Active FOP and FOE fractions were analyzed by RP-HPLC on a XTerra C18 column (4.6 mm∗100 mm, 3.5 mm) using a Waters 2696 system equipped with a 600 PDA and Empower software. Solvents A and B were H2O and MeCN supplemented with 0.08% (v/v) TFA. Linear 0–60% gradients of B into A over 45 min, 60–95% B into A for1 min followedby 95% A isocratic gradient during 10 min were used for elution at 1 mL/min flow rate, with UV detection at 214, 268, 280, and 220 nm. For separation, preparative RP-HPLC was performed usingan XTerra C18 column (19 mm × 300 mm, 10 mm, Waters) with the solvents A and B, 0.1% TFA (v/v) in H2O and 0.08% TFA (v/v) in MeCN, respectively. For elution we used linear 0–60% gradients of B into A over 40 min, 60–95% B into A for1 min followedby an isocratic gradient during 10 min with 95% A and 5% B at 10 mL/min flow rate, with UV detection at 214 nm (Solecki et al., 2015 (link)).
Xterra c18 column
Manufactured by Waters Corporation
Sourced in United States, Japan
The XTerra C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with C18 functionalized ligands, which provides excellent retention and selectivity for a variety of analytes. The column is suitable for reversed-phase HPLC applications and can be used with both aqueous and organic mobile phases.
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Lab products found in correlation
11c r pk11195, by Horiba (2 mentions)
Triart c18 column, by YMC (2 mentions)
Empower software, by Waters Corporation (2 mentions)
Escalab 250xi, by Thermo Fisher Scientific (2 mentions)
Sigma 300, by Zeiss (2 mentions)
Prominence lc system, by Shimadzu (1 mentions)
Securityguard c18 guard column, by Phenomenex (1 mentions)
Alliance 2965, by Waters Corporation (1 mentions)
Aryl sulfatase, by Merck Group (1 mentions)
β glucuronidase, by Merck Group (1 mentions)
Sep pak spe cartridge, by Waters Corporation (1 mentions)
Tsq quantum mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
1260 series hplc system, by Agilent Technologies (1 mentions)
1100 hplc system, by Waters Corporation (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
Alkaline phosphatase, by Promega (1 mentions)
6460 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Nuclease p1, by US Biological (1 mentions)
996 pda detector, by Waters Corporation (1 mentions)
47 protocols using xterra c18 column
1
Analytical and Preparative RP-HPLC Profiling
2
Synthesis and Characterization of Peptide Substrates
3
HPLC Quantification of FPV
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The FPV quantification was performed by HPLC (Waters 2690 Alliance, Shimadzu, Japan) supplied with a Waters 996 photodiode array detector using a C-18 Xterra column (4.6 × 250 mm and 5 μm particle size). The mobile phase consisting of 5:95 v/v acetonitrile: water and 0.1% phosphoric acid was used. The HPLC system conditions were set as follows: UV detector wavelength of 320 nm, a flow rate of 1 mL/min, and sample injection of 10 µL. Samples were purified using a 0.22 µm syringe filter. The retention and running times of the sample were 9.5 and 13 min, respectively.
4
Tomato Nicotianamine Extraction and Analysis
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Nicotianamine was extracted from 1 g of homogenized fresh tomato by adding 10 mL of water. After stirring and centrifugation for 10 min (6000 rpm, 25 °C), the supernatant was filtered (0.45 μm hydrophilic PTFE filter from Millipore) and diluted with a 0.1% (v/v) formic acid aqueous solution by a factor of 25. A 5-µL volume of the final extract was injected into the HPLC-ESI-MS/MS system. Chromatography was performed using a C18 XTerra column (150 × 2.1 mm, 3.5 µm from Waters) kept at 35 °C. Mobile phases consisted water (phase A) and water/acetonitrile (30:70) (phase B), both of them containing 0.1% (v/v) formic acid. The flow rate of the mobile phase was 140 µL/min and it was entirely introduced into the mass spectrometer. The applied gradient was: t0−0.5 min, 100% A; t0.5−15 min, 100% B.
The ESI detection was performed in positive ionization. The working MS parameters were the following ones: the capillary voltage at 5500 V; the probe temperature at 450 °C; the curtain gas was high-purity nitrogen at 5 L/min; the collision gas high-purity nitrogen at 4 mTorr; the nebulizer gas was air at 2 L/min; the drying gas was air at 20 L/min.
The ESI detection was performed in positive ionization. The working MS parameters were the following ones: the capillary voltage at 5500 V; the probe temperature at 450 °C; the curtain gas was high-purity nitrogen at 5 L/min; the collision gas high-purity nitrogen at 4 mTorr; the nebulizer gas was air at 2 L/min; the drying gas was air at 20 L/min.
5
Synthesis of Ac-VLTK-AOMK Peptide Inhibitor
6
Radiochemical Purity and pKa Determination
7
LC-MS Analysis of SAG Analog
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A 1 mL SAG analog culture sample was centrifuged and 50 μL of supernatant was used for analysis. LC-MS was performed using an API 3000 Triple Quad LC-MS with a Turbo Ion Spray source (PE Sciex) coupled with a Shimadzu Prominence LC system. Chromatography was performed through a Waters XTerra C18 column (5 mm, 2.1 mm × 250 mm) and MS analysis was conducted in positive ion mode. Following a 3 μL injection from the 50 μL sample, a linear gradient of 5–95% acetonitrile (balance water; both solutions containing 0.1% formic acid) was used for 20 min at a flow rate of 0.2 mL/min.
8
Paclitaxel Quantification in Tumors and Plasma
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Tumors and plasma samples were prepared and paclitaxel concentrations were analyzed by LC-MS/MS. Briefly, the samples were extracted with 100% acetonitrile containing carbamazepine as an internal standard, and chromatography was conducted on an Xterra C18 column (50 × 2.1 mm i.d., 5 μm, Waters, Milford, MA, USA) with a SecurityGuard C18 guard column (2.0 × 4.0 mm i.d., Phenomenex, Torrance, CA, USA) maintained at room temperature. The mobile phase was 95% v/v solvent A (deionized water containing 0.1% v/v formic acid)/5% v/v solvent B (acetonitrile containing 0.1% v/v formic acid) at a flow rate of 0.4 mL/min. A linear gradient of the two solvents was used: start at 95% A and hold for 0.5 min, ramp to 5% A to 0.6 min, and hold until 4 min. The flow rate was 0.4 mL/min throughout the gradient. The retention times of paclitaxel and the internal standard (IS) were 3.0 and 2.8 min, respectively. The electrospray ionization source was operated at 5500 V and 550°C. The samples were analyzed via multiple reaction monitoring. The monitoring ions were set as m/z 876 → 308 for paclitaxel and m/z 237 → 194 for the IS. The scan dwell time was 0.1 sec for each channel. Acquisition and analysis of data were performed using the Analyst software ver. 1.5.2 (Applied Biosystems, Foster City, CA, USA).
9
Synthesis of (R)-4-Amino-N-(1-(4-Chlorophenyl)-3-Hydroxypropyl)-1-(7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)Piperidine-4-Carboxamide
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Example 15
HCl (4M in dioxane) (0.378 mL, 1.51 mmol) was added to (R)-tert-butyl 4-(1-(4-chlorophenyl)-3-hydroxypropylcarbamoyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-ylcarbamate (Intermediate 42) (0.160 g, 0.30 mmol) in DCM (3 mL). The resulting suspension was stirred at 20° C. for 3 hours. The reaction mixture was evaporated. The crude product was purified by preparative HPLC (Waters XTerra C18 column, 5 μm silica, 19 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% ammonia) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (R)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (0.051 g, 39.3%) as a white solid.
1H NMR (399.9 MHz, DMSO-d6) δ 1.46 (2H, d), 1.86 (1H, d), 1.90-1.93 (1H, m), 2.10 (2H, m), 3.37 (1H, t), 3.55 (2H, d), 4.40 (2H, d), 4.53 (2H, m), 4.88 (1H, d), 6.58 (1H, t), 7.16 (1H, t), 7.32-7.38 (4H, m), 8.14 (1H, d), 8.43 (1H, d), 11.63 (1H, s) no visible NH2. MS m/e MH+ 429; HPLC tR=1.46 min.
10
Synthesis of 4-Amino-N-(3-Amino-1-(4-Chlorophenyl)-3-Oxopropyl)-1-(7H-Pyrrolo[2,3-d]Pyrimidin-4-yl)Piperidine-4-Carboxamide
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Example 66
HCl in 1, 4-dioxane (4M) (0.228 mL, 0.91 mmol) was added to tert-butyl 4-(3-amino-1-(4-chlorophenyl)-3-oxopropylcarbamoyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-ylcarbamate (Intermediate 108) (99 mg, 0.18 mmol) in DCM (4 mL) at 20° C. The resulting solution was stirred at 20° C. for 3 hours. The reaction mixture was filtered through a PTFE cup and the collected solid was purified by preparative HPLC (Waters XTerra C18 column, 5μ silica, 30 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% NH3) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford 4-amino-N-(3-amino-1-(4-chlorophenyl)-3-oxopropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (9.00 mg, 11.1%) as a cream solid.
1H NMR (399.9 MHz, DMSO-d6) δ 1.40-1.43 (2H, m), 1.93-1.96 (2H, m), 2.18 (2H, s), 3.54-3.56 (2H, m), 4.36-4.40 (2H, m), 5.12 (1H, d), 6.58 (1H, d), 6.81 (1H, s), 7.15-7.16 (1H, m), 7.32-7.37 (5H, m), 8.13 (1H, s), 8.76 (1H, d), 11.63 (1H, s), (no NH2 visible).
m/z (ESI+) (M+H)+=442; HPLC tR=1.47 min.
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