Aquasil c18 column

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Aquasil C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a silica-based stationary phase with C18 alkyl chains, which provides a hydrophobic interaction with the analytes. The column's dimensions and particle size can vary depending on the specific application requirements.

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C18 column (178 citations) Aquasil C18 (13 citations) Aquasil C18 pre-column (2 citations) AQUASIL C18 140×4.6 mm column (2 citations)

18 protocols using aquasil c18 column

Phenolic Profiling via LC-ESI-MS Analysis

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The phenolic profile was determined viaLC–ESI–MS analysis using a Shimadzu UFLC XR system (Kyoto, Japan), equipped with a SIL-20AXR auto-sampler, a CTO-20 AC column oven, a LC-20ADXR binary pump, and a quadripole 2020 detector system.For analysis, an Aquasil C18 column (Thermo Electron, Dreieich, Germany) (150 mm × 3 mm, 3 μm) preceded by an Aquasil C18 guard column (10 mm × 3 mm, 3 μm, Thermo Electron) was used. The mobile phase was composed of A (0.2% acetic acid in 5% MeOH and 95% H₂O, v/v) and B (0.2% acetic acid in 50% CAN and 50% H₂O, v/v) with a linear gradient elution: 0–45 min, 10–100% B; 45–55 min, 100% B. Re-equilibrate duration was 5 min between individual runs. The injection volume was 20 μL, the flow rate of the mobile phase was 0.4 mL/min, and the temperature of the column was maintained at 40 °C. Spectra were monitored in selected-ion-monitoring (SIM) mode and processed using Shimadzu LabSolutions LC–MS software. The mass spectrometer was operated in negative ion mode with a capillary voltage of −3.5 V, a nebulizing gas flow of 1.5 L/min, a dry gas flow rate of 12 L/min, a dissolving line (DL) temperature of 250 °C, a block source temperature of 400 °C, a voltage detector of 1.2 V, and the full scan spectra from 50 to 2000 m/z. Phenolic compound identification was achieved throughcomparison with retention times of standard compounds [31 (link)].

Lekmine S., Boussekine S., Akkal S., Martín-García A.I., Boumegoura A., Kadi K., Djeghim H., Mekersi N., Bendjedid S., Bensouici C, & Nieto G. (2021). Investigation of Photoprotective, Anti-Inflammatory, Antioxidant Capacities and LC–ESI–MS Phenolic Profile of Astragalus gombiformis Pomel. Foods, 10(8), 1937.

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Quantitative Metabolite Profiling by UPLC-MS/MS

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The separation procedure was carried out by ultra-performance liquid chromatography (UPLC) using an Aquasil C18 column (Thermo Electron, Dreieich, Germany) with a column size (150 mm × 3 mm, particle size 3 µm). The mobile phase (A) formic acid 0.1% in water and (B) formic acid 0.1% in acetonitrile was used in gradient manner. The elution was 90% A: 10% B (0-20 min); 100% B (20-20.1 min); 90% A: 10% B (20.1-25 min), with running re-equilibration time every 5 min. The mobile phase flow rate was 0.4 mL/min, and the injection volume was 10 µL. The column temperature was maintained at room temperature.
LC-ESI-MS/MS analysis was performed using a triple quadrupole mass spectrophotometer (Waters, Acquity UPLC I-Class with Xevo G2-XF QTof) equipped with electrospray ionization (ESI). The mass spectrometer operated in positive ion mode, full scan spectrum from 100-1300, cone voltage 30 V, Capillary Voltage 3.0 kV, and Source temp 500 • C. All LC-MS data were processed, peaked, and analyzed using the UNIFI informatics platform. The intensity of each ion produces a matrix consisting of the m/z value, retention time (RT), and peak area. The variables of interest were then identified using the UNIFI software [23] (link).

Ruslin R., Yamin Y., Rahma N.A., Irnawati I., & Rohman A. (2022). UPLC MS/MS Profile and Antioxidant Activities from Nonpolar Fraction of Patiwala (Lantana camara) Leaves Extract. Separations, 9(3), 75-75.

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Rapid UHPLC-MS/MS Quantification

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Chromatographic partition was achieved by using a Thermo Scientific, Aquasil C₁₈ column (100×2.1 mm, 5 µm particle size) attached to Thermo Dionex Ultimate 3000 UHPLC with a quaternary pump, autosampler, solvent manager, and an MS-MS detector (Thermo Scientific LCQ Fleet, Ion Trap mass spectrometer, Serial# LCF 10356, San Jose, CA, USA). Ammonium acetate buffer (1 mM, pH 4.4) and methanol (20:80, v/v) were used as the mobile phase in nongradient elution mode at a flow rate of 300 µL/min. The injected sample volume was 10 µL. The column temperature was maintained at 40°C±2°C, and temperature of autosampler was maintained at 4°C±2°C. The chromatographic run time was 3 minutes.

Al Asmari A.K., Ullah Z., Tariq M, & Fatani A. (2016). Preparation, characterization, and in vivo evaluation of intranasally administered liposomal formulation of donepezil. Drug Design, Development and Therapy, 10, 205-215.

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Microsomal P450 Activity Assay

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Liver microsomal P450 methoxyresorufin O-demethylation (MROD) activities were determined using the buffer and cofactor conditions previously described employing microsomal protein (0.25 mg/mL) and methoxyresorufin (10 µM) in a final volume of 0.5 mL.^{38 (link)} Resorufin formation was measured by fluorescence employing a Cary Eclipse fluorescence spectrophotometer (Agilent Technology) with excitation at 530 nm and emission at 585 nm, using a slit width of 5 nm.^{40 (link),41}Metabolism studies with AαC, MeIQx, or PhIP (100 µM) used microsomal protein (1.0 mg/mL) in 100 mM potassium phosphate (pH 7.6) containing 0.5 mM EDTA, and 1 mM NADPH. Incubations were conducted at 37 °C for up to 30 min and terminated as previously described.^{38 (link)} The supernatant was assayed by HPLC with UV detection monitoring at 274 (MeIQx), 315 (PhIP) and 336 nm (AαC). HPLC was done with an Agilent Technology 1260 Infinity System (Santa Clara, CA) with an Aquasil C18 column (4.6 × 150 mm, 5-µm particle size) from Thermo Scientific. The gradient elution started from 99% of 10 mM NH₄CH₃CO₂ (pH 6.8, containing 5% CH₃CN), increased to 40% CH₃CN in 16 min, and increased to 100% CH₃N at 25 min with holding for 2 min, then returned to 1% CH₃CN at 29 min.

Turesky R.J., Konorev D., Fan X., Tang Y., Yao L., Ding X., Xie F., Zhu Y, & Zhang Q.Y. (2015). The Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-Pyrido[2,3-b]Indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice. Chemical research in toxicology, 28(12), 2400-2410.

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Quantitative LC-MS Analysis of Metabolites

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An LC-LTQ Orbitrap XL (Thermo Scientific, Tewksbury, MA, USA) equipped with an ESI source, Dionex Ultimate® 3000 LC system and XCalibur® 2.0.7 data package was employed. Samples were resolved on a Thermo AQUASIL C-18 column (250 × 1 mm, 5 μm) using a binary gradient in which mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in ACN. The flow rate was 40 μL/min and the column oven was maintained at 31ºC. The gradient was 6% B from 0–4 min, ramped up to 95% B from 4–17 min, and held at 95% B from 17–48 min.

Mungalachetty P., Kulkarni P., Wang P, & Giese R. (2022). A High Specificity Aniline-based Mass Tag for Aldehyde Detection. Rapid communications in mass spectrometry : RCM, 36(15), e9322.

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Quantifying Folate Stability in Foods

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Folate content and stability in intervention foods were repeatedly quantified in duplicate throughout the trial using HPLC-UV/fluorescence detection⁽²⁰ ⁾. Food samples were extracted using tri-enzyme treatment for beans and di-enzyme treatment for bread, juice and cookies. After purification, folates were quantified using reversed-phase-HPLC-UV/fluorescence detection (Shimadzu LC10) after separation on an Aquasil C18 column (3 µm, 150 × 4·6 mm; Thermo Scientific) based on an external multilevel (n 8) calibration curve.

Hefni M.E., Witthöft C.M, & Moazzami A.A. (2018). Plasma metabolite profiles in healthy women differ after intervention with supplemental folic acid v. folate-rich foods. Journal of Nutritional Science, 7, e32.

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Quantification of Remdesivir and Metabolites

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For RDV, chromatographic separation was achieved on a Phenomenex Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm). The two eluents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The mobile phase was delivered at a flow rate of 0.5 mL/min using a gradient of A and B as follows: from 0 to 1 min, 5% to 90% (v/v) B; from 1 to 2.8 min, 90% (v/v) B; from 2.8 to 3 min, 90% to 5% (v/v) B; from 3 to 6 min, 5% (v/v) B. RDV was detected by positive electrospray ionization (ESI) ion source with multiple reaction monitoring (MRM) transitions of m/z 603.3 to 402.3 and m/z 603.3 to 318.2. For the alanine metabolite (Ala-met) and mono-oxidation metabolite, chromatographic separation was achieved on a Thermo Aquasil C18 column (150 mm × 2.1 mm, 2.6 μm). Same eluents were used as in the determination of RDV. The mobile phase was delivered at a flow rate of 0.3 mL/min using a gradient of A and B as follows: from 0 to 1.5 min, 3% to 90% (v/v) B; from 1.5 to 3.8 min, 90% (v/v) B; from 3.8 to 4 min, 90% to 3% (v/v) B; from 4 to 7.5 min, 3% (v/v) B. The Ala-met and mono-oxidation metabolite were detected by ESI ion source with fast polarity switch and MRM transitions of m/z 441.1 to 150.0 and m/z 619.3 to 200.1, respectively.

Xie J, & Wang Z. (2021). Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An in vitro and in vivo DMPK assessment. Acta Pharmaceutica Sinica. B, 11(6), 1607-1616.

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Hawthorn Flavonoid Extraction and Purification

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Hawthorn fruit (Crataegus pinnatifida, variety of Mengyin dajinxing) was collected from the National Germplasm Resources Garden of Hawthorn located at Shenyang Agricultural University. After removing the core, the hawthorn was freeze-dried and ground as powder with a high-speed grinder and sieved through 40 mesh. Hawthorn flavonoids were extracted from hawthorn powder with 60% ethanol at 50 °C.
Vitexin-rhamnoside was isolated and purified by HP-20 macroporous resin (column, 2 × 20 cm; eluent, 70% ethanol) and semi-preparative liquid chromatography (LC-3000 liquid chromatography system (Shimadzu, Japan) equipped with a SHIMADZU ODS column (20 × 250 mm); mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); the elution program was 0–55 min of 95%–0% phase A and 55–60 min of 100% phase B. The purity of VR was more than 95% measured by high-pressure liquid chromatography (Waters H-Class High-Performance Liquid Chromatograph (Waters, USA); a Thermo AQUASIL C18 column (4.6 × 250 mm) was used with the solvent of 0.1% formic acid in water and 0.1% formic acid; the elution program was same as the condition of semi-preparative liquid chromatography mentioned above).
Zein was purchased from Yifa Biotech Co., Ltd. Apple, citrus, and sunflower pectin were obtained from Fuyuan pectin Co., Ltd. Other chemical reagents used were of analytical grade.

Huang X., Li T, & Li S. (2022). Encapsulation of vitexin-rhamnoside based on zein/pectin nanoparticles improved its stability and bioavailability. Current Research in Food Science, 6, 100419.

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Quantification of 4-AP Conversion Yield

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A conversion yield from 4-NP to 4-AP was determined using RP-HPLC. Shimadzu RP-HPLC system (CBM-20A) was composed of an autosampler (SIL-20 AC), a pump (LC-20AT), a column oven (CTO-20A), a UV detector (SPD-M20A), and a degassing unit (DGU-20A_5R). A Thermo AQUASIL C18 column (150 mm length × 4.6 mm i.d., 5 μm particle size) was used with an isocratic elution. Mobile phase was consisted of 10% acetonitrile in ammonium bicarbonate buffer (10 mM, pH 8.11). The injection volume was 10 μL with a flow rate of 0.8 mL/min. Column oven temperature was set at 30 °C, and UV detection wavelength was at 254 nm. The 4-AP standard was dissolved in deionized water to produce a standard stock solution (50 mM). A calibration curve was established based on six concentrations of 4-AP standard solutions in the range of 0.0125 to 0.2 mM by serial dilution of the standard stock solution with deionized water. A linearity was observed in a concentration range of 0.0125 to 0.2 mM with a regression equation of y = 840160 × + 14095 (r² = 0.999). After completion of the reduction reaction of 4-NP to 4-AP by cf-CA-AuNPs, the reaction mixture was filtered using Minisart RC syringe filters (hydrophilic, 0.2 μm) prior to RP-HPLC injection. To calculate the conversion yield, cf-CA-AuNPs that were synthesized using the lowest caffeic acid concentration (0.08 mM) were employed as a catalyst.

Seo Y.S., Ahn E.Y., Park J., Kim T.Y., Hong J.E., Kim K., Park Y, & Park Y. (2017). Catalytic reduction of 4-nitrophenol with gold nanoparticles synthesized by caffeic acid. Nanoscale Research Letters, 12, 7.

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Sirtuin Deacetylation Assay Protocol

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Deacetylation assay was performed using human recombinant HDAC (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) (Active Motif, Carlsbad, CA) and a synthetic peptide substrate for each enzyme. Sirtuin utilizes NAD + as a cofactor to remove the acetyl group from the peptide substrate. This reaction ultimately generates deacetylated peptide, nicotinamide (NAM), and a novel metabolite O-acetyl-ADPribose (AADPR). A typical reaction was performed in 100 mM phosphate buffer pH 7.5 in a total volume of 50 μL. Each reaction contained 500 μM of NAD⁺, 500 μM of substrate, and either 0, 10, 100, or 500 μM of the candidate compound. Control experiments were run at the same time: one contained 500 μM of NAD⁺ alone; the other contained 500 μM of NAD⁺ and 500 μM of candidate compound only. Reactions were initiated with the addition of 10 μM of sirtuin. The reactions were incubated at 37 °C for 2 hours before being quenched by 8 μL of 10% trifluoroacetic acid (TFA). The samples were then injected on an HPLC fitted to a Thermo Scientific Aquasil C18 column. AADPR, NAD⁺ and NAM peaks were resolved using a gradient of 0 to 10% methanol in 20 mM ammonium acetate. Chromatograms were analyzed at 260 nm. Reactions were quantified by integrating areas of peaks corresponding to NAD⁺ and deacetylation product AADPR.

Carraway H.E., Malkaram S.A., Cen Y., Shatnawi A., Fan J., Ali H.E., Abd Elmageed Z.Y., Buttolph T., Denvir J., Primerano D.A, & Fandy T.E. (2020). Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia. Scientific Reports, 10, 10325.

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