A 100 μl aliquot of urine was used for MDA analysis. Briefly, we add 700 μl of 1% orthophosphoric acid and 200 μl of thiobarbituric acid (42 mm ) to the sample and then heated it for 60 min in a water bath at 100 °C; the sample was cooled on ice immediately after. Two hundred microliters were transferred to a 2 ml tube containing 200 μl of sodium hydroxide–methanol (1:12 v/v). The sample was vortex-mixed for 10 s and centrifuged for 3 min at 13 000 g. We transferred 200 μl of the supernatant to a 300 μl glass vial, and injected 50 μl onto the column. The HPLC system used was a Shimadzu LC-10AD system (Shimadzu Corporation, Kyoto, Japan), with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), a Shimadzu RF-535 fluorescence detector (excitation: 525 nm, emission 551 nm), and 0.5 ml min−1 flow of phosphate buffer (KH2PO4 1 mm , pH 6.8). MDA was quantified by area determination of the peaks in the chromatograms relative to a standard curve of known concentrations.
Rf 535 fluorescence detector
Manufactured by Shimadzu
Sourced in United States, Germany
The RF-535 is a fluorescence detector designed for high-performance liquid chromatography (HPLC) applications. It features a xenon lamp as the excitation source and a monochromator for wavelength selection. The detector measures the intensity of fluorescent light emitted by analytes in the sample, providing sensitive and selective detection for a wide range of compounds.
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Lab products found in correlation
8 protocols using rf 535 fluorescence detector
1
Urine MDA Quantification by HPLC
2
Nutritional Analysis of Animal Feed
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Feed and feces samples were analyzed for dry matter (EU 71/393), Kjeldahl nitrogen (N) (EU 93/28), lipid (HCl hydrolysis and diethyl ether extraction (EU 98/64)), and starch (AOAC enzymatic method 996.11). Gross energy was measured by bomb calorimetry (Parr 1271 bomb calorimeter, Parr, Moline, IL). Amino acids (except tryptophan) analysis of all samples were performed with a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, U.K.) after hydrolysis, according to EC Commission Directive 98/64/EC. Tryptophan was analyzed on a Dionex Summit HPLC system, with a Shimadzu RF-535 fluorescence detector. Yttrium oxide concentrations in feed and feces were determined by inductively coupled plasma mass spectroscopy (ICPMS) as previously described [25 ].
3
Tocopherol Quantification in Pumpkin Seed Oil
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The determination of tocopherols in PSO was conducted with slight modifications to the method described by Constantin et al. [20 ]. Briefly, PSO samples were saponified before the hexane-soluble compounds (containing tocopherols) were collected for drying by nitrogen gas. The tests were conducted using a HPLC (2695 Waters, Milford, MA, USA) equipped with a reversed-phase Nucleosil 50-5 C18 column and an RF-535 fluorescence detector (Shimadzu, Tokyo, Japan). The HPLC system was operated with an excitation wavelength of λ = 290 nm and an emission wavelength of λ = 330 nm. The relative retention time and maximum absorption values at the given relative retention time were used for the identification of tocopherols in the oil samples.
4
Quantification of Malondialdehyde via HPLC
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Homogenate (100 μL) was used for MDA analysis. Briefly, 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM) were added to the sample. Then, the sample was boiled for 60 min in a water bath, and afterwards, it was immediately cooled on ice. Two hundred microliters was transferred to a 2 mL tube containing 200 μL sodium hydroxide/methanol (1:12 v/v). The sample was vortex-mixed for 10 s, and centrifuged for 3 min at 13,000× g. The supernatant (200 μL) was transferred to a 300 μL glass vial and 50 μL injected onto the column. The HPLC was a Shimadzu LC-10AD system (Kyoto, Japan) equipped with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), a Shimadzu RF-535 fluorescence detector (excitation 525 nm, emission 551 nm), and 0.5 mL/min flow of phosphate buffer (KH2PO4 1mM, pH 6.8) [32 (link)]. MDA was quantified by determination of the peak area in the chromatograms relative to a standard curve of known concentrations.
5
Profiling Egg Yolk Phospholipids
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Eggs collected at 48 wk of hen age were analyzed for total lipids, PC, and PE contents estimation. The PE and PC contents of egg yolk were determined as per Chen and Kou (1982) , with modifications described in Balazs et al. (1996) . Phospholipid classes were separated and quantified using a Shimadzu LC-2010 HT high performance liquid chromatograph with an LC-2010 AHT High Speed Autosampler and a Shimadzu RF-535 fluorescence detector (Shimadzu, Columbia, MD). A Cosmosil 5SL-II, 250 × 4.6 mm packed column and guard column were used (Nacalai Tesque Inc., Japan). The mobile phase was acetonitrile:methanol: phosphoric acid (100:30:0.05 vol/vol/vol), oven temperature set at 40°C, the flow rate 1 mL/min., and wavelength 205 nm. Twenty five mL of lipid extract was evaporated under nitrogen gas and resuspended in 800 μL of methanol. Samples were passed through 0.45 μm nylon membrane syringe filters and 10 μL injected into the chromatograph for analysis. Standard curves were generated with a polar lipid mixture (1127, Matreya, Inc., PA).
6
Amino Acid Analysis of Freeze-Dried Yeast
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Analysis of the content of amino acids (except tryptophan) in freeze–dried yeast was performed according to EC regulation No: 152/2009 (pp. 23–32) using a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, UK). Tryptophan was analyzed according to EC regulation No: 152/2009 (pp. 32–37) using a Dionex Ultimate 3000 HPLC system (Dionex Softron GmbH, Germering, Germany) connected to a RF-535 fluorescence detector (Shimadzu., Kyoto, Japan). All amino acids were quantified by using external standards (Dionex Ltd., Surrey, UK).
7
Amino Acid Analysis of Dried Yeast
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Analysis of the content of amino acids (except tryptophan) in freeze-dried yeast was performed according to EC regulation No: 152/2009 (pp. 23–32) using a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, UK). Tryptophan was analyzed according to EC regulation No: 152/2009 (pp. 32–37) using a Dionex Ultimate 3000 HPLC system (Dionex Softron GmbH, Germering, Germany) connected to a RF-535 fluorescence detector (Shimadzu., Kyoto, Japan). All amino acids were quantified using external standards (Dionex Ltd., Surrey, UK).
8
Muscle Homogenate MDA Analysis
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A 100 μL aliquot of soleus muscle homogenate was used for MDA analysis. Briefly, we added 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM) to the sample and then boiled it for 60 min in a water bath; the sample was cooled on ice immediately after that. Two hundred μL was transferred to a 2 mL tube containing 200 μL sodium hydroxide-methanol (1 : 12 v/v). The sample was vortex-mixed for 10 s and centrifuged for 3 min at 13,000 ×g. The supernatant (200 μL) was transferred to a 300 μL glass vial and 50 μL injected onto the column. The HPLC was a Shimadzu LC-10AD system (Kyoto, Japan) equipped with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), a Shimadzu RF-535 fluorescence detector (excitation: 525 nm, emission 551 nm), and 0.5 mL/min flow of phosphate buffer (KH2PO4 1 mM, pH 6.8) [14 (link)]. MDA was quantified by area determination of the peaks in the chromatograms relative to a standard curve of known concentrations.
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