A 6530 QTOF LC/MS (Agilent Technologies) equipped with an autosampler (G7129A), a quat pump (G7104C) and a column comp (G7116A) was used for chromatographic separation. The injection volume was 6 μL. The analytes were separated in a Zorbax RP-18 column (Agilent Technologies; dimensions: 150 mm × 3 mm, dp = 2.7 μm) at a flow rate of 0.230 mL/min. The mobile phase consisted of a combination of solvent A (0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid). The gradient elution was as follows: 0–20 min (98–90% A), 20–50 min (90–80% A), 50–70 min (80–50% A), 70–90 min (50–30% A), 90–110 min (30–10% A) and 110–120 min (10–0% A) [27 (link)]. Mass spectra were simultaneously acquired using ESI in (+,−) ionisation modes, with a capillary voltage of 5500 V. The mass spectra were recorded in the m/z range of 100–1000 m/z. The gas temperature and drying gas flow were 190 °C and 6 L·/min, respectively. The skimmer and fragmentator voltages were set to 65 V and 130 V, respectively, and collision energy was 10 V. The nebulisation pressure was 25 psi g.
Zorbax rp 18 column
Manufactured by Agilent Technologies
The Zorbax RP-18 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It features a C18 stationary phase and is designed for the separation and analysis of a wide range of organic compounds.
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Lab products found in correlation
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Efv drug powder, by Merck Group (1 mentions)
1200 hplc system, by Agilent Technologies (1 mentions)
6530 q tof lc ms, by Agilent Technologies (1 mentions)
1260 infinity hplc system, by Agilent Technologies (1 mentions)
6530 qtof ms, by Agilent Technologies (1 mentions)
6 protocols using zorbax rp 18 column
1
Comprehensive QTOF LC/MS Analysis of Compounds
2
Quantitative HPLC-MS/MS Analysis of HIV-1 Drug
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Human neuroblastoma cells (SK-N-MC) (ATCC Cat # HTB-10) were cultured in Eagle’s minimum essential medium (MEM) (ATCC catalog # 30–2003) supplemented with fetal bovine serum to a final concentration of 10% (ATCC catalog # 30–2020). These cells and media were obtained from American Type Culture Collection, Manassas, VA and 1% antibiotic and antimycotic solution (catalog # A5955) was obtained from Sigma-Aldrich, St. Louis, MO. HIV-1 Ba-L (clade B) (Cat. # 510) was obtained through AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. ND powder (3–6 nm, purity 97 + %) was purchased from Nanostructured and Amorphous Materials Inc. (Garland, TX, USA). EFV drug powder and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). An Agilent 1200 HPLC system (Palo Alto, CA) coupled to an Applied Biosystem 4000 Q TRAP quadrupole linear ion trap hybrid mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) was used for drug analysis. The HPLC-MSMS system is controlled by ChemStation and Analyst 1.4.2 software, respectively. All chromatographic separations were performed on an Agilent ZORBAX RP 18 column (3.5 µ, 150 mm × 0.5 mm) (Palo Alto, CA).
3
Metabolite Profiling by Q-TOF-LC/MS
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Tentative metabolite assignments were obtained by comparing mass spectral data of the identified compounds in both negative and positive ionization modes with previously reported data, as well as data from online public databases to which references were added Table 3 .
The Q-TOF-LC/MS system, 6530 (Agilent Technologies) equipped with an autosampler (G7129A), a Quat. Pump (G7104C) and a column comp (G7116A) were used for chromatographic separation. The injection volume was 8 µL. The analytes were separated on a Zorbax RP-18 column from Agilent Technologies (dimensions: 150 mm × 3 mm, dp = 2.7 µm) in a flow rate of 0.3 mL/min. The mobile phase consisted of a combination of solvent A Water (0.1 formic acid) and solvent B (acetonitrile + 0.1% formic acid). The gradient elution was as follows: t = 0 min, 3% B; t = 15 min, 10% B; t = 40 min, 20% B; t = 70 min, 40% B; t = 90 min, 60% B; t = 110 min, 80% B; t = 120 min, 90% B and t = 135 min, 100% B. Mass spectra were simultaneously acquired using ESI in positive ionization mode with a capillary voltage of 4000 V. The mass spectra were recorded in the m/z range of 40 to 1500 m/z. The gas temperature and drying gas flow were 350 °C and 10 L/min, respectively.
The Q-TOF-LC/MS system, 6530 (Agilent Technologies) equipped with an autosampler (G7129A), a Quat. Pump (G7104C) and a column comp (G7116A) were used for chromatographic separation. The injection volume was 8 µL. The analytes were separated on a Zorbax RP-18 column from Agilent Technologies (dimensions: 150 mm × 3 mm, dp = 2.7 µm) in a flow rate of 0.3 mL/min. The mobile phase consisted of a combination of solvent A Water (0.1 formic acid) and solvent B (acetonitrile + 0.1% formic acid). The gradient elution was as follows: t = 0 min, 3% B; t = 15 min, 10% B; t = 40 min, 20% B; t = 70 min, 40% B; t = 90 min, 60% B; t = 110 min, 80% B; t = 120 min, 90% B and t = 135 min, 100% B. Mass spectra were simultaneously acquired using ESI in positive ionization mode with a capillary voltage of 4000 V. The mass spectra were recorded in the m/z range of 40 to 1500 m/z. The gas temperature and drying gas flow were 350 °C and 10 L/min, respectively.
4
Optimized LC-MS/MS Analysis of Compounds
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The LC-MS/MS experiments were carried out at the Faculty of Pharmacy, Fayoum University, using a 6530 Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) outfitted with an autosampler (G7129A), a quat. pump (G7104C), and a column comp (G7116A). The extracts were separated on an Agilent Technologies Zorbax RP-18 column (150 mm 3 mm, dp = 2.7 m). The flow rate was 0.23 mL/min, and the injection volume was 2 L. The parameters were adjusted as previously described, and mass spectra were acquired using ESI in both ionization modes. A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) served as the solvents. The following gradient elution times: 0–6 min; 5–50 min; 55–75 min; isocratic elution of 50% A: 50% B; and 75–140 min, were used for the linear gradient from 50% A: 50% B to 100% B.
5
HPLC Analysis of Bioactive Compounds
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HPLC analysis was performed using the 1260 Infinity HPLC system (Agilent, Waldbronn, Germany), consisting of a quaternary pump, an autosampler, a thermostatted column compartment and a diode array detector (DAD). The HPLC system was operated by ChemStation software. HPLC analysis was carried out on a Zorbax RP18 Column (4.6 mm × 250 mm I.D., 5 μm particle size, Agilent). The column temperature was set to 25 °C. Mobile phase A [o-phosphoric acid-water (0.1:99.9, v/v)] and mobile phase B (acetonitrile) were degassed and filtered before analyses. The following gradient elution was applied: 15–18% B (0–5 min), 18–41% B (5–15 min), 41–55% B (15–25 min), 55–80% B (25–27 min), 80% B (27–29 min), and 80–15% B (29–31 min). The flow rate was 1 mL/min. The injection volume was 10 μL. Three different acquisition wavelengths were used for quantitative analyses: (1) 260 nm for quercetin-3-O-β-glucopyranosyl-(1→2)-β-galactopyranoside and afzelin; (2) 310 nm for platanoside; (3) 330 nm for chlorogenic acid and 3,5-dicaffeoylquinic acid. This newly developed HPLC method was validated according to the International Conference on Harmonisation (ICH) 1995 guidelines [29 ]. The validated method was then applied for the quantification of all five compounds investigated in DSTSs.
6
HPLC-MS Analysis of C. volkensii Extract
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The methanol extract of C. volkensii (CVM) was analyzed on an HPLC-MS system consisting of a 1260 Infinity II Flexible HPLC coupled to a 6530 Q-TOF-MS (Agilent Technologies, USA). An injection of 8 μL from the processed crude extract was introduced into a Zorbax RP-18 column provided by Agilent Technologies (150 mm × 3 mm, 2.7 μm), maintained at a temperature of 40 °C, with a flow rate set at 0.5 mL min−1. The mobile phase system comprised a combination of solvent A (0.1% formic acid) and a gradual gradient increase from 0 to 100% of solvent B (acetonitrile + 0.1% formic acid) over a duration of 120 min. Mass spectra were acquired in both positive and negative electrospray ionization (ESI) modes, employing a capillary voltage of 4000 V. The recorded spectra encompassed the m/z values range from 100 to 1500. Parameters such as the capillary temperature and drying gas flow were adjusted to 320 °C and 10 L min−1, respectively. The collision energy and the nebulization pressure were adjusted to 18–45 eV and 40 psi respectively. The LC-MS/MS data acquired was transformed using Proteowizard msconvert, followed by processing and analysis through MZmine.
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