LC-MS for metabolomic analysis was performed using an HPLC apparatus (Agilent) coupled to an LTQ Orbitrap XL-MS system (Thermo Fisher Scientific Inc., San Jose, CA, USA), equipped with an electrospray source in APCI positive-ion mode. The spray capillary temperature was 200°C. Scan event 1 was performed in full mass mode (analyzer; orbitrap), and scan events 2–5 were operated in MS/MS mode (analyzer; ion trap, act type; collision-induced dissociation, normalized collision energy: 35.0). The tomato extract was injected and analyzed by LC, using the same LC analysis program as that used for HPLC. The data were obtained using Xcalibur (Thermo Fisher Scientific Inc.), Compound Discoverer 2.1 (Thermo Fisher Scientific Inc.), and PowerGet software (Kazusa DNA Research Institute, Chiba, Japan) using previously described methods [31 (link)–33 (link)]. Compound Discoverer software and PowerGet software were linked to the metabolite databases (KEGG, ChemSpider, and PubChem).
Hplc apparatus
Manufactured by Agilent Technologies
Sourced in United States
The HPLC (High-Performance Liquid Chromatography) apparatus is a laboratory instrument used for the separation, identification, and quantification of various chemical compounds. It consists of a solvent delivery system, a sample injection port, a separation column, a detector, and a data processing unit. The core function of the HPLC apparatus is to efficiently separate and analyze complex mixtures of substances.
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Lab products found in correlation
Compound discoverer 2, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Api 4000 triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
C18 column, by Waters Corporation (1 mentions)
Galaxie chromatography manager software, by Agilent Technologies (1 mentions)
200 2031 metachem online degasser, by Agilent Technologies (1 mentions)
Galaxie chromatography data system, by Agilent Technologies (1 mentions)
9 protocols using hplc apparatus
1
Metabolomic Analysis by LC-MS
2
Quantitative LC-MS/MS Analysis of SCY-078
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All biological samples were assayed by a validated LC-MS/MS method using an HPLC apparatus (Agilent Technologies, Inc., Santa Clara, CA) and an API-4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a turbo-ion electrospray source operated in positive ionization mode. In summary, treated samples were loaded onto a Phenomenex (Torrance, CA) Kinetix C18 analytical column (2.6 μm, 50 by 2.1 mm) and eluted with a gradient mobile phase. Mobile phases consisted of 0.1% (vol/vol) formic acid in water (A) and 0.1% (vol/vol) formic acid in acetonitrile (B). The initial mobile phase composition of 5% B was held for 1.5 min, followed by a linear gradient from 5% to 80% B over 2.5 min. Chromatography was optimized to ensure that PEG400 did not coelute with SCY-078. Test compounds eluted at approximately 3.7 min, and the total run time was 5 min. Instrumental conditions for the mass spectrometer and precursor-to-product ion transitions were selected and optimized for sensitivity and selectivity. The MS/MS transitions for SCY-078 and d9-SCY-078 were 730.7/584.5 and 739.7/593.5, respectively. Data acquisition and reduction was performed using Analyst v1.4.2 software (Applied Biosystems).
3
Quantitative Analysis of Compounds in LRO
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To investigate how the main compounds in LRO changed during the different growth periods, the contents of eight compounds, namely, hyperoside, emodin, rhein, aloe-emodin, myricitrin, kaempferol, and isoorientin, were determined using an Agilent Technologies HPLC apparatus (1,290 Infinity II, Agilent, United States). Before the test, LRO (3 g) was added to methanol (50 mL) and then ultrasonicated for 30 min. After the mixture was filtered through a 0.22 μm filter, the supernatant was used as the final solution for quantitative analysis, which was based on the retention times and UV spectra of commercial compounds. The solvent system was composed of 0.1% formic acid solution (A) and acetonitrile (B). The following gradient elution method was applied: 0–10 min 95–82% A; 10–40 min 92–35% A; 40–53 min 35–10% A; and 53–55 min 90–95% A with a flow rate of 0.8 mL/min. The total run time and UV detector were set to 55 min and 280 nm, respectively. Symmetry (C18, 4.6 mm × 150 mm, 5 μm, Waters, Ireland) was maintained at ambient temperature (30.0°C).
4
HPLC-based PAH Quantification Protocol
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The PAH analyses were performed based on the method described by Farhadian et al. [28 (link)]. PAH analysis was carried out using an HPLC apparatus (Agilent, Santa Clara, CA, USA) equipped with a 600 controller pump, fluorescence detector (G1312, Agilent, USA) and a 20 μL loop injector. A PAH column (250 mm × 4.6 mm, 5 μm particle size) (Agilent, Santa Clara, CA, USA) was used. The mobile phase was constituted of acetonitrile and water. The elution conditions applied were: 0–3 min, 60% of acetonitrile isocratic; 3–15 min, 60–100% of acetonitrile; 15–46 min, 100% of acetonitrile isocratic; 46–53, 100–60% of acetonitrile, gradient. The flow rate was 1.0 mL min−1. A fluorescence detector operated at excitation/emission wavelength 265/327 nm for NAP, 285/320 nm for Ac, 256/300 for FLU, 275/450 nm for FLT, 274/382 nm for BaA, 260/360 for CHR, 283/430 for BbFA and BkFA, 285/410 for BaP, 284/395 for DBahA, 290/410 for BPE, and 301/480 for IPY. Separation was performed under isocratic conditions. Each PAH sample solution was passed through a 0.22 μm filter before injection into the HPLC system. The quantification of PAHs was performed using an external calibration curve method. The quantification of 12 PAHs was carried out through the external standard method.
5
Eicosanoid Extraction Methodology
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Seven milliliters of venous blood were obtained from patients and collected into adequate tubes containing EDTA as an anticoagulant. The blood was then centrifuged and separated into individual Eppendorf tubes for differentiation of morphotic components. The samples were stored at −80 °C in a freezer. The eicosanoid extraction methodology was performed on a liquid chromatography (HPLC) apparatus (Agilent Technologies, Cheshire, UK).
6
Quantifying Malondialdehyde in Red Blood Cells
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Briefly, 250 µL thiobarbituric acid (42 mM), 750 µL H3PO4 (0.44 M), and 450 µL distilled water were added to the 50-µL RBC suspension in a test tube and the mixture was incubated for 60 min in a boiling water bath (total volume: 1.5 mL). After cooling in ice water, the same volume of alkaline methanol (50 mL methanol + 4.5 mL of 1 M NaOH) (1/1 v/v) was added to the test tube and centrifuged at 3000 × g for 3 min. The supernatant (200 µL) was transferred to a vial and injected into an HPLC apparatus (Agilent, Boblingen, Germany). An RP-18 column (150 ×4.6 mm) and 5 -μm particle size were used. The mobile phase consisted of a mixture of 400 mL of 50-mM phosphate buffer (pH 6.8) and 600 mL of methanol (total volume 1 L). The flow rate of the mobile phase and the injection volume of samples were set to 0.8 mL/min and 20 µL, respectively. Measurements were made against standard samples of different concentrations at an excitation wavelength of 527 nm and an emission wavelength of 551 nm.21 (link) MDA levels are expressed as nmol/gHb.
7
Quantifying Serum Retinol by HPLC
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Serum retinol concentrations were measured by high-performance liquid chromatography (HPLC). 17 (link) Serum (200 μL) was deproteinized with absolute alcohol, and the retinol was extracted with hexane and evaporated to dryness with nitrogen gas. The mobile phase was a double-distilled water mixture (96:4). The mobile phase mixture (100 µL) was added to dilute the precipitate, and a portion (20 µL) of the sample was injected into a bottle that was inserted into a C18 column (5 μm, 4.6 × 150 mm, Waters, Milford, USA) on the HPLC apparatus (Agilent, Palo Alto, USA). The retinol concentrations were determined by spectrophotometry at 315 nm.
8
HPLC Quantification of Iloprost Enantiomers
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Iloprost solution was carried out using HPLC apparatus (Varian Inc., Palo Alto, USA) equipped with 200-2031 Metachem online degasser, a M210 binary pump, a ProStart 410 auto sampler, a G1316A thermostatic column, a 25 μl CSL20 Cheminert Sample Loop injector. Data was acquired and processed using Galaxie chromatography manager software (Varian Inc., Palo Alto, USA). A GraceSmart RP C18 column (4.6 × 250 mm, 5 μm, Alltech Grom GmbH, Rottenburg-Hailfingen, Germany) was used as a stationary phase during analysis. Deionized water (HPLC grade) acidified with 0.1% v/v trifluoroacetic acid (TFA, HPLC grade) and acetonitrile (HPLC grade) 60:40% v/v was used as the mobile phase. The flow rate was 1 ml/min; while, Iloprost solution was acquired at a maximum wavelength of 205 nm. The drug was separated as an enantiomer at the retention times of 20.5 and 22 min, respectively. Enantiomers were widely separated during analysis and no interference peaks or overlapping of compounds were observed (Supplementary Fig. 1
AUC = 0.5899x + 0.0234
where, x is the Iloprost concentration (μg/ml) and AUC (mAU × min) is the area under the curve. A linear correlation with R2 = 0.9998 was carried out in the range from 0.01 to 10 μg/ml.
AUC = 0.5899x + 0.0234
where, x is the Iloprost concentration (μg/ml) and AUC (mAU × min) is the area under the curve. A linear correlation with R2 = 0.9998 was carried out in the range from 0.01 to 10 μg/ml.
9
HPLC Quantification of Polysaccharides
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The concentration of PSs was evaluated by high-performance liquid chromatography (HPLC), according to the method reported in Romani et al. [55 (link)]. After filtration through 0.45 μm nitrocellulose membranes, 20 μL of sample were injected into the HPLC apparatus (Varian Inc., Palo Alto, CA, USA) equipped with a 410 series autosampler, a 210 series pump, and a 356-LC refractive index detector. Isocratic separation was performed on a TSK G-OLIGO-PW (808031) column (30 cm × 7.8 mm i.d.) and a TSK-GEL OLIGO (808034) guard column (4 cm × 6 mm i.d.) (Supelco, Bellefonte, PA, USA). The mobile phase was 0.2M NaCl, at a flow rate of 0.8 mL/min. Peaks were quantified by comparison with an external calibration curve of mannan (Sigma-Aldrich, Milan, Italy) from 50 mg/L to 1000 mg/L. The analysis of the peaks was performed using the software Galaxie Chromatography Data System (version 1.9.302.530) (Varian Inc., Palo Alto, CA, USA). All the analyses were carried out in duplicate.
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