High performance chromatographic analyses were performed using an Agilent 1260 Infinity chromatograph coupled with a DAD detector. The biotransformation of an equimolar ratio of substrates A and B2 was monitored by reverse-phase C18 HPLC using a Phenomenex Kinetex Polar C18 (4.6 × 100 mm, 2.6 µm) column. The isocratic elution was prepared using methanol (eluent A) and 50 mM formate buffer (eluent B) in the ratio of 45% of eluent A (v/v). The pH of eluent B was adjusted to 4.1 using 1 M NaOH. The eluent flow rate was set at 1 mL min−1 and the separation column was maintained at 40 °C. Each 3-µL sample was injected using an autosampler. The total run time of each analysis was 20 min. The elution of compounds was monitored at a wavelength of 280 and 500 nm. Agilent Open Lab software (Agilent OpenLab CDS Chem Station product version A.02.10 [026]) was used for data processing and reporting. Identification of substrate peaks was achieved by comparing retention times with the standards.
Kinetex polar c18
Manufactured by Phenomenex
The Kinetex Polar C18 is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed to provide efficient and reliable separation of polar and ionizable analytes. The column features a proprietary bonded phase that enhances the retention and selectivity for polar compounds.
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Lab products found in correlation
Maxis hd, by Bruker (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Ultimate 3 000 uplc system, by Bruker (1 mentions)
Oasis hlb, by Waters Corporation (1 mentions)
Strata x, by Phenomenex (1 mentions)
Oasis prime hlb, by Waters Corporation (1 mentions)
Kinetex xb c18, by Phenomenex (1 mentions)
Zorbax bonus rp, by Agilent Technologies (1 mentions)
Luna omega polar, by Phenomenex (1 mentions)
Zorbax eclipse plus c18, by Agilent Technologies (1 mentions)
5 protocols using kinetex polar c18
1
Biotransformation Monitoring via HPLC
2
Liquid Chromatography-Mass Spectrometry Analysis
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The reaction mixture was concentrated in vacuo, then the residue was redissolved by sonication in 1.0 ml CH2Cl2 and diluted 20× for analysis. LC–MS/MS analysis was performed using a Thermo UltiMate 3000 UPLC system coupled to a high-resolution Q-ToF mass spectrometer (Bruker Daltonics MaXis HD). A polar C18 column (Kinetex polar C18, 100 × 2.1 mm, 2.6 µm particle size, 100 Å pore size; Phenomenex) was used as the stationary phase, and a high-pressure binary gradient pump was used to deliver the mobile phase, which consisted of solvent A (100% H2O + 0.1% FA) and solvent B (100% acetonitrile + 0.1% FA). The flow rate was set to 0.5 ml min–1 and the injection volume for each sample was 10 µl. Following injection, samples were eluted using the following gradient: 0–1.0 min, 5% B; 9.0–11.0 min, 100%; 11.5–14.0 min, 5%.
3
Targeted Metabolite Profiling by LC-MS/MS
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The reaction mixture was concentrated in vacuo, then the residue was reconstituted by sonication in 1.5 ml CH2Cl2–methanol (2:1). An aliquot of 150 µl was transferred from each sample and concentrated in vacuo. The dried extract was resuspended in 1 ml of HPLC-grade MeOH–H2O (1:1) and diluted for analysis. LC–MS/MS analysis was performed using a Thermo UltiMate 3000 UPLC system coupled to a high-resolution Q-ToF mass spectrometer (Bruker Daltonics MaXis HD). A polar C18 column (Kinetex polar C18, 100 × 2.1 mm, 2.6 µm particle size, 100 Å pore size; Phenomenex) was used as the stationary phase, and a high-pressure binary gradient pump was used to deliver the mobile phase, which consisted of solvent A (100% H2O + 0.1 % formic acid (FA)) and solvent B (100% acetonitrile + 0.1% FA). The flow rate was set to 0.5 ml min–1 and the injection volume for each sample was 10 µl. Following injection, samples were eluted using the following gradient: 0–1.0 min, 5% B; 9.0–11.0 min, 100%; 11.5–14.0 min, 5%.
4
Comprehensive Analytical Method Development
5
Liquid Chromatography-Mass Spectrometry Protocol
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Samples were analyzed by liquid chromatography (Ultimate 3000 RSLC nano system, Thermo Fisher Scientific) interfaced with a Bruker Impact II quadrupole time-of-flight mass spectrometer equipped with an electrospray source (Bruker Daltoniks, Bremen). Chromatographic separation was performed on a column Kinetex Polar C18, 2.6µm, 100Å, and 100 × 2.1mm (Phenomenex) at 45°C. The gradient was as follows: 1 min 5% phase B, then in 5 min to 50% phase B, next in 4 min to 100% phase B, held for 8 min at 100% phase B, subsequently in 1 min to 0% phase B and held for 6 min at 0% phase B. Internal calibration was achieved with a sodium formate solution introduced to the ion source via a 20μL loop at the beginning of each analysis using a six-port valve. Calibration was then performed using high-precision calibration mode (HPC). The Mass Spectrometer was operated in positive ionization mode on the full scan mode and data was acquired in the mass range from m/z 50 to 1000 with a spectra rate of 1 Hz. The capillary was set at 4500 V, the End Plate offset at 500 V, the Nebulizer gas (N2) at 4 Bar and the Dry gas (N2) at 8 L/min at 200°C. To evaluate the performance of the instrument, quality control pools (QCpool, n=3; made by pooling 5µl of upper and lower extracted phases) were injected every 7 to 10 samples.
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