applied
Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. 1H NMR spectra were performed on a Bruker AV-400 spectrometer at 400
MHz or a Bruker AV-500 spectrometer at 500 MHz. 13C NMR
spectra were performed on a Bruker AV-500 spectrometer at 125 MHz.
Coupling constants (J) were expressed in hertz (Hz).
Chemical shifts (δ) of NMR were reported in parts per million
units relative to an internal standard (tetramethylsilane). Purity
of the compounds was determined by reverse-phase high-performance
liquid chromatography (HPLC) analysis to be >95%. HPLC instrument:
Dionex Summit HPLC (column: Diamonsil C18, 5.0 μm, 4.6 ×
250 mm2 (Dikma Technologies); detector: PDA-100 photodiode
array; injector: ASI-100 autoinjector; pump: p-680A). A flow rate
of 1.0 mL/min was used with mobile phase of MeOH in H2O
with a 0.1% modifier (ammonia, v/v).
from commercial sources and used directedly. Flash chromatography
was performed using 300-mesh silica gel. Reactions were monitored
by thin-layer chromatography using silica gel plates with fluorescence
F254 and UV light visualization. Low-resolution electrospray
ionization mass spectrometry (ESI-MS) was performed on an Agilent
1200 high-performance liquid chromatography (HPLC)-mass selective
detector mass spectrometer and high-resolution ESI-MS on an Applied
Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. 1H NMR spectra were performed on a Bruker AV-400 spectrometer at 400
MHz or a Bruker AV-500 spectrometer at 500 MHz. 13C NMR
spectra were performed on a Bruker AV-500 spectrometer at 125 MHz.
Coupling constants (J) were expressed in hertz (Hz).
Chemical shifts (δ) of NMR were reported in parts per million
units relative to an internal standard (tetramethylsilane). Purity
of the compounds was determined by reverse-phase high-performance
liquid chromatography (HPLC) analysis to be >95%. HPLC instrument:
Dionex Summit HPLC (column: Diamonsil C18, 5.0 μm, 4.6 ×
250 mm2 (Dikma Technologies); detector: PDA-100 photodiode
array; injector: ASI-100 autoinjector; pump: p-680A). A flow rate
of 1.0 mL/min was used with mobile phase of MeOH in H2O
with a 0.1% modifier (ammonia, v/v).
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