Freeze-dried samples of radish roots (0.5 g) were extracted with 90% methanol (10 ml, 30 min, 70°C, shaken every 5 min). After this process, the samples were filtered and centrifuged (10 min, 15,000 rpm). The supernatants were separated; methanol was evaporated from the mixtures. Then, the samples were dissolved in water (1 ml), and the LC-MS analyses were carried out by means of reversed-phase high-performance liquid chromatography (HPLC Shimadzu Prominence-i LC-2030C, Kyoto, Japan), equipped with a PDA detector coupled to a triple quadrupole mass spectrometer (Shimadzu LCMS-8045). Glucosinolates present in the radish roots were semi-quantified, using the LC-MS-tq apparatus in the MRMs mode (Table 1 ). The separation of the desired compounds was performed, using the following mobile phase: water with 0.1% TFA (eluent A) and acetonitrile with 0.1% TFA (eluent B). The flow rate was set at 0.25 ml·min−1, and the gradient was as follows: starting at 1% solvent B for 3 min, then reaching 20% up to 20 min, 30% up to 23 min, and 0.1% B at 35 min. The Kinetex C18 100A column (100 × 3 mm, 2.6 μm particle size, Phenomenex, Germany) was used. Singrin was used as an external standard (Ciska et al., 2000 (link); Ediage et al., 2011 (link); Maldini et al., 2017 (link)).
Prominence i lc 2030c
Manufactured by Shimadzu
Sourced in Japan
The Prominence-i LC-2030C is a compact high-performance liquid chromatography (HPLC) system designed for routine analysis. It features an integrated solvent delivery unit, autosampler, and column oven, providing a complete HPLC solution in a small footprint.
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Lab products found in correlation
Kinetex c18 100a column, by Phenomenex (2 mentions)
Lcms 8045, by Shimadzu (2 mentions)
Lichrospher 100 rp 18e, by Merck Group (1 mentions)
Supelcosil c18 column, by Merck Group (1 mentions)
C18 analytical column, by Shimadzu (1 mentions)
Gist c18 column, by Shimadzu (1 mentions)
Universal 32r, by Hettich (1 mentions)
11 protocols using prominence i lc 2030c
1
Quantifying Glucosinolates in Radish Roots
2
RP-HPLC-DAD Analysis of Aloin A and Aloenin A
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The RP-HPLC-DAD method was applied to confirm the identity of the material. For this purpose, two standard substances were used as standards—aloin A and aloenin A. In the study, the HPLC Shimadzu Prominence-i LC-2030C equipped with DAD detector was used (Shimadzu, Tokyo, Japan). The software was LabSolution DB/CS version 6.50 (Shimadzu, Tokyo, Japan). The stationary phase was LiChrospher 100 RP-18e (250 mm × 4.6 mm, 5 µm) HPLC column (Merck, Warsaw, Poland), the mobile phase comprised of methanol (phase A) and water (phase B) in a gradient (Table S2, Supplementary Materials ) the injection volume was 20 µL, and a flow rate was 1 mL/min. The detection was performed at the wavelength λmax = 295 nm.
3
pH-dependent AF Release from PEG-FTn
4
Photosynthetic Pigment Extraction and Quantification
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Photosynthetic pigments were extracted using methods detailed in Cruz et al. [29 (link)]. Briefly, samples were extracted in 95% cold buffered methanol with 2% ammonium acetate. Samples were ground with a plastic rod and sonicated for 30 s and vortexed. Samples were then transferred to −20 °C for 20 min in the dark. Extracts were filtered through 0.2 μm PTFE membrane filters and immediately injected into a Prominence-i LC 2030C High-performance liquid chromatography (HPLC) system (Shimadzu, Japan) with a photodiode array detector. Chromatographic separation was carried out using a Supelcosil C18 column (250 mm length; 4.6 mm diameter; 5 μm particles; Sigma-Aldrich, Burlington, MA, USA) for reverse phase chromatography and a 35 min elution program [41 (link)]. Photosynthetic pigments were identified from retention times and absorbance spectra, and concentrations calculated from the signals in the photodiode array detector. Pigment calibration was done using pure crystalline standards from DHI (Hørsolm, Denmark).
5
Validated HPLC Method for Drug Analysis
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Analysis was performed on a Shimadzu Prominence-i LC-2030C HPLC system using a gradient method for all the drugs except probucol. Column Xbridge® C18 5 µm (2.1 × 50 mm) at 30 °C, mobile phase A 10 mM ammonium formate pH 3 (adjusted with formic acid) in water, and mobile phase B 10 mM ammonium formate in acetonitrile:water (9:1), flow rate 1 mL/min (except carvedilol 0.7 mL/min), gradient start 70:30 (A:B), 3 min 0:100, 4 min 0:100, 4.5 min 70:30 total run time 8 min. The retention time, analysis wavelength and injection volume for each drug are provided in Table 2 . For probucol an isocratic method was used [17] (link) mobile phase ACN, MeOH, and water 45:45:10 and the column was Speck and Burke, ODS-H optimal 5 µm (30 × 150 mm). For each drug a concentration curve was prepared using five or six standards that bracketed all the measurement concentrations, for all drugs correlation coefficient > 0.99.
HPLC conditions.
| Drug | Retention time (min) | Wave-length (nm) | Injection volume (µL) |
|---|---|---|---|
| Naproxen | 1.6 | 254 | 10 |
| Indomethacin | 2.1 | 254 | 10 |
| Phenytoin | 1.1 | 254 | 20 |
| Piroxicam | 1.07 | 254 | 10 |
| Aprepitant | 2.27 | 254 | 50 |
| Carvedilol | 1.6 | 254 | 10 |
| Zafirlukast | 2.6 | 254 | 25 |
| Tadalafil | 1.4 | 291 | 50 |
| Fenofibrate | 3 | 291 | 10 |
| Felodipine | 2.4 | 254 | 10 |
| Griseofulvin | 1.5 | 291 | 10 |
| Probucol | 4.87 | 220 | 100 |
6
HPLC Analysis of Compound Separation
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HPLC analysis was performed with a Shimadzu Prominence-i LC-2030C instrument equipped (Shimadzu, Kyoto, Japan) with a Sunniest C18 analytical column (5 µm, 4.6 × 250 mm). Water/formic acid (1000:1, v/v) and methanol/formic acid (1000:1, v/v) were used as mobile phases A and B, respectively. Linear gradient elution was performed based on the procedure described below: 0–5 min 10% solution B; 5–20 min 10–30% solution B; 20–45 min 30–100% solution B; 45–50 min 100% solution B; 50–60 min 10–100% solution B; flow rate: 1.0 mL/min; detection wavelength: 254 nm; column oven: 40 °C.
7
Quantification of Glycosides by LC-MS
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Freeze-dried samples of 1 g were extracted with 70% methanol (10 mL; 30 min, 70 °C). During the extraction process, samples were shaken on a vortex every 3 min, then filtered and centrifuged (10 min, 15,000 rpm). The supernatant was separated, methanol was removed from the mixture by evaporation on a vacuum evaporator. Next, samples were prepared by dissolving in water (1 mL), and the LC-MS analysis was performed using reversed-phase high-performance liquid chromatography (HPLC Shimadzu Prominence-i LC-2030C, Kyoto, Japan) equipped with a PDA detector coupled to a triple quadrupole mass spectrometer (Shimadzu LCMS-8045). Glycosides were separated using the following mobile phase: water with 0.1% TFA (eluent A) and acetonitrile with 0.1% TFA (eluent B). The flow rate was set at 0.25 mL·min−1, and the gradient was as follows: starting at 1% solvent B for 3 min, then reaching 20% up to 20 min, 30% up to 23 min, and 0.1% B at 35 min. Separation was obtained on a Kinetex C18 100A column (100 × 3 mm, 2.6 µm particle size, Phenomenex, Germany). Singrin and neoglucobrassicin were used as external standards for the analysis [49 (link)].
8
HPLC Analysis of Drug Formulations
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Content uniformity, drug diffusion, and drug release have been analyzed by Shimadzu Prominence-i LC 2030C HPLC instrument with an isocratic method. The mobile phases were composed of phosphate buffer (pH 7.4) and acetonitrile with a ratio of 30:70 respectively. The column used was ACS C18 (3 mm, 15 cm) with a flow rate of 1 mL/min at 27°C. All absorbance measurements were done under 245 and 290 nm wavelengths for both samples and calibration curves. Calibration curves resulted in a correlation coefficient of > 0.99.
9
HPLC Analysis of Sulfamethazine in Extracts
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The concentrations of SMZ in the prepared and cleaned-up extracts were analyzed by using high-performance liquid chromatography (HPLC; Prominence-i, LC-2030C, Shimadzu, Kyoto, Japan), with a reversed-phase Raptor C18 column (100 mm × 21 mm, 2.7 μm particle size) and PDA detector. Acetonitrile and phosphoric acid (pH = 3.0) were used as mobile phase A and B, respectively. The mobile phase A was maintained at 93% for an initial 5.0 min, then changed to 70% from 5.0–18 min, and held at 70% till 25 min. Then, the phosphoric acid: acetonitrile ratio was changed to 93:7.0 from 25–30 min and held up to 35 min. A calibration curve (R2 > 0.99) was constructed between SMZ concentrations in standards versus absorbance (%) by the HPLC. The standards were also fed as unknown samples for quality assurance. The samples with known concentrations of SMZ were used to estimate percent recovery, which was found to be 67.47 ± 4.55%.
10
Pharmacokinetics and Biodistribution of CLP Nanoparticles
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BALB/c mice (6 to 8 weeks) were intravenously injected with 0.1 ml of saline containing CLP nanoparticles at 25 mg/kg. At predetermined time periods, whole blood and major organs were collected after mice were euthanized. The plasma was obtained by centrifugation at 2000g for 10 min. After the proteins were precipitated via methanol, the plasma CLP concentrations were quantified by high-performance liquid chromatography (HPLC; Prominence-i LC-2030C, Shimadzu), using a Shim-pack GIST C18 column (250 mm by 4.6 mm; particle size, 5 μm) and an RF-20A fluorescence detector. The excitation wavelength was set at 401 nm, while the emission wavelength was set at 660 nm. The mobile phase consisted of water and methanol at a volume ratio of 10:90, with a flow rate of 1.0 ml/min. The column temperature was 40°C. For different organ tissues, they were homogenized in PBS and centrifuged, and the supernatant was collected. The CLP concentrations were also determined by HPLC.
In another study, excretion of CLP was examined in BALB/c mice that were housed individually in metabolic cages. Briefly, after CLP nanoparticles were administered via intravenous injection at 25 mg/kg, urine and feces were separately collected at predefined time points. Urine samples were precipitated via methanol, while feces were extracted with methanol. The CLP concentrations were quantified by HPLC.
In another study, excretion of CLP was examined in BALB/c mice that were housed individually in metabolic cages. Briefly, after CLP nanoparticles were administered via intravenous injection at 25 mg/kg, urine and feces were separately collected at predefined time points. Urine samples were precipitated via methanol, while feces were extracted with methanol. The CLP concentrations were quantified by HPLC.
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