The SHIMADZU HPLC system consisted of a CBM-20A communication bus module, a DGU-20A3R degassing unit, and two LC-20AD pumps. Electrochemical detection was performed using an amperometric detector ED723 coupled with three electrodes including a diamond working electrode with a surface area of 1.44 cm2, an Ag/AgCl reference electrode and a stainless steel counter electrode. The flow cell is a thin layer type with the volume of 1.5 μL. Separation of analytes was performed on a Hypersil ODS2 column (250 mm × 4.6 mm, 5.0 μm particle size, Elite analysis instrument co., LTD, Dalian, China) fitted with a C18 security guard cartridge (phenomenex, American) at a flow rate of 1.0 mL/min. The mobile phase was composed of an aqueous solution and acetonitrile in the ratio of 90/10. The aqueous portion contained 25 mmol/L sodium acetate, 25 mmol/L citric acid, 0.01 mmol/L EDTA-2Na and 1.0 mmol/L OSA, adjusting pH to 3.5 with acetic acid. The mobile phase was vacuum-filtered through a 0.22 μm cellulose acetate membrane and degassed for 10 min. The column oven temperature was set at 30 °C and the injection volume was 20 μL. The analytical potential of the detector was set at 700 mV within the output range of 10 μA. The chromatograms were integrated with Shimadzu Software.
C18 security guard cartridge
Manufactured by Phenomenex
Sourced in United States, Germany
The C18 security guard cartridge is a laboratory equipment product designed to protect analytical columns from contaminants. It functions as a pre-column filtration device, capturing unwanted substances before they reach the primary analytical column, thereby extending the column's lifespan and improving the quality of analytical results.
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Lab products found in correlation
Ltq xl linear ion trap instrument, by Thermo Fisher Scientific (2 mentions)
Finnigan surveyor plus system, by Thermo Fisher Scientific (2 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Kinetex c18 column, by Phenomenex (2 mentions)
Hplc grade, by Merck Group (2 mentions)
Luna c18 2, by Phenomenex (2 mentions)
Hp 1100 lc ms system, by Agilent Technologies (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
C18 column, by Phenomenex (1 mentions)
Stereotaxic instrument, by Stoelting (1 mentions)
Hplc system, by Hitachi (1 mentions)
Cosmosil 5c18 ar 2 column, by Nacalai Tesque (1 mentions)
1260 infinity 2 lc system, by Agilent Technologies (1 mentions)
Eclipse xdb c18, by Agilent Technologies (1 mentions)
Gemini c18 column, by Phenomenex (1 mentions)
1290 series uhplc system, by Agilent Technologies (1 mentions)
Hypersil ods, by Agilent Technologies (1 mentions)
Amx300 nmr spectrometer, by Bruker (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
19 protocols using c18 security guard cartridge
1
HPLC-ECD Analysis of Analytes
2
Silybin and Conjugates Quantification
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The analysis for the determination of the concentration of silybin and its glucuronides and sulfates was performed on an Agilent HP 1100 LC-MS system (Agilent, CA, United States) with Waters ACQUITY UPLC® BEH C18 Column (1.7 μm, 2.1 × 50 mm, Waters Corp., Ireland) protected by a C18 Security Guard cartridge (4 × 2.0 mm i.d., Phenomenex, Torrance, CA, United States). Silybin A and B were well separated using optimized gradient elution with 0.1% formic acid in water (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.4 ml/min within 10 min at room temperature (22°C). The gradient elution was performed as follows (time: mobile phase B percentage): 0 min: 25%, 6 min: 40%, 6.5 min: 25%.
Mass spectrometry parameters: capillary voltage, -4000 V; drying gas, 9 L/min; drying gas temperature, 325°C; nebulizer pressure, 40 psi; fragment voltage, 35 V; dwell time, 200 ms; scan mode, selective ion monitoring (SIM) with [M-H]- for silybin (m/z 481), mono-glucuronide (m/z 657), and NG (m/z 271). The LC-MS data were collected by Agilent ChemStation Software.
Mass spectrometry parameters: capillary voltage, -4000 V; drying gas, 9 L/min; drying gas temperature, 325°C; nebulizer pressure, 40 psi; fragment voltage, 35 V; dwell time, 200 ms; scan mode, selective ion monitoring (SIM) with [M-H]- for silybin (m/z 481), mono-glucuronide (m/z 657), and NG (m/z 271). The LC-MS data were collected by Agilent ChemStation Software.
3
Depletion of High Abundance Proteins
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The preparation and use of the immunoaffinity column is described in [17 ]. To deplete samples of the 7 high abundance proteins, 100 μL of RPMI supernatant was diluted with 100 μL Tris-buffered saline (TBS, 0.1 M TRIS-base containing 0.1 M NaCl, pH 8.0), and the solution was injected into a TBS solution with a flow of 0.2 mL/min. The nonretained proteins were trapped on a 4 mm × 2.0 mm (inner diameter, i.d.), C18 security guard cartridge with 300 Å pore size (Phenomenex, Teknolab, Norway) and were eluted by backflushing the security guard cartridge with ethanol at a flow of 0.3 mL/min. The affinity column was washed using 0.1 M glycine at pH 2.5 with a flow of 1.2 mL/min. Both columns were reequilibrated with TBS at a flow of 0.2 mL/min for 5 minutes. The pH adjustments were done using 6 M HCl.
4
Mycotoxin Quantification in Extracts
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Sample preparation, detection and quantification were performed as described by Malachová et al. (2014 ). Briefly, the extraction solvent (acetonitrile/water/acetic acid; 79/20/1) was added to dried ethyl acetate extract and after shaking and centrifugation, the extract was injected into LC-MS/MS equipped with a TurboV electrospray ionization (ESI) source. The Phenomenex C18-column (150 × 4.6 mm, 5 μm) fitted with a C18 security guard cartridge (4 × 3 mm) was used to separate the compounds. The mobile phases consisted of methanol/water/acetic acid with the ratio of 10/89/1 (v/v/v) for eluent A and 92/2/1 (v/v/v) for eluent B. Both eluents contain 5 mM ammonium acetate.
5
HPLC Analysis of Primary Amino Acids
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Primary amino acids were analyzed using a modified version of a previously reported method [167] (link). A Hewlett-Packard (Agilent Technologies Massy, France®) 1100 179 series HPLC instrument was used, with a G1321A fluorescence detector set at excitation and emission wavelengths of 330 nm and 440 nm, respectively. Separations were carried out on a 150 mm×3 mm Macherey Nagel Durabond® column 5 µm dp, protected by a 1 mm C18 SecurityGuard® cartridge supplied by Phenomenex (France). Mobile phase A consisted of 95% 0.05 M acetate buffer, pH 6.5 and 5% methanol:acetonitrile [1∶1] filtered under vacuum using a 0.22 µm nylon membrane. Mobile phase B consisted of methanol:acetonitrile [1∶1]. Separations were carried out at 40°C with a flow rate of 0.5 ml/min. As proline does not react with OPA, a new high-throughput spectrophotometric method was developed and validated for its analysis. Briefly, the method involves reacting the sample with ninhydrin in DMSO and formic acid at 100°C for 15 minutes to yield a salmon pink reaction product. Under these conditions, primary amino acids do not react with ninhydrin and thanks to the particular solvent composition, the extraction and centrifugation steps reported in similar methods are avoided.
6
Hippocampal Adenosine Measurement via Microdialysis
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The microdialysis experiment was performed according to the methods described by Lee et al. (2018) (link). Mice were anesthetized by i.p. injection with ketamine/xylazine and fixed on a stereotaxic instrument (Stoelting, Wood Dale, IL, United States). A vertical guide cannula was stereotaxically implanted into the hippocampus (anteroposterior, ±2.8 mm; mediolateral, ±3.0 mm; dorsoventral, −2.3 mm). After 3 days cannulation, a microdialysis probe (MAB 10.8.2.Cu, Microbiotech/se AB, Stockholm, Swedish) was inserted into the mouse brain through the guide cannula and infused with Ringer’s solution (1 μl/min) for 4 h. The brain outflow was collected every 30 min. The samples were frozen at −20°C until assayed. For adenosine measurements, the adenosine was first converted to fluorescent 1,N6-etheno-adenine derivatives. The supernatant was then injected into an HPLC system (Hitachi, Tokyo, Japan) and tested using a COSMOSIL 5C18-AR-II column (5 μm, 250 × 4.6 mm, Nacalai Tesque, Inc., Kyoto, Japan) equipped with a C18 SecurityGuard cartridge (Phenomenex, Torrance, CA, United States).
7
HPLC Analysis of Fasudil Quantification
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Fasudil was quantified by high performance liquid chromatography (HPLC) using a 1260 Infinity II LC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a binary pump and a diode array detector. Analyses were performed using a reverse phase C18 column (150 mm × 5 mm; 5-µm microsphere size; Eclipse XDB-C18, Agilent Technologies, Inc., Santa Clara, CA, USA) preceded by a C18 security guard cartridge (Phenomenex, Inc., Torrance, CA, USA) at 40 °C. The mobile phase was composed of ultrapure water (eluent A) and methanol (eluent B; HPLC grade, Merck, Darmstadt, Germany), both containing 0.03% (v/v) trifluoroacetic acid (HPLC grade, Sigma-Aldrich GmbH, Taufkirchen, Germany). Fasudil was eluted by a gradient at a flow rate of 0.8 mL/min. The gradient used was as follows: 0.0–0.5 min constant at 85% (v/v) eluent A; 0.5–6.5 min 85–40% (v/v) eluent A (linear gradient); 6.5–7.0 min changed to 5% (v/v) eluent A; 7.0–17.0 min constant at 5% (v/v) eluent A; 17.0–17.10 min changed to 85% (v/v) eluent A; 17.10–27.10 min constant at 85% (v/v) eluent A. The injection volume was 5 µL for encapsulation efficiency (% EE) measurements and 10 to 12 µL for in vitro release studies. Each sample was injected three times. The absorbance of fasudil was measured at λ = 320 nm.
8
Quantitative UHPLC-MS/MS analysis
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The samples derived from YES plates was followed by dilution of 1 + 9 in acetonitrile/water/acetic acid 49.5/49.5/1 (v/v/v). Further dilutions of 1 + 49 and 1 + 999 were performed and re-analyzed in cases of distorted peak shapes due to column overloading caused by large analyte concentrations. The method used in this study is an extension of the version described in detail elsewhere [30 (link),31 (link),32 ]. Briefly, a QTrap 5500 MS/MS system (Sciex, Foster City, CA, USA) equipped with a TurboV electrospray ionization (ESI) source was coupled to a 1290 series UHPLC system (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25 °C on a Gemini C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (both Phenomenex, Torrance, CA, USA). Two MS/MS transitions were acquired per analyte with the exception of moniliformin and 3-nitropropionic acid that yield only one product ion. For confirmation of a positive identification, the ion ratio had to agree with the related values of the related authentic standard within 30% as stated in official guidelines [32 ], whereas for the retention time, a more strict in-house criterion of ± 0.03 min was applied.
9
LC–MS Analysis of Memantine and Amantadine
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LC–MS analysis was performed using a Finnigan Surveyor plus system (Thermo Scientific, San Jose, CA, USA), equipped with a quaternary MS pump with an integrated degasser and an autosampler with an integrated column oven. The chromatographic separation was performed on a Kinetex C18 column (5 µm, 50 mm × 4.6 mm ID) preceded by a C18 security guard cartridge (4.0 mm × 3.0 mm ID), both from Phenomenex (Torrance, CA, USA). Separation was attained using an isocratic elution with the flow rate of 0.5 ml/min. The column and tray temperature was set at 25°C and 10°C, respectively. The mobile phase consisted of 0.5% formic acid in water and methanol (45:55 v/v). The run time of the analysis was 3 min.
Mass spectrometry was performed on an LTQ XL linear ion trap instrument (Thermo Scientific, San Jose, CA, USA), coupled with heated electrospray ionization (HESI-II) probe operated in the positive ion mode. After optimization, the parameters in the source were set as follows: source voltage of 4.5 kV, source heater temperature of 250°C, sheath gas flow of 30 arb, and auxiliary gas flow of 10 arb. The mass spectrometer was operated in the selected reaction monitoring (SRM) channels. Fragment ion m/z 180 → 163 was used for memantine quantification, while m/z 152 → 135 was used for amantadine quantification. Thermo Fisher Xcalibur software was used for the analysis.
Mass spectrometry was performed on an LTQ XL linear ion trap instrument (Thermo Scientific, San Jose, CA, USA), coupled with heated electrospray ionization (HESI-II) probe operated in the positive ion mode. After optimization, the parameters in the source were set as follows: source voltage of 4.5 kV, source heater temperature of 250°C, sheath gas flow of 30 arb, and auxiliary gas flow of 10 arb. The mass spectrometer was operated in the selected reaction monitoring (SRM) channels. Fragment ion m/z 180 → 163 was used for memantine quantification, while m/z 152 → 135 was used for amantadine quantification. Thermo Fisher Xcalibur software was used for the analysis.
10
LC-MS Analysis of Memantine and Amantadine
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LC-MS analysis was carried out using a Finnigan Surveyor plus system (Thermo Scientific, San Jose, CA, USA), consisting of a quaternary MS pump with integrated degasser and an auto sampler with integrated column oven. The chromatographic separation was performed on a Kinetex C18 column (5 μm, 50 mm × 4.6 mm I.D.) preceded by a C18 security guard cartridge (4.0 mm × 3.0 mm I.D.) both by Phenomenex (Torrance, CA, USA). Separation was attained using an isocratic elution with a flow rate of 0.5 mL.min-1. The column and tray temperature were set at 25 and 10°C, respectively. The mobile phase consisted of 0.5% formic acid in water and methanol (45:55 v/v). The run time of analysis was 3 min.
Mass spectrometry was performed on an LTQ XL linear ion trap instrument (Thermo Scientific, San Jose, CA, USA), coupled with heated electrospray ionization (HESI-II) probe operated in positive mode. After optimization, the parameters in the source were set as follows: source voltage 4.5 kV, source heater temperature 250°C, sheath gas flow 30 arb and auxiliary gas flow 10 arb. The mass spectrometer was operated in selected reaction monitoring (SRM) mode. The fragment ions m/z 180 → 163 and m/z 152 → 135 were used for quantification of memantine and amantadine, respectively. Thermo Fisher Xcalibur software was used for the analysis.
Mass spectrometry was performed on an LTQ XL linear ion trap instrument (Thermo Scientific, San Jose, CA, USA), coupled with heated electrospray ionization (HESI-II) probe operated in positive mode. After optimization, the parameters in the source were set as follows: source voltage 4.5 kV, source heater temperature 250°C, sheath gas flow 30 arb and auxiliary gas flow 10 arb. The mass spectrometer was operated in selected reaction monitoring (SRM) mode. The fragment ions m/z 180 → 163 and m/z 152 → 135 were used for quantification of memantine and amantadine, respectively. Thermo Fisher Xcalibur software was used for the analysis.
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