Samples were analyzed by HPLC on a gradient-type analytical HPLC system (Gilson, Inc.) equipped with a Waters 2996 photodiode-array detector. Oligonucleotides were loaded on a µBondasphere C18 column (Waters Co.) at 1.0 mL min−1 and 30 °C, and eluted over a linear, 20-minute gradient of 6–14% acetonitrile in 0.1 M triethylammonium acetate pH 7.0. In contrast, nucleosides from digested oligonucleotides were loaded on an Inertsil ODS-3 column (GL Science Inc.) at 1.0 mL min−1 and ambient temperature, and eluted over 30 min along a linear gradient of 2.5–20% acetonitrile in 0.1 M triethylammonium acetate, pH 7.0.
2996 photodiode array detector
Manufactured by Waters Corporation
Sourced in United States, Ireland
The 2996 Photodiode Array Detector is a high-performance liquid chromatography (HPLC) detector developed by Waters Corporation. It utilizes a photodiode array to collect a full UV-visible spectrum for each sample. The detector provides accurate and precise measurements of analyte concentrations within a sample.
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Lab products found in correlation
2695 separation module, by Waters Corporation (9 mentions)
717 plus autosampler, by Waters Corporation (7 mentions)
2695 separations module, by Waters Corporation (7 mentions)
1525 binary hplc pump, by Waters Corporation (7 mentions)
600 hplc pump, by Waters Corporation (4 mentions)
Waters 2695 high performance liquid chromatograph, by Waters Corporation (3 mentions)
2795 separation module, by Waters Corporation (3 mentions)
600 controller, by Waters Corporation (3 mentions)
Symmetry c18 column, by Waters Corporation (3 mentions)
Waters hplc system, by Waters Corporation (3 mentions)
Sephadex lh 20, by Cytiva (3 mentions)
2475 multi λ fluorescence detector, by Waters Corporation (3 mentions)
717 autosampler, by Waters Corporation (3 mentions)
2767 sample manager, by Waters Corporation (3 mentions)
3100 mass spectrometer, by Waters Corporation (3 mentions)
In line degasser af, by Waters Corporation (3 mentions)
Surveyor hplc system, by Thermo Fisher Scientific (3 mentions)
C18 column, by Waters Corporation (2 mentions)
Empower software, by Waters Corporation (2 mentions)
Zearalatest column, by VICAM (2 mentions)
91 protocols using 2996 photodiode array detector
1
Oligonucleotide Analysis by HPLC
2
HPLC Determination of KCR in Plasma
3
Zearalenone Quantification by HPLC
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After incubation time, 1 mL of each reaction mixture was homogenized for 3 min with 5 mL of acetonitrile:water (90:10, v/v). ZEA was extracted and purified on a ZearalaTest column (Vicam, Milford, CT, USA) according to a procedure described in detail previously (Goliński et al., 2010 (link)). The elute was evaporated to dryness at 40°C under a stream of nitrogen. Dry residue was stored at -20°C until HPLC analyses. Evaporated extracts were dissolved in a 200 μL mixture of acetonitrile:methanol:water (70:20:10, v/v/v), homogenized in an ultrasonic bath (Ultron, type U-505, Dywity, Poland), filtered through a syringe filter of 0.2 μm mesh and applied onto the chromatographic column.
The chromatographic system used in the study consisted of a Waters 2695 high-performance liquid chromatograph (Waters, Milford, CT, USA) with detectors – Waters 2475 Multi aaa Fluorescence Detector (aaaex = 274 nm, aaaem = 440 nm) and Waters 2996 Photodiode Array Detector – and a Nova Pak C-18 column (150 × 3.9 mm). Data were processed using the Empower software (Waters, Milford, CT, USA). Quantification of ZEA was performed by measuring the peak areas at the retention time according to the relevant calibration curve. A Photodiode Array Detector (PDA) was used to confirm the presence of ZEA on the basis characteristic spectra of this compound. The limit of detection was 0.01 μg m L-1.
The chromatographic system used in the study consisted of a Waters 2695 high-performance liquid chromatograph (Waters, Milford, CT, USA) with detectors – Waters 2475 Multi aaa Fluorescence Detector (aaaex = 274 nm, aaaem = 440 nm) and Waters 2996 Photodiode Array Detector – and a Nova Pak C-18 column (150 × 3.9 mm). Data were processed using the Empower software (Waters, Milford, CT, USA). Quantification of ZEA was performed by measuring the peak areas at the retention time according to the relevant calibration curve. A Photodiode Array Detector (PDA) was used to confirm the presence of ZEA on the basis characteristic spectra of this compound. The limit of detection was 0.01 μg m L-1.
4
HPLC Quantification of DNPH Samples
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Before each run the syringe and the injection port loop were rinsed 3 times with MeOH and 3 times with MeCN. DNPH samples were analyzed and quantified using a Waters 1525 Binary HPLC Pump and a Waters 2996 Photodiode Array Detector. Analysis conditions: two SUPELCOSIL C-18, 25 cm × 4.6 mm, 5 µm particle size columns connected in series with a column heater at 40 °C. The mobile phase comprised of MeCN/H2O with a gradient system as follows: 0 min. 60/40; 7 min. 60/40; 25 min. 100/0, at a combined flow rate of 1 mL/min, with a 360 nm detection setting. The sample injection volume was 20 µL.
5
Analytical Characterization of Compounds
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The melting points were determined in open capillaries on electrothermal stuart SMP3 advanced melting point apparatus; IR spectra were obtained from a KBr matrix (4000–400 cm−1) using a Perkin-Elmer, Spectrum 100, FT-IR spectrometer.
The LC/ESI-MS/MS system used was Agilent 1200 series liquid chromatography coupled with a 6520 accurate mass quadruple-time of flight mass spectrometer (Q-TOF LC/MS). The analysis was performed in the positive electrospray ionization mode. The m/z range for MS scans was 100–1600 Da. The high pressure liquid chromatography (HPLC) system consisted of an Alliance 2695 module coupled with 2996 Photodiode array detector from Waters (Germany). Data acquisition and control were carried out using Empower 2 software (Waters, Germany). Analytes were separated on a 4.6 mm × 150 mm XBridge C18 column (5 μm particle size) used in conjunction with a 4.6 × 20 mm, XBridge C18 guard column. Microfilters of 0.45 μm porosity were normally used (Acrodisc GHP, Waters).
1H-NMR experiments were performed with a Bruker AvanceII 500 spectrometer equipped with a 5 mm BBO probe. pH values were recorded on pH meter model HM-30G: TOA electronics; thin-layer chromatography (TLC) was carried out on TLC plastic sheets silica gel, 20 × 20 cm, layer thickness 0.2 mm, and the spots on the plates were localized by exposure to UV light.
The LC/ESI-MS/MS system used was Agilent 1200 series liquid chromatography coupled with a 6520 accurate mass quadruple-time of flight mass spectrometer (Q-TOF LC/MS). The analysis was performed in the positive electrospray ionization mode. The m/z range for MS scans was 100–1600 Da. The high pressure liquid chromatography (HPLC) system consisted of an Alliance 2695 module coupled with 2996 Photodiode array detector from Waters (Germany). Data acquisition and control were carried out using Empower 2 software (Waters, Germany). Analytes were separated on a 4.6 mm × 150 mm XBridge C18 column (5 μm particle size) used in conjunction with a 4.6 × 20 mm, XBridge C18 guard column. Microfilters of 0.45 μm porosity were normally used (Acrodisc GHP, Waters).
1H-NMR experiments were performed with a Bruker AvanceII 500 spectrometer equipped with a 5 mm BBO probe. pH values were recorded on pH meter model HM-30G: TOA electronics; thin-layer chromatography (TLC) was carried out on TLC plastic sheets silica gel, 20 × 20 cm, layer thickness 0.2 mm, and the spots on the plates were localized by exposure to UV light.
6
HPLC Assay for Clofazimine Nanosuspension
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The assay method was modified by Kaur et al. and validated according to ICH Q2 [26 (link)]. The analysis was performed on a Waters Alliance HPLC 2695 separations module connected to a Waters 2996 Photodiode Array Detector set at 290 nm. Data acquisition was performed using Empower® 3 software. Chromatographic analysis was carried out using a C18 ACE column (250 mm × 4.5 mm, 5 mm). The mobile phase comprises 0.1% v/v ortho-phosphoric acid and acetonitrile in the ratio of 45:55 (v/v). The mobile phase was degassed for 20 min by sonication using an ultrasonic bath (Elmasonic S 180, Singen, Germany). The injection volume was 10 µL with a flow rate of 1 mL/min; the column oven temperature was 30 °C ± 2 °C and the sample temperature of 25 °C ± 2 °C.
Nanosuspension equivalent to 10 mg CFZ was dissolved with mobile phase in a volumetric flask and samples were prepared at 100% concentration (16.7 µg/mL), sonicated and then filtered with 0.20 µm RC filter (Sartorius, AG, Goettingen, Germany). The analysis was performed in triplicate. The drug content was calculated using the following equation:
Nanosuspension equivalent to 10 mg CFZ was dissolved with mobile phase in a volumetric flask and samples were prepared at 100% concentration (16.7 µg/mL), sonicated and then filtered with 0.20 µm RC filter (Sartorius, AG, Goettingen, Germany). The analysis was performed in triplicate. The drug content was calculated using the following equation:
7
HPLC Analysis of Anthocyanin C-3-G
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The C-3-G content was analyzed using a Waters 2695 high-performance liquid chromatograph linked to a Waters 2996 photodiode array detector. All samples were filtered through a 0.45-μm Millipore membrane filter before injection. A 10-μL aliquot was separated using a SunFire-C18 column (250 mm × 4.6 mm, 5 μm; Waters, Milford, MA, USA) at 30 °C. The mobile phase consisted of solvent A (2% hydrochloric acid in methyl alcohol) and solvent B (water, methyl alcohol, acetonitrile, and acetic acid at a ratio of 160:90:90:40, v/v/v/v). Using an isocratic elution with the ratio of A:B as 93:7 at a flow rate of 1.0 mL/min, the C-3-G content was detected at 530 nm.
8
Synthesis and Characterization of Cytotoxic Compounds
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The known cytotoxic agents 1 (35 (link)) and seco-5 40 (link) were prepared as previously described. These
compounds were coupled
to known trioxolane29 (link) and dioxolane30 (link) intermediates via activated nitrophenyl carbonate
or isocyanate intermediates as we have described previously29 (link)−31 (link) and as further detailed in theSupporting Information .
All compounds tested in cells or animals were judged to be
of >95%
purity as determined using a Waters Micromass ZQTM, equipped with
Waters 2795 separation module, Waters 2996 photodiode array detector
(254 nm), and Waters 2424 ELS detector. Separations were carried out
with an XTerra MS C18, 5 μm, 4.6 mm × 50 mm column, at
ambient temperature (unregulated) using a mobile phase of water–methanol
containing a constant 0.10% formic acid. Representative LC chromatograms
are provided in theSupporting Information .
Mammalian cell lines were maintained in an atmosphere of
5% CO2 in RPMI 1640 media purchased from HyClone supplemented
with
10% FBS (Gibco), Pen/Strep (1× final concentration, Gemini Bio-Products),
and nonessential amino acids (UCSF Cell Culture Facility). Unless
otherwise noted, cell lines were obtained from ATCC and verified by
STR profiling. Graphing and analysis of data were done using GraphPad
Prism 6 software and Microsoft Excel 2010. Figures were prepared with
Adobe Design Standard CS6 software.
compounds were coupled
to known trioxolane29 (link) and dioxolane30 (link) intermediates via activated nitrophenyl carbonate
or isocyanate intermediates as we have described previously29 (link)−31 (link) and as further detailed in the
All compounds tested in cells or animals were judged to be
of >95%
purity as determined using a Waters Micromass ZQTM, equipped with
Waters 2795 separation module, Waters 2996 photodiode array detector
(254 nm), and Waters 2424 ELS detector. Separations were carried out
with an XTerra MS C18, 5 μm, 4.6 mm × 50 mm column, at
ambient temperature (unregulated) using a mobile phase of water–methanol
containing a constant 0.10% formic acid. Representative LC chromatograms
are provided in the
Mammalian cell lines were maintained in an atmosphere of
5% CO2 in RPMI 1640 media purchased from HyClone supplemented
with
10% FBS (Gibco), Pen/Strep (1× final concentration, Gemini Bio-Products),
and nonessential amino acids (UCSF Cell Culture Facility). Unless
otherwise noted, cell lines were obtained from ATCC and verified by
STR profiling. Graphing and analysis of data were done using GraphPad
Prism 6 software and Microsoft Excel 2010. Figures were prepared with
Adobe Design Standard CS6 software.
9
Isolation and Purification of Compound 2
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Four litres of fermentation broth of SUKA34::rapH/pKU503rapΔM9AT::M6ATm was centrifuged to obtain a mycelial cake, which was extracted twice with 500 ml of acetone. The acetone was removed in vacuo, and the residual aqueous layer was extracted twice with ethyl acetate (EtOAc). The resultant EtOAc layer was concentrated in vacuo to afford 1.9 g of crude extract. The crude extract was subjected to medium-pressure liquid chromatography (MPLC) on silica gel (SNAP Ultra 25 g, Biotage, Uppsala, Sweden) eluted with a gradient system of n-hexane–EtOAc (0-25% EtOAc) followed by a stepwise solvent system of chloroform (CHCl3)–methanol (MeOH) (0, 1, 3, 5, 10, 50 and 90% MeOH). The 3% MeOH fraction (242 mg) was collected and subjected to silica gel MPLC (SNAP Ultra 25 g) with isocratic elution with 3% MeOH in CHCl3. The fractions were monitored by UPLC analysis, and fractions containing 2 were collected (48.1 mg). The sample was further purified by preparative reversed-phase HPLC using a CAPCELL PAK MG-II C18 column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan). 2 was detected using a 2996 photodiode array detector (Waters) and a 3100 mass detector (Waters) following elution with 80% aqueous acetonitrile, and 4.4 mg of 2 was obtained.
10
HPLC Separation of Grape Leaf Phenolics
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