Mouse plasma and brain samples were analyzed for EVG concentration, using the same LC–MS/MS method as described before [30 (link)]. Briefly, EVG and internal standard (RTV) were quantified, using a tandem mass spectrometer AB SCIEX Triple Quad 5500 that was equipped with electron spray ionization. The isolation of analyzed compounds was conducted in a Shimadzu liquid chromatographic system (Kyoto, Japan). The sample extraction was performed by adding 9-volume of cold acetonitrile to an aliquot of plasma or brain homogenate. The multiple reactions monitoring (MRM) transitions (m/z) Q1/Q3 selected for quantitative analyses were 447.9/343.8 for EVG and 721.3/296.1 for the RTV.
Liquid chromatographic system
Manufactured by Shimadzu
Sourced in Japan
The Liquid chromatographic system is a laboratory instrument used for the separation, identification, and quantification of chemical compounds in a liquid mixture. It functions by passing the sample mixture through a column filled with a stationary phase, where the compounds are separated based on their interactions with the stationary phase and a mobile phase. The separated compounds are then detected and quantified by various detection methods, such as UV-Vis, mass spectrometry, or refractive index detection.
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Lab products found in correlation
Sil 20a ht, by Shimadzu (1 mentions)
Luna c18 column, by Thermo Fisher Scientific (1 mentions)
Cbm 20a system controller, by Shimadzu (1 mentions)
Cto 10 as vp column, by Shimadzu (1 mentions)
Lc 20ad solvent, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Lc 20at pump, by Shimadzu (1 mentions)
Zorbax extend c18 column, by Agilent Technologies (1 mentions)
Lc 20at solvent delivery module, by Shimadzu (1 mentions)
1800 double beam uv vis spectrophotometer, by Shimadzu (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
6 protocols using liquid chromatographic system
1
Quantification of EVG in Mouse Plasma and Brain
2
Quantification of MS using HPLC-UV
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Samples were analysed using a liquid chromatographic system (Shimadzu, Kyoto, Japan) equipped with a CBM-20A system controller connected to an LC-20AD solvent delivery unit, DGU-20A3 degasser system, CTO-10ASVP column oven, SIL-20AHT autosampler and SPD-20A ultraviolet-visible detector. The isocratic mobile phase used was methanol and 0.1% of trifluoroacetic acid in distilled water at a volume ratio of 85:15 and a flow rate of 1 mL/min. The mobile phase was filtered through a 0.45 µm nylon membrane filter under a vacuum condition and sonicated before use. A Phenomenex® Luna C18 column (250 × 4.6 mm ID, 5 μm; Torrance, CA, USA) fitted with a Thermo Scientific Uniguard™ guard column (Waltham, Boston, MA, USA) with an ODS C18 cartridge (10 × 4 mm ID, 5 μm) was used as stationary phase. UV detection was set at 305 nm and a sample injection volume of 40 μL was used with a column temperature of 40 °C. An internal standard of MS was used during method development and validation. The retention time of MS was ~4.6 min. The limits of detection and quantification for MS were 0.08 and 0.25 µg/mL for a low drug concentration range of 0.25–7 µg/mL, respectively, as well as 4.20 and 12.72 µg/mL for a high drug concentration range of 5–150 µg/mL, respectively.
3
HPLC Profiling of Phytochemicals
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HPLC profiling was performed in the Shimadzu Liquid Chromatographic System (Tokyo, Japan), consisting of an LC-20AT pump and a double UV-VIS spectrophotometric detector. The eluents were monitored at 330 nm at 30°C. The isocratic mobile phase consisted of methanol −0.1% formic acid (80 : 20, v/v) and ran at a flow rate of 1.0 ml/min. Chromatographic separation was performed on a Diamonsil C18 column (4.6 mm × 250 mm, particle size 5 μm, Beijing Dikma Science and Technology Co., Ltd., Beijing, China).
4
Quantification of AS-IV in Huang Qi Injection
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The concentration of AS-IV in Huang Qi injection (HQI) was determined using a liquid chromatographic system from Shimadzu Instruments (Kyoto, Japan). The system consisted of an LC-10AVP binary pump and an evaporative light-scattering detector, connected to a computer system (for data acquisition) from LC-Solution. The analytical column used was the Agilent Zorbax Extend C18 column (250 mm × 4.6 mm, 5 μm) from Agilent Corporation. The mobile phase was a mixture of acetonitrile and water in a ratio of 36 : 64 at a flow rate of 1.0 mL/min. The injection volume, detector temperature, and pressure were 10 μL, 40°C, and 0.35 MPa, respectively.
5
Liquid Chromatography Analytical Protocol
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Liquid chromatographic system (Shimadzu Corporation, Japan) equipped with dual LC-20 AT solvent delivery modules connected with DGU-20A3/20A5 on-line degasser, tted with rheodyne manual injector connected with SPD-20A/20AV UV/VIS detector and Shimadzu CBM-20A communication bus module.
Data acquisition was performed on LC solution GPC Chromatographic software (version 1.25). λ max was measured on Shimadzu-1800 double beam UV/vis spectrophotometer. Rotary evaporator (Heidolph G 3 Germany) was used to concentrate the sample.
Data acquisition was performed on LC solution GPC Chromatographic software (version 1.25). λ max was measured on Shimadzu-1800 double beam UV/vis spectrophotometer. Rotary evaporator (Heidolph G 3 Germany) was used to concentrate the sample.
6
HPLC-DAD Analysis of Citrus Flavonoids
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HPLC-DAD analysis was performed on a Shimadzu liquid chromatographic system (Kyoto, Japan) equipped with a Rheodyne injection valve with a 1 μL fixed loop, an SCL-10 AVP pump, and an autosampler. The flow rate was 0.3 mL/min, and a thermostated column oven was used with a reverse-phase C18 Purospher STAR tracer 3 μm (55 × 4 mm) (Merck, Germany) column with sample and column temperatures of 5 and 25°C, respectively. Compounds were monitored at 240 and 280 nm using a UV-Vis photodiode array detector (Shimadzu model SPD-MD), and UV spectra were recorded from 200 to 400 nm and were controlled by a GPC/LC solution workstation (version 1.21 SP1).
A gradient mobile phase consisting of methanol (solvent A) and acid H2O (1.0% acetic acid) (solvent B) was used for HPLC analysis. The conditions were as follows: initial conditions of 20% A for 5 min, followed by an increase to 60% A for 10 min, 80% A for 20 min, and 60% A in the following 40 min, and then back to the initial conditions for 10 min. The total run time was 50 min at 0.3 mL/min. Compounds were identified by comparing their retention times (Rt NAR = 9.0 min; Rt HESP = 15.0 min; Rt PON = 17.0 min; Rt NARG = 22.0) and their UV-vis spectra with corresponding standards. Concentrations were determined using an external standard curve.
A gradient mobile phase consisting of methanol (solvent A) and acid H2O (1.0% acetic acid) (solvent B) was used for HPLC analysis. The conditions were as follows: initial conditions of 20% A for 5 min, followed by an increase to 60% A for 10 min, 80% A for 20 min, and 60% A in the following 40 min, and then back to the initial conditions for 10 min. The total run time was 50 min at 0.3 mL/min. Compounds were identified by comparing their retention times (Rt NAR = 9.0 min; Rt HESP = 15.0 min; Rt PON = 17.0 min; Rt NARG = 22.0) and their UV-vis spectra with corresponding standards. Concentrations were determined using an external standard curve.
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