For extraction of nicotianamine, leaf tissue was homogenized in liquid nitrogen and extracted in ddH2O, incubated at 80 °C for 30 min and after centrifugation for 10 min at 16,000× g the supernatant was filtered and used for HPLC analysis, similarly as in Mendoza-Cózatl et al. [61 (link)]. For histidine determination, leaf tissue was homogenized in liquid nitrogen and extracted in 50% methanol, centrifuged at 4 °C and the filtered supernatant was used for analysis. Analysis of NA and His was performed according to Vasanits et al. [62 (link)] using o-phthaldialdehyde and 3-mercaptopropionic acid (OPA/MPA). Ten times diluted samples were derivatized with OPA/MPA for 5 min and immediately loaded on a reversed phase column (5.0 μm, 250 mm × 4.6 mm Luna C18 (2); Phenomenex Ltd., Torrance, CA, USA) using a Shimadzu LC-20AB Prominence liquid chromatograph (Shimadzu, Kyoto, Japan). The elution gradient was as described in Vasanits et al. [62 (link)] and the flow rate was 1.2 mL min−1 at 40 °C. The fluorescence intensity of OPA/MPA/AA derivative was measured at excitation and emission wavelengths of 337 and 454 nm using RF-10-AXL, fluorescence detector (Prominence, Shimadzu, Kyoto, Japan). The NA and His standards were obtained from Toronto Research Chemicals (North York, ON, Canada) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
Rf 10axl fluorescence detector
Manufactured by Shimadzu
Sourced in Japan, Germany, United States
The RF-10AXL fluorescence detector is a laboratory instrument designed to measure the fluorescence intensity of samples. It is capable of detecting fluorescent compounds and can be used in various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
Pcp cy3, by Jena Biosciences (2 mentions)
Class vp software, by Shimadzu (2 mentions)
Lc 20ab prominence liquid chromatograph, by Shimadzu (1 mentions)
Luna c18 2, by Phenomenex (1 mentions)
Cadenza cd c18 column, by Imtakt (1 mentions)
Whatman, by Cytiva (1 mentions)
Mini uniprep, by Cytiva (1 mentions)
Lc 20ad hplc system, by Shimadzu (1 mentions)
F 7000 fluorescence spectrophotometer, by Hitachi (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
Rid 10a refractive index detector, by Shimadzu (1 mentions)
Lc 10advp pump, by Shimadzu (1 mentions)
C 18 reversed phase column, by Macherey-Nagel (1 mentions)
Tryptophan, by Merck Group (1 mentions)
Xanthurenic acid, by Merck Group (1 mentions)
Uv 1601 pc spectrophotometer, by Shimadzu (1 mentions)
Rf 5301 pc fluorimeter, by Shimadzu (1 mentions)
3 hydroxykynurenine, by Merck Group (1 mentions)
Kynurenine, by Merck Group (1 mentions)
37 protocols using rf 10axl fluorescence detector
1
Nicotianamine and Histidine Quantification
2
LVFX Quantification in Plasma using HPLC
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After mixing 300 μL of acetonitrile solution containing 10.0 μg/mL norfloxacin as the internal standard with 300 μL of plasma, the solution was centrifuged at 10 600 × g for 10 min at 4 °C and the supernatant was separated. Then, 300 μL of water was added to 150 μL of the supernatant, and the mixture was filtered using Whatman Mini-UniPrep (0.45 μm; J.G Finneran Associates, Inc., USA). Subsequently, 25 μL of this filtrate was subjected to high-performance liquid chromatography (HPLC) to measure the concentration of LVFX. The conditions for HPLC analysis were as follows: column, Cadenza CD-C18 column (4.6 mm × 150 mm, 3 μm; Imtakt Corp., Kyoto, Japan); mobile phase, 10 mM phosphate buffer solution containing 2% trimethylamine (pH 7.0)/acetonitrile = 85:15 (v/v); and flow rate, 1.0 mL/min. The measurements were taken at excitation and emission wavelengths of 292 and 494 nm, respectively, using Shimadzu, RF-10AXL fluorescence detector (Shimadzu Corp.). The retention times based on these conditions were 3.5 and 10 min for norfloxacin and LVFX. In addition, the Cmax was defined as the maximum plasma LVFX concentration at 180 min after oral administration and Tmax was defined as the time required to reach the Cmax.
3
HPLC Separation of Doxorubicin and Prodrug
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The HPLC separation was conducted on an LC-20AD HPLC system (Shimadzu, Japan) with an RF-10AXL fluorescence detector (Shimadzu, Japan), and a manual injector matched up with a 20 μL sample loop. The separation was conducted on a Sepax Technologies Ameththyst C18-H column (4.6 mm×250 mm, 5 μm). The mobile phase consisted of 0.1% formic acid (aqueous) and 0.1% formic acid in acetonitrile (organic solvent). A gradient elution was used with a 1.0 mL/min flow rate, where initially 5% organic solvents (acetonitrile contained formic acid) was increased linearly to 65% over 20 min, and finally decreased to 5% in 20.1 min, where it was held until the end of the 30 min run. The fluorescence detector was set for excitation at 490 nm and emission at 550 nm for detection of DOX and PDOX. All analyses were performed at 30 °C. The mobile phase was filtered through 0.45 μm nylon filter membranes (Millipore, Milford, USA) and degassed in an ultrasonic bath before use.
4
SEC Analysis of Biomacromolecules
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SEC measurements were performed using a Shimadzu HPLC system (Model no. LC-20AD, Kyoto, Japan) equipped with TSK gel columns (G3000PW and G5000PW) (Tosoh, Minato-Ku, Tokyo, Japan), an RID-10A refractive index detector, and an RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan). Elution was performed with 0.5 M acetic acid aqueous solution containing tetramethylammonium hydroxide pentahydrate (28 mM) at a flow rate of 0.5 mL min−1 at 25 °C. The fluorescence at an excitation/emission wavelength of 400/635 nm was recorded using an RF-10AXL fluorescence detector. 1H NMR spectra were recorded at room temperature on a JEOL-400YH spectrometer at 400 MHz (JEOL, Tokyo, Japan) using D2O. The UV–vis spectra were collected using a DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA). The fluorescence spectra were collected on an F-7000 fluorescence spectrophotometer (HITACHI, Tokyo, Japan).
5
HPLC Analysis of Agmatine with OPA-ME Derivatization
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The HPLC system consisted of a pump and multi-solvent delivery system (Shimadzu HPLC CLASS-VP, Japan), a RF-10Axl fluorescence detector (the excitation wavelength of 325 nm and the emission wavelength of 425 nm; Shimadzu, Japan), and a Hypersil GOLD 150×2.1, 5-µm column (ThermoFisherScientific). Potassium borate buffer (final concentration 0.2 M, pH 9.4 at 20℃) was prepared by dissolving boric acid in water and adjusting the pH with a saturated solution of potassium hydroxide in a final volume of 250 ml. The buffer was passed through a 0.22-µm filter (Gelman Sciences, MI, USA) and stored at 4℃. The OPA-ME derivatizing reagent was prepared by dissolving 50 mg OPA in 1 ml of methanol and adding 53 µl of ME and 9 ml of 0.2 M potassium borate buffer (pH 9.4); the solution was stored at 4℃ for not more than three days before use. Agmatine was measured by derivatizing with OPA-ME. The mobile phase consisted of a mixture of 46% 10 mM potassium dihydrogen phosphate containing 3 mM octylsulfate sodium salt in water (pH 5.93), 34% acetonitrile, and 20% methanol. The mobile phase was filtrated (FHLP04700, 0.45 µm FH, Millipore) and degassed before use.
6
Quantitative Analysis of Aflatoxins
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Aflatoxins analyses were performed using the methodology described by Bragulat et al. (2001 (link)). The strains were incubated in Petri dishes containing yeast extract sucrose (YES agar, Katsurayama et al. 2018 (link)) at 25ºC for 14 days in the dark. Then, 3 plugs from each Petri dish were transferred to an Eppendorf tube and 1 ml of methanol was added. After 1 hour, the solution was filtered with a Millipore filter (Ø 0.22 mm), an aliquot (200 µl) was derivatised with 700 µl trifluoroacetic acid: acetic acid: water (20:10:70, v/v/v). The derivatised solution was analysed using a reverse phase HPLC consisting of a Shimadzu LC-10ADvp pump, a RF-10Axl fluorescence detector (Shimadzu; excitation and emission wavelength of 360 nm and 440 nm, respectively), and a C18 reversed-phase column (150 mm x 4.6 mm i.d., 5 µm particle size; Nucleodur®, Macherey-Nagel, Düren, Germany) connected to a pre-column Security Guard (8 mm x 4 mm i.d., 5 µm particle size; Nucleodur®, Macherey-Nagel, Düren, Germany). The mobile phase was water: methanol: acetonitrile (4:1:1, v/v/v) at a flow rate of 1.5 ml min-1. The injection volume was 20 µl. Aflatoxins production was measured in ng g-1 of culture medium. The limit of detection was 1 ng g-1 of AFB1 and AFG1, and 0.8 ng g-1 of AFB2 and AFG2.
7
HPLC Analysis of DPM Quantification
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DPM was quantified using an HPLC system consisting of Series 20AD machine (Shimadzu Corporation, Kyoto, Japan). RF-10AXL fluorescence detector (version 3.20, Kyoto, Japan) was used for the analysis of in vitro and ex vivo samples while samples from solubility studies were analysed using a photodiode array detector (SPD-10AP, Shimadzu Corporation, Kyoto, Japan). The stationary phase was Lichrosphere® C18 (200 × 4.6 mm, 5 µm) reversed-phase column and the temperature was maintained at 40 °C. The mobile phase was composed of orthophosphoric acid (pH 4.6) and methanol (30%:70% v/v) run in an isocratic mode. DPM was eluted through the stationary phase at a flow rate of 1 mL/min at a retention time of 11 min and detected at an excitation wavelength of 285 nm and an emission wavelength of 470 nm.
HPLC method was validated for linearity, repeatability, reproducibility, and accuracy. The linearity was attained over a concentration range of 100–8000 ng/mL with a correlation coefficient of 0.999. The repeatability and reproducibility were confirmed by coefficients of variation of less than 2%.
HPLC method was validated for linearity, repeatability, reproducibility, and accuracy. The linearity was attained over a concentration range of 100–8000 ng/mL with a correlation coefficient of 0.999. The repeatability and reproducibility were confirmed by coefficients of variation of less than 2%.
8
Glutamate Quantification via OPA Derivatization
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Samples were diluted in 20 mM borate buffer at pH 9.0 and were derivatized for 1 min with N-tert-butyloxycarbonyl-L-cysteine and o-phthalaldehyde. Samples were then separated in a 5-mm C18 reverse-phase column (220 × 4.6 mm) Sheri-5 (Brownlee), and glutamate separation was monitored by fluorescence (excitation at 334 nm and emission at 433 nm) using an RF-10AXL fluorescence detector (Shimadzu).
9
HPLC Analysis of Squid Ommochromes
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Isolated ommochromes from squid were analyzed by high performance liquid chromatography (HPLC) using a Knauer chromatograph (Berlin, Germany) on a Diasphere 120 C18 column (4 × 250 mm; particle size, 5 μm). Solvent A was 10% aqueous acetonitrile containing 0.5% formic acid; solvent B was 100% acetonitrile containing 0.5% formic acid. The pigments were fractionated in a linear gradient (0–40%) of solution B in solution A for 60 min at a flow rate of 0.4 mL/min at 24 °C. Eluted pigments were registered with a Knauer K-2501 UV/Vis detector and an RF-10A-xl fluorescence detector (Shimadzu, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
10
Measuring Antioxidant Potential via Fenton Reaction
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The purified and commercial betanin were analyzed as previously described by Silva et al. [35 (link)]. Aliquots of 900 µL (16 mg·mL−1) were diluted in water to achieve the same absorbance, transferred to amber vials, and incubated at 37 °C for 10 min with a solution containing 1 mM Fe2+, 10 mM H2O2, and 1 mM terephthalic acid (TPA) in 50 mM phosphate buffer (pH 7.4). The hydroxyterephthalic acid (HTPA) mimics the OH· radical, was detected employing an HPLC system equipped with a reverse-phase C18 Kromasil® (Göteborg, Sweden) column (5 µm, 250 × 4.6 mm ID, Kromasil®) and RF-10AXL fluorescence detector (Shimadzu®) with excitation and emission wavelengths set at 312 nm and 428 nm, respectively. The mobile phase comprised 100 mM sodium phosphate buffer (pH 6.6) at 1.0 mL·min−1. The TAP value was estimated by the percentage difference of the HTPA generated by the Fenton reaction, according to Equation (2):
SHTPA: surface area of the chromatogram in the Fenton reaction with the sample;
S0HTPA: surface area of the chromatogram in the Fenton reaction without sample.
SHTPA: surface area of the chromatogram in the Fenton reaction with the sample;
S0HTPA: surface area of the chromatogram in the Fenton reaction without sample.
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