We stored the liver, kidney, adrenal glands, gastrocnemius muscle, and WAT at −80 °C to avoid 11β-Hsd1 and 11β-Hsd2 activity. This method was based on that described in a previous report [25 (link)]. Briefly, the tissues were homogenized in 30 mM Tris buffer (pH = 7.4) containing 0.9 mM ethylenediaminetetraacetic acid and 0.3 mM sucrose to produce 40 mg/mL homogenates. The 11b-Hsd1 and 11b-Hsd2 activities were measured in the presence of cortisone and cortisol, respectively. Steroids were extracted with chloroform and 50 µM dexamethasone was added as an internal control. Reverse-phase HPLC analysis was performed on a 5C18-MS-II column (Nacalai Tesque Inc., Kyoto, Japan) at a column oven temperature of 30 °C and a detection wavelength of 254 nm. The isocratic mobile phase was methanol/water (60:40, v/v), and its flow rate was 0.6 mL/min. The analytes were identified using the following standards: cortisone (Merck, Darmstadt, Germany), cortisol (VWR International Ltd., Leuven, Belgium), and dexamethasone (Merck, Darmstadt, Germany). The HPLC measurement system used in the present study was linear in the range of 2–40 nmol for cortisol and cortisone levels, and all measured values were within this range. 11β-Hsd1 activity was calculated from the cortisol levels, and 11β-Hsd2 activity was calculated from the cortisone levels, which were adjusted to dexamethasone levels.
5c18 ms 2 column
Manufactured by Nacalai Tesque
Sourced in Japan
The 5C18-MS-II column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a reversed-phase C18 stationary phase and is suitable for use in various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Cortisone, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Pu 2089 pump, by Jasco (1 mentions)
Av 600 spectrometer, by Bruker (1 mentions)
Ohpak sb 804 hq column, by Resonac (1 mentions)
Microtof q 3 spectrometer, by Bruker (1 mentions)
Ri 2031 refractive index detector, by Jasco (1 mentions)
Fp 6500 fluorometer, by Jasco (1 mentions)
Co 2065 column oven, by Jasco (1 mentions)
Uh5300 uv vis double beam spectrophotometer, by Hitachi (1 mentions)
Autopol 4 polarimeter, by Rudolph Research Analytical (1 mentions)
Q exactive orbitrap lc ms ms system, by Thermo Fisher Scientific (1 mentions)
Organic solvents, by Sinopharm (1 mentions)
Lc 10at, by Shimadzu (1 mentions)
Agilent 1260 hplc system, by Agilent Technologies (1 mentions)
Avance 3 700 spectrometer, by Bruker (1 mentions)
Axima lnr, by Shimadzu (1 mentions)
Jem 2100 tem, by JEOL (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
Cto 10acvp column oven, by Shimadzu (1 mentions)
11 protocols using 5c18 ms 2 column
1
Measuring 11β-HSD Enzyme Activities
2
Analytical Characterization of Compounds
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The NMR spectra were recorded using Bruker BioSpin AV-300 and AV-600 spectrometers. The ESI-MS spectra were recorded using a Bruker Daltonics micrOTOF Q-III spectrometer (Bruker Daltonics, Billerica, MA, USA). The HPLC and GPC measurements were conducted using a system consisting of a JASCO PU-2089 pump and a JASCO CO-2065 column oven (JASCO Corporation, Tokyo, Japan). A JASCO UV-2075 ultraviolet detector and a JASCO RI-2031 refractive index detector were used for the HPLC and GPC analyses, respectively. A 5C18-MS-II column (ɸ4.6 × 250 mm, Nacalai Tesque, INC.) was used for the HPLC analysis. 5, 4, 3, and 10 % MeCN-containing water were used as the eluents at a flow rate of 1.0 mL/min at 30 °C to analyze the enzymatic reaction with 1a – d , respectively. A Shodex OHpak SB-804 HQ column (ɸ8.0 × 300 mm, Showa Denko K.K., Tokyo, Japan) was used for the GPC analysis of 3a – c using a phosphate buffer (20 mM, pH 7.0) as the eluent at a flow rate of 0.5 mL/min at 30 °C. Pullulan samples were used as standards. A Shodex KD-804 column (ɸ8.0 × 300 mm, Showa Denko K.K.) was used for the GPC analysis of 3d using N , N -dimethylformamide (DMF) containing 10 mM lithium bromide as the eluent at a flow rate of 0.5 mL/min at 50 °C. Poly(methylmethacrylate) samples were used as standards. The fluorescence intensity was recorded using a JASCO FP-6500 fluorometer for the lectin binding tests.
3
Spectroscopic Analysis of Compounds
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The optical rotation was measured in MeOH by an Autopol IV polarimeter (Rudolph Research Analytical, Hackettstown, NJ, United States). UV spectra were obtained by a UH5300 UV–VIS double beam spectrophotometer (Hitachi Co., Tokyo, Japan). 1D and 2D NMR spectra were obtained by a Bruker AVANCE IIITM 500 and 600 MHz spectrometers (Bruker, Ettlingen, Germany) in methanol-d4, DMSO-d6 using TMS as internal standard. HR-ESI-MS data was obtained on a Thermo Scientific Q Exactive Orbitrap LC-MS/MS System (Thermo Scientific, Waltham, MA, United States). An Ultimate 3000 HPLC system (Dionex Co., Sunnyvale, CA, United States) with an UltiMate 3000 pump and UltiMate 3000 variable wavelength detector was employed to carry out semi-preparative HPLC, with a Nacalai Tesque 5C18-MS-II column (250 × 10 mm, 5 μm). Silica gel (200–300 mesh and 300–400 mesh) for open column chromatography (CC) was purchased from Qingdao Haiyang Chemical Group Co., Ltd. (Qingdao, China). The organic solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Reagents used for α-glucosidase inhibitory assay (α-glucosidase, 4-nitrophenyl-α-D-glucopyranoside, and acarbose) were purchased from Shanghai Yuanye Biology Co., Ltd. (Shanghai, China), and the absorbance was measured by a full-wavelength microplate reader (Thermo Fisher Scientific Shier Technology Co., Ltd.).
4
HPLC Assay for FT and NT Quantification
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The quantitative determination of FT and NT was performed by the HPLC method on an LC-10AT (Shimadzu, Kyoto, Japan) using a 5C18-MS-II column (4.6 × 150 mm, 5 μm; Nacalai Tesque, Kyoto, Japan) with a mobile phase containing a mixture of acetonitrile and water (9:1, v/v) at a flow rate of 0.5 mL/min. A sample was dissolved in ethanol to a suitable drug concentration before injection. The sample injection volume was 20 μL, and the detection of both drugs was performed using a UV detector at a wavelength of 295 nm.
5
Quantitative Analysis of Erlotinib in Mice
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Fourty C57BL/6 mice (male, 20-30g) were randomly divided into two groups: erlotinib (45 mg/kg p.o. 3d) group and erlotinib (45 mg/kg i.p.) group. Mice blood samples were collected 0.5, 1, 2, 4, 6 and 12 h post-dose. The blood samples were centrifuged at 10 000 g for 10 min and the supernatant (plasma) was collected. The plasma 90 ul were mixed with 350 μl methanol then add 10 μl grfitinib (20 μg/ml, as the internal standard), followed by vortex and centrifugation (15 min, 13000 g), The supernatant was collected and dried, then redissolve by 200 μl 50% acetonitrile-water, followed by vortex, sonicated (10 min) and centrifuged at 13000 g (10 min), A 20 μl aliquot of the supernatant was subjected to HPLC analysis. The separation was performed using the Agilent 1260 HPLC system. Chromatographic elution was performed on the 5C18-MS-II column (20-250mm, Cosmosil) using an isocratic gradient of 35% acetonitrile in water. The detection wavelength was at 210 nm.
6
Analytical and Characterization Techniques
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Example 2
Solvents and starting materials were purchased by the standard suppliers and used without further purification. Analytical thin-layer chromatography (TLC) was performed on 0.2 mm silica 60 coated on plates with F254 indicator. Flash column chromatography was performed on Wako gel C-200 silica gel. High performance liquid chromatography (HPLC) was performed by a Gilson 811C Dynamic Mixer system with a UV detector set at 254 nm using a Cosmosil 5C18-MS-II column (150×20 mm) with a dual solvent system of 0.1% HOAc/H2O (Solvent A) and MCCN (Solvent B). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 700 spectrometer at 21±3° C. unless otherwise indicated. Chemical shifts (δ) are reported in parts per million (ppm). Coupling constants (J) were reported in Hertz. 1H NMR chemical shifts were referenced to the residual solvent peak at 3.31 ppm in CD3OD-d4. 13C NMR chemical shifts were referenced to the center solvent peak at 49.00 ppm for CD3OD. ESI mass spectra were recorded on a Thermo LTQ Orbitrap XL mass spectrometer.
7
Synthesis and Characterization of Fluorescent ODNs
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A coupling reaction between amino-modified ODNs (100 μM) and Alexa Fluor 488 NHS ester or succinimidyl derivative of protein tag substrates (1 mM) was carried out in 100 mM phosphate buffer (pH 8.0) for 8 h at ambient temperature. The modified ODNs were purified by reversed-phase HPLC on a Cosmosil 5C18-MS II column (4.6 × 150 mm, eluted with 100 mM triethylammonium acetate buffer, pH 7.0, with a linear gradient over 30 min from 2.5% to 30% acetonitrile at flow rate of 1.0 mL min–1) and characterized by MALDI-TOF mass spectrometry (AXIMA-LNR, Shimadzu, HPA matrix). A488-ODN-ZF: m/z calcd 11 397, observed 11 395; A488-ODN-AZ: m/z calcd 11 400, observed 11 399; A488-ODN-AP: m/z calcd 12 011, observed 12 014. ODN-AP-2BC: m/z calcd 12 290, observed 12 290, ODN-8G-AP-2BC: m/z calcd 21 577, observed 21 578, ODN-11G-ZF-BC: m/z calcd 21 160, observed 21 167, ODN-ZF-BC: m/z calcd 14 287, observed 14 287, ODN-ZF(G/T)-BC: m/z calcd 14 286, observed 14 289, ODN-ZF(G/C)-BC: m/z calcd 14 287, observed 14 282, ODN-ZF(GG/TC)-BC: m/z calcd 14 286, observed 14 288, and ODN-ZF(GG/TC)-BC: m/z calcd 14 286, observed 14 291. The method for preparing ODN-AZ-BC, ODN-AZ-BG, and ODN-24D-AZ-BC was described in a previous report.15 (link)
8
Purity Analysis of Compound 3a
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The purity of compound 3a was analyzed on a COSMOSIL 5C18-MS-II column (250 mm × 10 mm) at 25 °C. We used H2O containing 0.1% trifluoroacetic acid (TFA) as mobile phase A and methanol containing 0.1% TFA as mobile phase B, with a flow rate of 3 mL/min. The elution program of compound 3a was H2O (0.1% TFA): MeOH (0.1% TFA) = 5:95, and the detection wavelengths were set to 251 nm and 254 nm.
9
Characterization of Drug-Loaded Nanoparticles
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The mean size, polydispersity index (PDI), and zeta potential of LT-NLC, LT-rHDL, GM3-rHDL, and LT-GM3-rHDL nanoparticles were measured by dynamic light scattering (DLS) Analyzer (Zetasizer nano zs90, Malvern, UK) as previously reported. The morphologies of LT-NLC, LT-rHDL, GM3-rHDL, and LT-GM3-rHDL nanoparticles were detected by a transmission electron microscope (JEOL JEM-2100 TEM, Japan) after staining with 2% (w/v) uranyl acetate.
The concentration of LT-loaded in nanoparticles was measured by the HPLC method. A COSMOSIL 5C18-MSII column (250 mm × 4.6 mm) was used at 30 °C. Approximately 80% methanol (v/v) of chromatographic grade was utilized as the mobile phase (flow rate: 1 mL/min). The detected wavelength was 238 nm. The standard curve was achieved by using standard LT solutions (1, 2, 4, 8, and 16 μg/mL). The entrapment efficiency (EE) and drug loading efficiency (DL) of LT-loading nanoparticles were calculated according to the following equations: EE (%) = W/Wt × 100% and DL (%) = Q/Qt × 100%, where W and Q are the amount of LT in each drug carrier whereas Wt and Qt are the total amount of the feeding LT and the feeding materials.
The concentration of LT-loaded in nanoparticles was measured by the HPLC method. A COSMOSIL 5C18-MSII column (250 mm × 4.6 mm) was used at 30 °C. Approximately 80% methanol (v/v) of chromatographic grade was utilized as the mobile phase (flow rate: 1 mL/min). The detected wavelength was 238 nm. The standard curve was achieved by using standard LT solutions (1, 2, 4, 8, and 16 μg/mL). The entrapment efficiency (EE) and drug loading efficiency (DL) of LT-loading nanoparticles were calculated according to the following equations: EE (%) = W/Wt × 100% and DL (%) = Q/Qt × 100%, where W and Q are the amount of LT in each drug carrier whereas Wt and Qt are the total amount of the feeding LT and the feeding materials.
10
Donor-Acceptor Molecule Synthesis
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All the donor-acceptor based molecules were synthesized using standard Suzuki and Stille-coupling protocols as detailed in Supplementary Information . Chemical structures and purity were confirmed by 1H NMR and 13C NMR spectroscopy using a Bruker Avance 400 MHz spectrometer in CDCl3 at room temperature. HPLC analysis was conducted using the Cosmosil 5C18-MS-II column to give the purity information.
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