Folic acid (0.88 g) in 0.1-L 50 mM Tris-HCI (pH 7.5) was incubated with 5 μg mL− 1Variovorax sp. F1 rPDA at 30 °C for 12 h. The rPDA was denatured with 1 M NaOH, then the pH of the reaction was reduced to < 4.0 with 1 M HCl to precipitate DFA with > 99% purity. Deaminopteroic acid was produced by incubating the DFA (~ 0.5 g) with 5 μg mL− 1 rCPG in 0.1-L 50 mM Tris-HCI (pH 7.5) containing 0.2 mM ZnSO4 at 30 °C for 6 h, then purified as described above to > 99%. The DFA and DPA were confirmed by liquid chromatography-mass spectrometry (LCMS). An LCMS 8030 spectrometer (Shimadzu Co., Kyoto, Japan) was equipped with a Purospher® STAR RP-18 endcapped column (particle size 5 μm, Merck KGaA, Darmstadt, Germany) and the flow rate of a 40-min linear gradient from 0 to 40% acetonitrile in 0.05% formic acid was 0.8 mL min− 1. Mass ions were detected in the negative mode under the following conditions: probe voltage, 3.5 kV; detection range, m/z = 10–500 for DFA (precursor m/z 441) and 10–400 (precursor m/z 312) for DPA; column temperature, 40 °C; desolvation line temperature, 250 °C; heat block temperature, 400 °C; nebulizer gas, 3 L min− 1; drying gas, 15 L min− 1.
Purospher star rp 18 endcapped column
Manufactured by Merck Group
Sourced in Germany, Japan, United States
The Purospher® STAR RP-18 endcapped column is a 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 octadecylsilyl (C18) functional groups, which provides high retention and selectivity for non-polar and moderately polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Lcms 8030 spectrometer, by Shimadzu (1 mentions)
Agilent 1260 infinity system, by Agilent Technologies (1 mentions)
Lcms 8030 system, by Shimadzu (1 mentions)
L 7455 diode array detector, by Hitachi (1 mentions)
L 7100 pump, by Hitachi (1 mentions)
L 7200 autosampler, by Hitachi (1 mentions)
Turbo ion spray source, by AB Sciex (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Guard column, by Merck Group (1 mentions)
C18 guard column, by Merck Group (1 mentions)
Glucose cii test kit, by Fujifilm (1 mentions)
1200 infinity series, by Agilent Technologies (1 mentions)
Uplc system, by Shimadzu (1 mentions)
Lcms 8030 triple quadrupole mass spectrometer, by Shimadzu (1 mentions)
Mc rr, by Enzo Life Sciences (1 mentions)
Mc lr, by Enzo Life Sciences (1 mentions)
Mc lf, by Enzo Life Sciences (1 mentions)
Mc la, by Enzo Life Sciences (1 mentions)
Mc lw, by Enzo Life Sciences (1 mentions)
Mc yr, by Enzo Life Sciences (1 mentions)
13 protocols using purospher star rp 18 endcapped column
1
Enzymatic Production of DFA and DPA
2
Quantitative Analysis of Microbial Metabolites
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Culture broth was diluted 10-fold with 80 mM KOH to solubilize cells and precipitates, and separated by centrifugation at 20,400 × g for 5 min at 4°C. Supernatants were analyzed by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Purospher STAR RP-18 end-capped column (Merck & Co. Inc., Kenilworth, NJ, USA), and absorption was monitored at 254 nm. The mobile phase comprised 10 mM sodium acetate (pH 4.5) and methanol (1:1 [vol/ vol]), with a flow rate of 0.8 mL min -1 . Analysis by LC-MS proceeded using an LCMS-8030 system (Shimadzu Corp., Kyoto, Japan) with the same column. The mobile phase comprised 10 mM ammonium formate (pH 4.0) and acetonitrile (1:4 [vol/ vol]), with a flow rate of 0.8 mL min -1 .
3
HPLC Analysis of Belinostat
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Belinostat was analyzed by high-performance liquid chromatography (HPLC). The HPLC system consisted of an L-7100 pump, L-7200 autosampler, L-7455 diode array detector at 265 nm (Hitachi, Tokyo, Japan) and a Purospher Star RP-18 endcapped column (250 × 4.6 mm, internal diameters 5 μm, Merck). The mobile phase was a mixture of 0.1% phosphoric acid and acetonitrile by gradient elution, and the flow rate was 1 mL/min. The column oven was set at 30 °C. Limits of detection and quantitation of belinostat were determined by dissolving belinostat at decreasing concentrations in distilled deionized water until the signal/noise ratios were 3 and 10, respectively. The linearity of the standard curves and intraday and interday precision and accuracy were established.
4
UPLC-MS/MS Analysis of Selegiline and Noscapine
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A Waters
Acquity UPLC system combined with a Purospher STAR RP-18 end-capped
column (100 mm × 2.1 mm, 2 μm, Merck KGaA, Darmstadt, Germany),
which was maintained in a column oven at approximately 40 °C,
was used for all analyses. The UPLC system was equipped with a Waters
Xevo tandem quadrupole mass spectrometer operated in positive electrospray
ionization mode. All parent ion transitions, product ion transitions,
cone voltages, and collision energies were optimized and modified
with the MassLynx 4.1 software data platform (Waters Acquity UPLC
system, Milford, USA). Chromatographic separation was carried out
with isocratic elution by a mobile phase consisting of 10 mM ammonium
formate (pH 6.40)/methanol [20:80 (v/v)]. The total run time was 4
min, the injection volume was 10 μL, and the flow rate was set
at 0.2 mL/min. The MS conditions were as follows: electrospray ionization,
positive mode; capillary voltage, 2.9 kV; cone voltage, 14 V; collision
energy, 12 V; desolvation temperature, 400 °C; source temperature,
150 °C; desolvation gas flow, 800 L/h; cone gas flow, 60 L/h;
and collision gas, argon. The monitored ion transitions were m/z 188.2 and 118.98 for selegiline and m/z 414.29 and 220.11 for noscapine (IS).
Acquity UPLC system combined with a Purospher STAR RP-18 end-capped
column (100 mm × 2.1 mm, 2 μm, Merck KGaA, Darmstadt, Germany),
which was maintained in a column oven at approximately 40 °C,
was used for all analyses. The UPLC system was equipped with a Waters
Xevo tandem quadrupole mass spectrometer operated in positive electrospray
ionization mode. All parent ion transitions, product ion transitions,
cone voltages, and collision energies were optimized and modified
with the MassLynx 4.1 software data platform (Waters Acquity UPLC
system, Milford, USA). Chromatographic separation was carried out
with isocratic elution by a mobile phase consisting of 10 mM ammonium
formate (pH 6.40)/methanol [20:80 (v/v)]. The total run time was 4
min, the injection volume was 10 μL, and the flow rate was set
at 0.2 mL/min. The MS conditions were as follows: electrospray ionization,
positive mode; capillary voltage, 2.9 kV; cone voltage, 14 V; collision
energy, 12 V; desolvation temperature, 400 °C; source temperature,
150 °C; desolvation gas flow, 800 L/h; cone gas flow, 60 L/h;
and collision gas, argon. The monitored ion transitions were m/z 188.2 and 118.98 for selegiline and m/z 414.29 and 220.11 for noscapine (IS).
5
Chromatographic Analysis of MNPL Additives
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The chromatographic separation to analyse the additives in the extracts of MNPLs was achieved with a C18 analytical column (Purospher® STAR RP-18 end-capped column (3 μm, 2.1 × 50 mm) from Merck) coupled to HRMS equipped with an ESI source, working in positive and negative ionization conditions by separate injections. The mobile phase consisted of (A) HPLC-water and (B) acetonitrile (in negative mode) or HPLC-water acidified with 0.05% formic acid (in positive conditions). The elution gradient conditions for the LC mobile phase started with 90% eluent A holding for 2 min and decreasing to 10% in 8 min, holding for two more min and rising to initial conditions (90% A) in one min and, finally, the re-equilibration of the system was achieved in 2 min. The flow rate was kept at 0.2 mL/min throughout the total chromatographic run of 15 min. The sample injection volume was set at 10 μL. Data were acquired in full scan (90–1500 Da) with an FWHM of 70,000 and, in parallel, in data-dependent scan at a resolution of 35,000 FWHM where the 10 most intense ions from the first full scan were further fragmented with an isolation of 1.0 Da and with a collision energy of 30 a.u.
The whole data were processed using Compound Discoverer 3.1.
The whole data were processed using Compound Discoverer 3.1.
6
LC-HRMS Analysis of Toluene Extracts
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The toluene extracts were re-analysed by LC(C18)-HRMS to investigate the organic additives and potential co-contaminants adsorbed onto the surface of the particles. In this case, the chromatographyc separation was achieved with the Acquity LC chromatograph equipped with Purospher® STAR RP-18 endcapped column (5 µm, 2 ×125 mm) from Merck. Toluene was used as mobile phase under isocratic mode at 0.5 mL/min. Injection volume was set at 20 μL, and the total chromatographyc run was 10 min. The chromatographic system was coupled to a QExactive equipped with an APPI source operating in negative and positive ionisation modes in two different injections. Data acquisition was performed in data dependant scan where the 10 most intense ions from full scan (m/z 50-750) were further fragmented with an isolation of 1.0 Da at a collision energy of 30 a.u. The data was acquired working at resolution of 35,000 FWHM.
7
Ion-pair LC-MS/MS Analysis of Ethyl Sulfate
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The analysis of EtS was performed with a previously described methodology based on ion-pair LC-MS/MS (Mastroianni et al., 2014) (link) using a Symbiosis TM Pico System (Spark Holland, Emmen, The Netherlands) equipped with a 100 µL sample loop. The LC system was coupled to a 4000QTRAP hybrid triple quadrupole-linear ion trap (QqLIT) mass spectrometer equipped with a Turbo Ion Spray source (AB-Sciex, Foster City, CA, USA) set in the negative ionization mode (ESI-). Chromatographic separation was performed with a Purospher Star RP-18 endcapped column (125 mm × 2 mm, particle size 5 µm) preceded by a guard column of the same packing material and particle size, both from Merck (Darmstad, Germany) and a mobile phase consisting of MeOH and water both containing 5 mM of dibutylammonium acetate (DBAA) at a constant flow rate of 0.3 mL/min. MS/MS detection was performed in selected reaction monitoring mode (SRM) recording 2 SRM transitions for EtS (125→97, 125→80) and one for EtS-d5 (130→98). Data acquisition and evaluation was performed with Analyst 1.5 software (AB-Sciex, Foster City, CA, USA). Quantification of the samples was based on the isotope dilution method.
8
Characterization of MC-Val-Cit-PAB-DOX
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1H-NMR measurements were performed in DMSO-d6 across the range of 0–15 ppm with a Varian 500 MHz instrument (Bruker, USA). The purity of MC-Val-Cit-PAB-DOX was determined by HPLC (Purospher STAR RP-18 endcapped column [Merck, Germany], 2:1 CH3OH/50 mM Et3N/HCO2H buffer [pH 2.8], flow rate 0.8 mL/min, λ=280 nm). The molecular weight was determined by mass spectrometry using a Waters ZQ 2000 (Waters, USA).
9
HPLC Analysis of Anthocyanin Compounds
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HPLC analysis of the anthocyanin compounds was based on a method developed by Azman et al. [39 (link)] using a Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA, USA) equipped with a Waters 2478 two-channel UV detector, two Waters 515 HPLC pumps, an auto-sampler, a column oven, and an online degasser. Analyses were carried out with a Purospher STAR RP18 end-capped column (250 mm × 4.6 mm i.d., particle size of 5 μm, Merck, Darmstadt, Germany) at 30 °C. The mobile phase consisted of 2% (v/v) of formic acid in water solution (solvent A) and 100% (v/v) methanol (solvent B). The gradient elution system was 15% (B) at 0 min, increasing to 35% (B) at 15 min and to 60% (B) at 30 min, reaching 80% (B) at 40 min. The flow rate was 1.0 mL/min and the injection volume was 20 µL. The duration of the analysis was 50 min.
The detection and quantification of the main anthocyanins present in the purified extract (D3R, C3R, D3G, and C3G) and the reaction samples were carried out at a wavelength of 520 nm, based on external standard solutions and their respective calibration curves.
The detection and quantification of the main anthocyanins present in the purified extract (D3R, C3R, D3G, and C3G) and the reaction samples were carried out at a wavelength of 520 nm, based on external standard solutions and their respective calibration curves.
10
Liquid Chromatography-Mass Spectrometry Quantification
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Instrumental analysis was performed by LC, using a Symbiosis™ Pico (SP104.002, Spark, Holland), connected in series with a 4000 QTRAP Hybrid Triple Quadrupole -Linear Ion Trap-MS equipped with a Turbo Ion Spray source (Applied Biosystems-Sciex, Foster City, CA, USA). Target compounds were separated on a Purospher Star RP-18 end-capped column (125 mm×2.0 mm, particle size 5 μm) with a C18 guard column (4×2.0 mm), both supplied by Merck (Darmstadt, Germany). The optimized separation conditions were as follows: solvent (A) water (0,1 % formic acid) and (B) methanol (10 mM ammonium acetate) at a flow rate of 0.25 mL/min. The gradient elution was: 50% (B) for initial and hold for 1 min; 80% (B) at 3 min and hold for 1 min; 90% (B) at 9 min and hold for 8 min; 100% (B) at 22 min and hold for 9 min. 50% (B) at 32 min and hold for 5 min to return to initial mode. The total chromatographic time was 37 min. The sample injection was 10 µL (Santín et al., 2016) .
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