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.
Lc 10ad system
Manufactured by Shimadzu
Sourced in Japan
The Shimadzu LC-10AD is a high-performance liquid chromatography (HPLC) system. It is designed to perform accurate and reliable liquid chromatography analysis. The LC-10AD system includes a pump, an autosampler, and a detector to enable the separation, identification, and quantification of chemical compounds in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Luna c18 column, by Phenomenex (4 mentions)
Rf 535 fluorescence detector, by Shimadzu (3 mentions)
Rf 535, by Shimadzu (1 mentions)
Inertsustain c18, by GL Sciences (1 mentions)
Xevo qtof, by Waters Corporation (1 mentions)
Ultra performance liquid chromatography (uplc), by Waters Corporation (1 mentions)
Drx 500 spectrometer, by Bruker (1 mentions)
Malvern zetasizer 3000 system, by Malvern Panalytical (1 mentions)
C18 column, by Shimadzu (1 mentions)
C18 analytical column, by Phenomenex (1 mentions)
Spd 10av, by Shimadzu (1 mentions)
7 protocols using lc 10ad system
1
Urine MDA Quantification by HPLC
2
Cardiac Lipid Oxidation Evaluation via MDA
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MDA is the main lipid peroxidation marker. It is considered an oxidative stress index [31 ] and associated with cardiovascular diseases [32 (link)]. Thus, MDA levels were used to evaluate the cardiac lipid oxidation as follow:
Cardiac tissue (±150 mg) was homogenized (ULTRA-TURRAX® T25 basic IKA® Werke Staufen/Germany) with 1.0 mL of cold phosphate buffered saline (PBS) pH 7.4, and centrifuged at 800 g at 4 °C for 10 min. Then, 100 µL from the supernatant was mixed with 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM). After this, the samples were kept at 100 °C for 60 min in a water bath, and immediately cooled on ice. In a 2 mL tube, 200 μL was mixed with 200 μL sodium hydroxide/methanol (1:12 v/v). After vortex, the samples were centrifuged for 3 min at 13,000 g. 200 μL from the supernatant was transferred to a glass vial and 50μL was injected into the column. The HPLC used was a Shimadzu LC-10AD system (Kyoto, Japan) with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), and a Shimadzu RF-535 fluorescence detector (excitation 525 nm, emission 551 nm), and 0.5mL/min phosphate buffer flow (KH2PO4 1mM, pH 6.8) [25 (link)]. The MDA levels considered the peak area determination in the chromatograms relative to the standard curve of known concentrations.
Cardiac tissue (±150 mg) was homogenized (ULTRA-TURRAX® T25 basic IKA® Werke Staufen/Germany) with 1.0 mL of cold phosphate buffered saline (PBS) pH 7.4, and centrifuged at 800 g at 4 °C for 10 min. Then, 100 µL from the supernatant was mixed with 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM). After this, the samples were kept at 100 °C for 60 min in a water bath, and immediately cooled on ice. In a 2 mL tube, 200 μL was mixed with 200 μL sodium hydroxide/methanol (1:12 v/v). After vortex, the samples were centrifuged for 3 min at 13,000 g. 200 μL from the supernatant was transferred to a glass vial and 50μL was injected into the column. The HPLC used was a Shimadzu LC-10AD system (Kyoto, Japan) with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), and a Shimadzu RF-535 fluorescence detector (excitation 525 nm, emission 551 nm), and 0.5mL/min phosphate buffer flow (KH2PO4 1mM, pH 6.8) [25 (link)]. The MDA levels considered the peak area determination in the chromatograms relative to the standard curve of known concentrations.
3
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.
4
NMR Spectroscopic and Mass Spectrometric Analysis
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1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX 500 spectrometer at 500 MHz for 1H NMR and 125 MHz for 13C NMR. Chemical shifts (δ ppm) in the 1H NMR spectra are expressed relative to the signal of tetramethylsilane adjusted to 0.00 ppm, and coupling constants are expressed in hertz. Liquid chromatography-mass spectrometry (LC-MS) was performed using a Waters Xevo QTOF (quadrupole time-of-flight) mass spectrometer and an ACQUITY UPLC (ultra-performance liquid chromatography) system.
Column chromatography was carried out using Amberlite XAD-1180N, Sephadex LH-20 (GE Healthcare), and Toyopearl HW 40F. Analytical and preparative HPLC were performed using a Shimadzu LC-10AD system with a reverse-phase HPLC column (InertSustain C18; 250 mm × 4.6 mm i.d., 3 μm particle size, GL Science, Japan).
Column chromatography was carried out using Amberlite XAD-1180N, Sephadex LH-20 (GE Healthcare), and Toyopearl HW 40F. Analytical and preparative HPLC were performed using a Shimadzu LC-10AD system with a reverse-phase HPLC column (InertSustain C18; 250 mm × 4.6 mm i.d., 3 μm particle size, GL Science, Japan).
5
Characterization of Multifunctional Liposomes
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The morphology of multifunctional liposomes was observed by transmission electron microscopy (TEM). The particle size, zeta potential, and polydispersity (PDI) were measured via a Malvern Zetasizer 3000 system (Malvern Instruments Ltd., UK). The HPLC was used to detect the drug encapsulation efficiency (EE). Briefly, various liposomes were respectively disrupted into water and equal volume of methanol was added. Subsequently, the solution was sonicated for 20 min and the supernatant was obtained after centrifugation at 10000 rpm for 10 minutes. The amounts of PTX and SOR were measured by HPLC (Shimadzu LC-10AD system, Kyoto, Japan) coupled with a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm) at a flow rate of 1 mL/min and the absorption wavelength of 227 and 266 nm. The mobile phase for PTX was methanol: water (75:25, v/v), while for SOR was a mixture of acetonitrile and disodium phosphate (buffer pH = 4 with phosphoric acid, 55:45, v/v) (Li et al., 2015 (link)). The EE was calculated using the following equations:
6
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.
7
Quantitative Analysis of NYST by HPLC
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Quantitative determination of NYST in the samples was carried out by HPLC. The apparatus consisted of a Shimadzu LC-10AD system with an UV SPD-10AV detection and a software Cromatoplus analyser. The injection valve was a Rheodyne with a capacity of 20µl. A Phenomenex C18 analytical column (4.60 × 100 mm, Luna, 3µ) was employed. The mobile phase consisted of MeOH:Water:DMF (60:30:10) mixture (flow rate 1.0 ml/min); the retention time and the detection wavelength were 7.5 min and 305nm, respectively. The limit of determination and quantification were 6.9 ng/ml and 2.0 ng/ml, respectively. The amount of drug in each sample was determined from standard curves, obtained by plotting the concentration of known solutions vs. the corresponding peak areas of HPLC chromatograms.
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