Biogenic amine concentrations were measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) 48 (link). Briefly, mice were sacrificed by decapitation and the striatum was quickly removed. Striatal tissue was weighed and sonicated in 0.2 ml ice cold 0.01 mM perchloric acid containing 0.01 % EDTA and 60 ng 3,4-dihydroxybenzylamine (DHBA) as an internal standard. After centrifugation (15,000 × g, 30 min, 4°C), the supernatant was passed through a 0.2 μm filter. Twenty microliters of the supernatant were analyzed in the HPLC column (3 mm x150 mm C-18 reverse phase column, Acclaim™ Polar Advantage II, Thermo Scientific, USA) by a dual channel coulochem III electrochemical detector (Model 5300, ESA, Inc Chelmsford, MA, USA). The protein concentrations of tissue homogenates were measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Data were normalized to protein concentrations and expressed in ng ug−1 protein.
Acclaim polar advantage 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Acclaim Polar Advantage II is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of polar and ionizable compounds. It features a proprietary stationary phase that provides enhanced selectivity and resolution for a wide range of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Savant rvt5105, by Thermo Fisher Scientific (1 mentions)
Savant sdp121 p, by Thermo Fisher Scientific (1 mentions)
Kojic acid, by Merck Group (1 mentions)
Chloroform d, by Merck Group (1 mentions)
Triart c18, by YMC (1 mentions)
Methanol d4, by Merck Group (1 mentions)
Acetone d6, by Merck Group (1 mentions)
N acetyl l tyrosine, by Merck Group (1 mentions)
L tyrosine, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Eclipse xdb c18, by Agilent Technologies (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Agilent 1290 hplc system, by Agilent Technologies (1 mentions)
Thermo scientific c18 column, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Compass dataanalysis 4, by Bruker (1 mentions)
Microtof q 3, by Bruker (1 mentions)
8 protocols using acclaim polar advantage 2
1
HPLC-ECD Biogenic Amine Quantification
2
HPLC-ECD Quantification of Biogenic Amines
3
Quantifying Neuronal Monoamine Levels
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Neuron culture medium was replaced by Hanks' balanced saline solution buffer with the addition of 56 mM KCl and incubated for 15 minutes at 37°C. The medium was collected and centrifuged to clear cell debris. Neuron pellet was also collected. Samples were immediately frozen in liquid nitrogen and stored at −80°C. For HPLC analysis, samples were thawed and concentrated using a vacuum (Savant SDP 121P, Thermo Fisher Scientific) connected with refrigerated vapor trap (Savant RVT 5105, Thermo Fisher Scientific), and the freeze‐dried samples were resuspended in 10 mM perchloric acid. Monoamines were analyzed by HPLC electrochemical detection by dual channel Coulochem III electrochemical detector (model 5300; ESA Inc., Chelmsford, MA, http://www.esainc.com ), and monoamines were separated by using a reverse phase C18 column (3‐mm 3 150‐mm C‐18 RP‐column; Acclaim Polar Advantage II; Thermo Fisher Scientific) with a flow rate of 0.600 ml/minute. Monoamine concentrations were quantified by comparison of the area under the curve to known standard dilutions.
4
Isolation and Characterization of Metabolites from A. fruticosa Seeds
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The
seeds of A. fruticosa (2 kg) [imported
from The People’s Republic of China] were purchased from a
market and extracted to isolate the metabolites. Organic solvents
for the extraction, separation, and isolation (water, methanol, butanol,
acetonitrile, ethyl acetate, chloroform, and n-hexane)
were bought from Duksan (Gyeonggi-do, South Korea). The solvents used
for HPLC and medium-pressure liquid chromatography (MPLC) were of
analytical grade. The analytical columns that were used for MPLC were
a Triart C18 (250 mm × 20.0 mm, S-5 μm, 12 nm, YMC, Kyoto,
Japan) and an Acclaim Polar Advantage II (250 mm × 20 mm, 5 μm,
Thermo Scientific, Waltham, MA), and for HPLC, an Eclipse XDB-C18
(150 mm × 4.6 mm, 5 μm, Agilent Technologies Inc., Santa
Clara, CA) column was used. Tyrosinase from mushroom,l -tyrosine, l -3,4-dihydroxyphenylalanine (l -DOPA), N-acetyl-l -tyrosine, kojic acid, DMSO, and NMR solvents (chloroform-d, methanol-d4, and acetone-d6) were purchased from Sigma Aldrich (St. Louis,
MO).
seeds of A. fruticosa (2 kg) [imported
from The People’s Republic of China] were purchased from a
market and extracted to isolate the metabolites. Organic solvents
for the extraction, separation, and isolation (water, methanol, butanol,
acetonitrile, ethyl acetate, chloroform, and n-hexane)
were bought from Duksan (Gyeonggi-do, South Korea). The solvents used
for HPLC and medium-pressure liquid chromatography (MPLC) were of
analytical grade. The analytical columns that were used for MPLC were
a Triart C18 (250 mm × 20.0 mm, S-5 μm, 12 nm, YMC, Kyoto,
Japan) and an Acclaim Polar Advantage II (250 mm × 20 mm, 5 μm,
Thermo Scientific, Waltham, MA), and for HPLC, an Eclipse XDB-C18
(150 mm × 4.6 mm, 5 μm, Agilent Technologies Inc., Santa
Clara, CA) column was used. Tyrosinase from mushroom,
MO).
5
Bark Extract Analysis via LC-MS
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350 mg of dried wood powder from bark samples were mixed separately at room temperature with 30 ml of methanol/water solution (ratio 4:1, v/v). After 3 h of stirring, the supernatant was recovered, then analyzed via LC-DAD (Ultimate 3000, Thermo Scientific), using an Acclaim Polar Advantage II (Thermo Scientific) in methanol (A)/water (B) gradient mode at 1 ml/min (injection volume 10 μl). The gradient elution program was set as follows: 0–9 min. (95% B), 9–16 min. (75% B), 16–25 min. (60% B), 25–35 min. (50% B), 35–52 min. (0.0% B), 52–62 min. (95% B). Then, the elution gradient was linearly ramped down to 60% A for 2 min and so maintained for 9 min to condition the column for the next injection. The column was connected to an electrospray hybrid linear ion Orbitap mass analyzer (LTQ Orbitrap Velos, Thermo-Fisher, Bremen, Germany). The electrospray spray voltage was 3.8 kV. The LC-MS analysis was used in negative ion detection mode. Data were analyzed with the Xcalibur software.
6
HPLC-DAD Method for Nitrosamine Quantification
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The N-nitrosamine
concentrations were measured using an Agilent 1290 HPLC system coupled
with a diode array detector (DAD) at 230 nm and a C18 column
(Acclaim Polar Advantage II, 120 Å, 5 μm, 4.6 mm ×
150 mm, Thermo Scientific).37 (link) The mobile
phase consisted of HPLC-grade acetonitrile (phase A) and 10 mM phosphoric
acid (phase B) at a flow rate of 0.8 mL/min. The gradient elution
procedure started from 98% A for the first 6 min, then decreased to
20% in the following 12 min, and was kept for 5 min; finally, it was
increased to 98% A again within 5 min and maintained for 4 min.
concentrations were measured using an Agilent 1290 HPLC system coupled
with a diode array detector (DAD) at 230 nm and a C18 column
(Acclaim Polar Advantage II, 120 Å, 5 μm, 4.6 mm ×
150 mm, Thermo Scientific).37 (link) The mobile
phase consisted of HPLC-grade acetonitrile (phase A) and 10 mM phosphoric
acid (phase B) at a flow rate of 0.8 mL/min. The gradient elution
procedure started from 98% A for the first 6 min, then decreased to
20% in the following 12 min, and was kept for 5 min; finally, it was
increased to 98% A again within 5 min and maintained for 4 min.
7
Biogenic Amine Quantification by HPLC-ECD
8
Ethanolic Extract Separation and Characterization
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A. sessilis ethanolic extract was separated using Thermo Scientific C18 Column (3 × 150 mm, 3 μm particle size; AcclaimTM Polar Advantage II, Thermo Scientific, USA) with an UltiMate 3000 UHPLC System (Dionex, Thermo Fisher Scientific, USA). Gradient elution was run at this setting: 0.4 mL/min and 40°C using water and 0.1% formic acid (A) and 100% acetonitrile (B) with a total run time of 22 mins. The sample was injected at a volume of 1 μL. The gradient began at 5% B (0–3 mins); 80% B (3–10 mins); 80% B (10–15 mins); and 5% B (15–22 mins). High-resolution mass spectrometry was performed with micrOTOF-QIII (Bruker Daltonics GmbH, Germany) using an ESI-positive ionization and the following conditions: capillary voltage: 4500 V; nebulizer pressure: 1.2 bar; and drying gas: 8 L/min at 200°C. The mass range was set at 50–1000 m/z. The mass data of molecular ions were then processed and analyzed using Compass Data Analysis 4.1 software (Bruker Daltonics GmbH, Germany).
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A. sessilis ethanolic extract was separated using Thermo Scientific C18 Column (3 × 150 mm, 3 μm particle size; AcclaimTM Polar Advantage II, Thermo Scientific, USA) with an UltiMate 3000 UHPLC System (Dionex, Thermo Fisher Scientific, USA). Gradient elution was run at this setting: 0.4 mL/min and 40°C using water and 0.1% formic acid (A) and 100% acetonitrile (B) with a total run time of 22 mins. The sample was injected at a volume of 1 μL. The gradient began at 5% B (0–3 mins); 80% B (3–10 mins); 80% B (10–15 mins); and 5% B (15–22 mins). High-resolution mass spectrometry was performed with micrOTOF-QIII (Bruker Daltonics GmbH, Germany) using an ESI-positive ionization and the following conditions: capillary voltage: 4500 V; nebulizer pressure: 1.2 bar; and drying gas: 8 L/min at 200°C. The mass range was set at 50–1000 m/z. The mass data of molecular ions were then processed and analyzed using Compass Data Analysis 4.1 software (Bruker Daltonics GmbH, Germany).
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