The separation was performed on a Nova-Pak C18 Column (150 mm × 3.9 mm, 4 μm; Waters, Milford, MA, USA) at 37 °C. The injection volume was 10 μL. The chromatographic conditions were chosen according to a previously reported method [23 (link)], using an Alliance 2695 HPLC system with a 2475 Multi λ Fluorescence detector (Waters, Milford, MA, USA). The concentration of amino acids from samples was identified and quantified by comparison with the amino acid mixture (AAS18; Sigma-Aldrich, St. Louis, MO, USA).
2475 multi λ fluorescence detector
Manufactured by Waters Corporation
Sourced in United States
The 2475 Multi λ Fluorescence Detector is a laboratory instrument designed to measure the fluorescence of samples. It is capable of detecting and quantifying multiple wavelengths of fluorescence simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Nova pak c18 column, by Waters Corporation (4 mentions)
E2695 separation module, by Waters Corporation (4 mentions)
2996 photodiode array detector, by Waters Corporation (3 mentions)
1525 binary hplc pump, by Waters Corporation (3 mentions)
Waters e2695, by Waters Corporation (3 mentions)
515 hplc pump, by Waters Corporation (3 mentions)
Alliance 2695 hplc system, by Waters Corporation (2 mentions)
Accq tag column, by Waters Corporation (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Zorbax rx sil column, by Agilent Technologies (2 mentions)
2695 separations module, by Waters Corporation (2 mentions)
2996 array detector, by Waters Corporation (2 mentions)
Alliance 2695 separations module, by Waters Corporation (2 mentions)
Cerezyme, by Sanofi (2 mentions)
Dpa 6s spe columns, by Merck Group (2 mentions)
2695 separation module, by Waters Corporation (2 mentions)
Alliance system, by Waters Corporation (2 mentions)
Waters 2695 high performance liquid chromatograph, by Waters Corporation (2 mentions)
C18 column, by Waters Corporation (2 mentions)
E2695 hplc system, by Waters Corporation (2 mentions)
45 protocols using 2475 multi λ fluorescence detector
1
Amino Acid Quantification by HPLC
2
Hop Extract Effects on GABA Receptor Expression
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Male ICR mice (25 g, 6-week-old) in the BDZ group were orally administered 0.2 mg/kg and 150 mg/kg of each hop extract (Saaz, Saphir, and hops mixture (Saaz:Saphir = 75:25)) for 3 weeks and sacrificed to measure the receptor expression and GABA levels.
Total RNA was extracted from the brain tissue using the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the Reverse Transcription System (Promega, Madison, WI, USA). The synthesized cDNA was subjected to real-time PCR using the TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA) and a real-time PCR system (7500, Applied Biosystems, Foster, CA, USA). The real-time PCR TaqMan probes used were as follows: GABAA receptor subunit gamma 2 (Gabrg2, NM_008073.3), GABAB receptor 1 (Gabbr1, NM_019439.3), GABAC receptor subunit rho 2 (Gabbr2, NM_008076.3), 5-hydroxytryptamine receptor 1A (Htr1a, NM_008308.4), and GAPDH (NM_001289726.1).
The GABA content in the brain was determined by a HPLC analysis using a Waters AccQ-Tag column (3.9 mm × 150 mm), a Waters 2475 Multi λ Fluorescence Detector (Milford, MA, USA) (250-nm excitation and 395-nm emission wavelengths), and a Waters AccQ-Tag system (Waters Corp., Milford, MA, USA). Mobile phases A, B, and C were water AccQ-tag eluent A (acetate–phosphate buffer), acetonitrile, and Milli-Q water, respectively [32 (link)].
Total RNA was extracted from the brain tissue using the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the Reverse Transcription System (Promega, Madison, WI, USA). The synthesized cDNA was subjected to real-time PCR using the TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA) and a real-time PCR system (7500, Applied Biosystems, Foster, CA, USA). The real-time PCR TaqMan probes used were as follows: GABAA receptor subunit gamma 2 (Gabrg2, NM_008073.3), GABAB receptor 1 (Gabbr1, NM_019439.3), GABAC receptor subunit rho 2 (Gabbr2, NM_008076.3), 5-hydroxytryptamine receptor 1A (Htr1a, NM_008308.4), and GAPDH (NM_001289726.1).
The GABA content in the brain was determined by a HPLC analysis using a Waters AccQ-Tag column (3.9 mm × 150 mm), a Waters 2475 Multi λ Fluorescence Detector (Milford, MA, USA) (250-nm excitation and 395-nm emission wavelengths), and a Waters AccQ-Tag system (Waters Corp., Milford, MA, USA). Mobile phases A, B, and C were water AccQ-tag eluent A (acetate–phosphate buffer), acetonitrile, and Milli-Q water, respectively [32 (link)].
3
Vitamin E Extraction and Quantification in Cake
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Vitamin E in cake samples was extracted and determined referring to Xie et al. [22 (link),23 (link)]. For the saponified cake samples, we extracted a mixture with n-hexane/ethyl acetate (9:1 v/v) and then collected an organic layer. After evaporation, the residues were dissolved in isopropanol (1%) n-hexane solution for HPLC. Equipped with a Waters 2475Multi λ Fluorescence Detector and Waters 515 HPLC pump, an NP-HPLC system was chosen for vitamin E determination. Additionally, an Agilent ZORBAX RX-SIL column was used. By the comparison of retention time between samples and standards, the identification of vitamin E was carried out. The results were reported as µg/100 g sample (mean ± SD, n = 3).
4
Serum Amino Acid Profiling by HPLC
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Serum-free amino acids were analyzed by high-performance liquid chromatography (HPLC) using a reverse-phase C18 column after derivatization with o-phthaldialdehyde reagent using the adjusted method described by Dai et al. [17 (link)]. Briefly, 50 μL of serum was acidified with 200 μL of 1.5 M HClO4, followed by addition of 100 μL of 2 M K2CO3. The neutralized extract was determined for pre-column derivatization with o-phthaldialdehyde on a Waters HPLC system (Model Alliance e2695 Separation Module, Waters, Milford, MA, USA). Fluorescence of amino acid–o-phthaldialdehyde derivatives was detected using a Waters 2475 Multi λ Fluorescence Detector.
5
HPLC Analysis of Zearalenone and Derivatives
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The concentrations of ZEN and its derivatives were determined by HPLC with the use of the Waters 2695 Separations Module, Waters 2475 Multi λ Fluorescence Detector and Waters 2996 Array Detector. Excitation and emission wavelengths were 274 and 440 nm, respectively. The reverse-phase column was C-18 Nova Pak (3.9 × 150 mm), and the mobile phase was acetonitrile-water-methanol (46:46:8, v/v/v) at a flow rate of 0.5 mL·min−1. The mycotoxins were quantified by measuring the retention time of the peaks based on the relevant calibration curves (correlation coefficient of 0.9996 for ZEN, 0.9989 for α-ZEL and 0.9976 for β-ZEL). Detection limits were determined at 0.001 µg·g−1 for ZEN, 0.003 µg·g−1 for α-ZEL and 0.002 µg·g−1 for β-ZEL.
6
Glycosphingolipid Quantification from Brain
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Glycosphingolipids (GSLs) from brain homogenates were extracted with chloroform:methanol (1:2, v/v) overnight at 4°C and further purified using solid-phase C18 columns (Telos, Kinesis, UK). GSLs were dried down under nitrogen and treated with either Cerezyme® (Genzyme, Cambridge, MA) to obtain glucose from GlcCer, or ceramide glycanase (prepared in house from the medicinal leech Hirudo medicinalis/verbena) to obtain oligosaccharides from other GSLs. Liberated glucose and free glycans were then fluorescently-labelled with anthranillic acid (2AA). Excess 2AA label was removed using DPA-6S SPE columns (Supelco, PA, USA). Purified 2AA-labelled glucose and 2AA-labelled glycans were separated and quantified by normal phase high-performance liquid chromatography (NP-HPLC) 34 (link). The NP-HPLC system consisted of a Waters Alliance 2695 separations module and an in-line Waters 2475 multi λ-fluorescence detector set at Ex λ360nm and Em λ425nm. The solid phase used was a 4.6 x 250mm TSK gel-Amide 80 column (Anachem, Luton, UK). Results were normalized to protein content determined using bicinchoninic acid (BCA) assay.
7
Murine Eye Lipid Extraction and HPLC
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Mouse eye cups (4–10 eyes/sample) were homogenized in Dulbecco's phosphate-buffered saline using a tissue grinder in the presence of chloroform/methanol (1:1). Extraction was performed according to a previously published protocol (49 ). Briefly, the sample was extracted three times with addition of chloroform and centrifuged at 1000g for 5 min. After passage through a reversed phase (C18 Sep-Pak; Millipore) cartridge with 0.1% trifluoroacetic acid in methanol, the extract was concentrated by evaporation of solvent under gas and was redissolved in 50% methanolic chloroform (up to 10 eyes/30 ml of solvent). An Alliance system (Waters Corp) equipped with 2695 Separation Module, 2996 Photodiode Array Detector, 2475 Multi λ Fluorescence Detector was used for HPLC analysis. For compound elution, an Atlantis dC18 (3 μm, 4.6 × 150 mm; Waters) reverse phase column was used for the stationary phase and for the mobile phase a gradient of acetonitrile in water with 0.1% trifluoroacetic acid, 75 to 90% acetonitrile (0–30 min), 90 to 100% acetonitrile (30–40 min). 100% acetonitrile (40–80 min) with a flow rate of 0.5 ml/min. Detection by photodiode array was set at 430 and 490 nm.
8
Plasma Metabolites and Oxidative Status
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Glucose (enzymatic-colorimetric method, sensitivity: 0.06 mmol/L) and urea (kinetic method, sensitivity: 0.056 mmol/L) concentrations were determined in plasma with an automatic analyzer (Gernon, RAL S.A, Barcelona, Spain). The mean intra- and interassay CV were 1.5% and 1.9% for glucose and 3.2% and 4.8% for urea, respectively. Plasma BHB (kinetic enzymatic method, sensitivity: 0.100 mmol/L) and NEFA (colorimetric method, sensitivity: 0.072 mmol/L) were determined using Randox kits (Randox Laboratories Ltd., Country Antrim, UK). The mean intra- and interassay CV were respectively 3.3% and 3.7% for NEFA and 6.2% in both cases for BHB. Oxidative status was determined using MDA as a biomarker of lipid peroxidation. This indicator was determined by liquid chromatography using an Acquity UPLC H-Class liquid chromatograph (Waters, Milford, MA, USA) equipped with a silica-based bonded phase column (Acquity UPLC HSS PFP, 100 mm × 2.1 mm × 1.8 μm, Waters), an absorbance detector (Acquity UPLC Photodiode Array PDA eλ detector, Waters) and a fluorescence detector (2475 Multi λ Fluorescence Detector, Waters). The quantification of MDA was done by fluorescence detection at ʎexcitation = 530 nm and ʎemission = 550 nm following the chromatographic conditions described in Bertolín et al. (2019) (link). The mean intra- and interassay CV were 4.6% and 7.3%, respectively.
9
HPLC Analysis of Vitamin A Content
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Vitamin A content was analyzed according to the method described by Cort et al. (1983) (link). The sample was solubilized using H2Cl2 containing 0.001% trimethylamine/acetonitrile/methanol (300/700/0.5) as the mobile phase. The filtrate was transferred to high-performance liquid chromatography (HPLC) equipment (Waters 2475 Multi λ Fluorescence Detector, USA). The flow rate and detection wavelength were kept constant at 0.3 mL/min and 280 nm, respectively. Vitamin A content was calculated from a calibration curve using a vitamin A standard.
10
Quantification of Biological Compounds
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The tissues were extracted and homogenized in ice-cold 0.1 M perchloric acid (v/w = 30:1) and immediately centrifuged at 15, 000 g for 15 min at 4 °C. The supernatant was collected and prepared for HPLC analysis or stored at -80 °C for later HPLC analysis with fluorometric detection. The column used was a REPROSIL-PUR 120 C18 5 μm (4.6 × 250.0 mm) from ThermoFisher. The mobile phase consisted of 0.05 M citric acid, 0.05 M sodium acetate and 0.5 mM sodium EDTA, 13% methanol, pH 3.1; the flow rate was 0.4 ml/min. The excitation wavelength used was 280 nm and the emission wavelength was 330 nm; the sample injection volume was 20 μL. The equipment included a Waters 717 Plus Autosampler System, a Waters 1525 Binary HPLC Pump and a Waters 2475 Multi λ Fluorescence Detector.
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