Protein samples were desalted with a Chromolith RP-18e column (Merck, Kenilworth, NJ). These samples in Acetonitrile:water + 0.1% Formic acid were introduced by electrospray ionisation into a Micromass LCT Premier XE orthogonal acceleration reflecting TOF mass spectrometer in positive ion mode (Micromass, Milford, MA). The resultant m/z spectra were converted to mass spectra by using the maximum entropy analysis MaxEnt in the MassLynx suite of programs.
Chromolith rp 18e column
Manufactured by Merck Group
The Chromolith RP-18e column is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with octadecyl (C18) functional groups, which provides efficient separation of both polar and non-polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 1200 liquid chromatograph, by Agilent Technologies (2 mentions)
Impact 2 lc q tof ms, by Bruker (2 mentions)
Kieselgel 60, by Merck Group (1 mentions)
Lc 20ad hplc system, by Shimadzu (1 mentions)
6470 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Advance 400, by Bruker (1 mentions)
6 protocols using chromolith rp 18e column
1
Protein Desalting and Mass Spectrometry
2
Characterization of Synthetic Compounds
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Commercially available solvents and reagents were used (Alfa Aesar, Panreac). Thin layer chromatography (TLC) was used for monitoring the reactions on silica gel-coated glass plates (Merck Kieselgel 60) until completion; the TLC plates were visualised by staining with iodine vapor. A Mel-Temp digital melting point electrothermal 9100 apparatus was used for determining the melting points, using open capillaries; the reported results have not been corrected. NMR spectra were recorded on a Bruker Avance. Chemical shifts (δ) were reported in parts per million (ppm, from the residual solvent peak). A Chromolith RP-18e column (Merck, Kenilworth, NJ, 50 mm) was used for UPLC analysis, using an Agilent 1200 Liquid Chromatograph (Agilent, Omaha, NE). The products were analysed on a Bruker Impact II LC Q-TOF MS equipped with electrospray ionisation (ESI) in positive mode.
3
Extraction of Unreacted Polymer Residues
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Polymer samples of approximately 1.5 g were immersed into 20 mL of PBS solution (pH 7.4) or into 20 mL of ethanol and shaken at 37 ± 1 °C in a laboratory shaker (Incubating Shaker IKA KS 4000i, 135 rpm, Fisher Scientific, Prague, Czech Republic). After 40 days, aliquots of the solutions were analyzed to determine the content of the extracted low molecular residues after polymerization, i.e., monomers, crosslinker and initiator. The samples obtained directly after polymerization (without acetone washing treatment) and samples washed after polymerization in acetone were examined in the extraction experiments.
The quantification of the extracted unreacted monomers was conducted by a HPLC-UV method using a Shimadzu LC-20AD (Shimadzu technology, Kyoto, Japan). The analysis was performed on a MERCK Chromolith RP-18e column (Merck, Prague, Czech republic) under the following conditions: Mobile phase ACN/water (75/25 v/v), flow rate 2.5 mL·min−1, and injection volume 10 µL. The wavelength of UV detection was set at 245 and 205 nm for styrene and MA, respectively. All experiments were made in triplicates and relative standard deviation was below 5%.
The quantification of the extracted unreacted monomers was conducted by a HPLC-UV method using a Shimadzu LC-20AD (Shimadzu technology, Kyoto, Japan). The analysis was performed on a MERCK Chromolith RP-18e column (Merck, Prague, Czech republic) under the following conditions: Mobile phase ACN/water (75/25 v/v), flow rate 2.5 mL·min−1, and injection volume 10 µL. The wavelength of UV detection was set at 245 and 205 nm for styrene and MA, respectively. All experiments were made in triplicates and relative standard deviation was below 5%.
4
JTTZ Formula Compound Analysis by HPLC
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The chemical composition of the JTTZ formula was analyzed using high-performance liquid chromatography (HPLC). The separation was carried out on a Merck Chromolith RP-18e column (4.6 × 100 mm inner diameter). For HPLC analysis, a 5 μL test sample was injected into the column and eluted at a constant flow rate of 2 mL/min. Water with 0.02% phosphoric acid (mobile phase A) and acetonitrile (mobile phase B) was used. Composition was determined at a wavelength of 237 nm. The JTTZ formula powder was dissolved in methanol. All solutions were filtered through 0.45 μm nylon membrane Millex syringe filters before use. Chemical structures of the eight major compounds (mangiferin, coptisine hydrochloride, jatrorrhizine hydrochloride, salvianolic acid B, aloin, berberine hydrochloride, palmatine hydrochloride, and lovastatin) were identified in the finished dose. Representative chromatograms of the JTTZ formula and the single granule and chemical structures of the major compounds are shown in Figure 1 .
5
Quantification of TPT Release Kinetics
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To determine the TPT release kinetics, TPT quantification was performed according to the HPLC-FLD method described earlier [46 (link)]. Briefly, a Shimadzu LC-20AD HPLC system (Shimadzu, Kyoto, Japan) fitted with a Chromolith RP-18e column (Merck, Prague, Czech Republic) was used under the following conditions: mobile phase ACN/water 70/30 v/v, flow rate 1.5 mL/min, injection volume 10 µL, and fluorescence detection at an excitation wavelength at 361 nm and an emission wavelength at 527 nm.
Round targets (8 mm discs) were cut from TPT-loaded mats, incubated in 2 mL of water (pH 6.7) and kept in the dark at 4 °C. At predetermined time intervals (5, 10, 15, 20, 25, 30, 45 min, 1, 1.5, 2, 3, 4, and 5 h), 2 mL of water was withdrawn and analyzed by HPLC-FLD. Then, fresh water with the same volume was added for replacement. The experiments were carried out in triplicate. The cumulative drug release (CR, %) was calculated using Equation (3):
where cn and cn−1 are the concentrations of drug (µg/mL) in the release medium after n and n − 1 withdrawing steps, respectively; n is the number of withdrawing steps; and V is the volume of release medium. The resulting CR is expressed as the average value ± SD.
Round targets (8 mm discs) were cut from TPT-loaded mats, incubated in 2 mL of water (pH 6.7) and kept in the dark at 4 °C. At predetermined time intervals (5, 10, 15, 20, 25, 30, 45 min, 1, 1.5, 2, 3, 4, and 5 h), 2 mL of water was withdrawn and analyzed by HPLC-FLD. Then, fresh water with the same volume was added for replacement. The experiments were carried out in triplicate. The cumulative drug release (CR, %) was calculated using Equation (3):
where cn and cn−1 are the concentrations of drug (µg/mL) in the release medium after n and n − 1 withdrawing steps, respectively; n is the number of withdrawing steps; and V is the volume of release medium. The resulting CR is expressed as the average value ± SD.
6
NMR and Mass Spectrometry Analysis
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1H spectra
were recorded at 400 MHz on a Bruker Advance 400 instrument. Molar
mass was determined with an Agilent 6470 triple quadrupole mass spectrometer.
RP-HPLC analyses were performed on a Chromolith RP-18e column (Merck,
Kenilworth, NJ, 50 mm), using an Agilent 1200 Liquid Chromatograph
(Agilent, Omaha, NE). All products were analyzed on a Bruker Impact
II LC Q-TOF MS equipped with electrospray ionization (ESI) in positive
mode.
were recorded at 400 MHz on a Bruker Advance 400 instrument. Molar
mass was determined with an Agilent 6470 triple quadrupole mass spectrometer.
RP-HPLC analyses were performed on a Chromolith RP-18e column (Merck,
Kenilworth, NJ, 50 mm), using an Agilent 1200 Liquid Chromatograph
(Agilent, Omaha, NE). All products were analyzed on a Bruker Impact
II LC Q-TOF MS equipped with electrospray ionization (ESI) in positive
mode.
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